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Giuseppe BIAGINI

presso: Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche


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Pubblicazioni

2022 - Neurosteroids and Status Epilepticus [Articolo su rivista]
Gol, Mohammad; Lucchi, Chiara; Biagini, Giuseppe
abstract

Status epilepticus (SE) is a common neurological emergency with considerable associated health-care costs, morbidity, and mortality. In about one-third of cases, SE is refractory towards first-line intravenous benzodiazepines. Allopregnanolone, a neurosteroid that positively modulates synaptic and extrasynaptic γ-aminobutyric acid type A receptors, has been evaluated as a possible novel treatment for SE. Notwithstanding the positive results obtained in the animal models of SE, the use of allopregnanolone for the treatment of benzodiazepine-resistant SE in humans resulted in controversial findings. Here, we summarize the main preclinical and clinical evidence about the effects of neurosteroids on SE to provide a possible pathophysiological background for their rational use.


2021 - Allopregnanolone and pregnanolone are reduced in the hippocampus of epileptic rats, but only allopregnanolone correlates with the seizure frequency [Articolo su rivista]
Lucchi, Chiara; Costa, Anna Maria; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Background: Neurosteroids modulate epileptic activity by interacting with the γ-aminobutyric acid type A receptor, but their brain levels are still undetermined. Objectives: We aimed to establish levels of neurosteroids in the neocortex and hippocampus by liquid chromatography–mass spectrometry in epileptic rats. Methods: Kainic acid-treated rats were continuously monitored up to 9 weeks to determine the seizure frequency by video electrocorticography (n=23), and compared to age-matched controls monitored in the same manner (n=11). Results: A reduction in allopregnanolone (-50%; p<0.05, Mann-Whitney test) and pregnanolone levels (-64%; p<0.01) was found in the hippocampus, whereas pregnenolone sulfate, pregnenolone, progesterone, and 5α-dihydroprogesterone were nonsignificantly reduced. No changes were found in the neocortex. Moreover, allopregnanolone (but not pregnanolone) levels were positively correlated with the seizure frequency (r2=0.4606, p<0.01). Conclusions: These findings indicate a selective reduction in hippocampal levels of 3α-reduced neurosteroids. This reduction was partially mitigated by seizures in the case of allopregnanolone.


2021 - Antiseizure Effects of Fully Characterized Non-Psychoactive Cannabis sativa L. Extracts in the Repeated 6-Hz Corneal Stimulation Test [Articolo su rivista]
Costa, Anna-Maria; Senn, Lara; Anceschi, Lisa; Brighenti, Virginia; Pellati, Federica; Biagini, Giuseppe
abstract

Compounds present in Cannabis sativa L. preparations have recently attracted much attention in the treatment of drug-resistant epilepsy. Here, we screened two olive oil extracts from a non-psychoactive C. sativa variety, fully characterized by high-performance liquid chromatography and gas chromatography. Particularly, hemp oils with different concentrations of terpenes were administered at the same dose of cannabidiol (25 mg/kg/day orally), 1 h before the 6-Hz corneal stimulation test (44 mA). Mice were stimulated once a day for 5 days and evaluated by videoelectrocorticographic recordings and behavioral analysis. Neuronal activation was assessed by FosB/DFosB immunoreactivity. Both oils significantly reduced the percentage of mice experiencing convulsive seizures in comparison to olive oil-treated mice (p < 0.050; Fisher’s exact test), but only the oil enriched with terpenes (K2) significantly accelerated full recovery from the seizure. These effects occurred in the presence of reduced power of delta rhythm, and, instead, increased power of theta rhythm, along with a lower FosB/deltaFosB expression in the subiculum (p < 0.050; Duncan’s method). The overall findings suggest that both cannabinoids and terpenes in oil extracts should be considered as potential therapeutic agents against epileptic seizures and epilepsy.


2021 - Editorial: Endocrine modulators of neurological processes: potential treatment targets of pediatric neurological diseases. [Articolo su rivista]
Ni, H.; Biagini, G.; Upadhya, D.; Capuano, A.
abstract

Editorial on the Research Topic Endocrine Modulators of Neurological Processes: Potential Treatment Targets of Pediatric Neurological Diseases.


2021 - Evaluation of e-health applications for paediatric patients with refractory epilepsy and maintained on ketogenic diet [Articolo su rivista]
Costa, Anna Maria; Marchio', Maddalena; Bruni, Giulia; Maria Bernabei, Silvia; Cavalieri, Silvia; Bondi, Marina; Biagini, Giuseppe
abstract

E-health technologies improve healthcare quality and disease management. The aim of this study was to develop a ketogenic diet management app as well as a website about this dietary treatment and to evaluate the benefits of giving caregivers access to various web materials designed for paediatric patients with refractory epilepsy. Forty families participated in the questionnaire survey, from January 2016 to March 2016. All caregivers were exposed to paper-based materials about the ketogenic diet, whereas only 22 received the app, called KetApp, and videos produced by dieticians. Caregivers with free access to web materials were more satisfied than the others with the informative material provided by the centre (p 0.001, Mann–Whitney test). Indeed, they showed a better attitude towards treatment, and they became more aware of dietary management in comparison to the control group (p 0.001). Moreover, caregivers provided with web materials were stimulated to pursue the treatment (p = 0.002) and to introduce it to their children and other people (p = 0.001). Additionally, caregivers supplied with web materials were more willing to help other families in choosing the ketogenic diet (p = 0.004). Overall, these findings indicate that web materials are beneficial for caregivers of paediatric patients with refractory epilepsy in our centres. Thus, the use of e-health applications could be a promising tool in the daily aspects of ketogenic diet management, and it is especially of value in the attempt to start or maintain the diet during the ongoing COVID-19 pandemic crisis.


2021 - HPLC-UV-HRMS analysis of cannabigerovarin and cannabigerobutol, the two impurities of cannabigerol extracted from hemp [Articolo su rivista]
Tolomeo, Francesco; Russo, Fabiana; Vandelli, Maria Angela; Biagini, Giuseppe; Capriotti, Anna Laura; Laganà, Aldo; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe; Citti, Cinzia
abstract

A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in "pure" commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01-1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.


2021 - Relationship between delta rhythm, seizure occurrence and allopregnanolone hippocampal levels in epileptic rats exposed to the rebound effect. [Articolo su rivista]
Costa, A. M.; Lucchi, C.; Malkoç, A.; Rustichelli, C.; Biagini, G.
abstract

Abstract: Abrupt withdrawal from antiepileptic drugs is followed by increased occurrence of epileptic seizures, a phenomenon known as the “rebound effect”. By stopping treatment with levetiracetam (LEV 300 mg/kg/day, n = 15; vs saline, n = 15), we investigated the rebound effect in adult male Sprague-Dawley rats. LEV was continuously administered using osmotic minipumps, 7 weeks after the intraperitoneal administration of kainic acid (15 mg/kg). The effects of LEV were determined by comparing time intervals, treatments, and interactions between these main factors. Seizures were evaluated by video-electrocorticographic recordings and power band spectrum analysis. Furthermore, we assessed endogenous neurosteroid levels by liquid chromatography-electrospray- tandem mass spectrometry. LEV significantly reduced the percentage of rats experiencing seizures, reduced the seizure duration, and altered cerebral levels of neurosteroids. In the first week of LEV discontinuation, seizures increased abruptly up to 700% (p = 0.002, Tukey’s test). The power of delta band in the seizure postictal component was related to the seizure occurrence after LEV withdrawal (r2 = 0.73, p < 0.001). Notably, allopregnanolone hippocampal levels were positively related to the seizure occurrence (r2 = 0.51, p = 0.02) and to the power of delta band (r2 = 0.67, p = 0.004). These findings suggest a role for the seizure postictal component in the rebound effect, which involves an imbalance of hippocampal neurosteroid levels.


2021 - Response letter to the Editorial: "Ketogenic diet in ADPKD patients" [Articolo su rivista]
Magistroni, Riccardo; Biagini, Giuseppe
abstract


2021 - The Pilocarpine Model of Mesial Temporal Lobe Epilepsy: Over One Decade Later, with More Rodent Species and New Investigative Approaches [Articolo su rivista]
Lévesque, Maxime; Biagini, Giuseppe; de Curtis, Marco; Gnatkovsky, Vadym; Pitsch, Julika; Wang, Siyan; Avoli, Massimo
abstract

Fundamental work on the mechanisms leading to focal epileptic discharges in mesial temporal lobe epilepsy (MTLE) often rests on the use of rodent models in which an initial status epilepticus (SE) is induced by kainic acid or pilocarpine. In 2008 we reviewed how, following systemic injection of pilocarpine, the main subsequent events are the initial SE, the latent period, and the chronic epileptic state. Up to a decade ago, rats were most often employed and they were frequently analysed only behaviorally. However, the use of transgenic mice has revealed novel information regarding this animal model. Here, we review recent findings showing the existence of specific neuronal events during both latent and chronic states, and how optogenetic activation of specific cell populations modulate spontaneous seizures. We also address neuronal damage induced by pilocarpine treatment, the role of neuroinflammation, and the influence of circadian and estrous cycles. Updating these findings leads us to propose that the rodent pilocarpine model continues to represent a valuable tool for identifying the basic pathophysiology of MTLE.


2021 - The novel heptyl phorolic acid cannabinoids content in different Cannabis sativa L. accessions [Articolo su rivista]
Linciano, Pasquale; Russo, Fabiana; Citti, Cinzia; Tolomeo, Francesco; Paris, Roberta; Fulvio, Flavia; Pecchioni, Nicola; Vandelli, Maria Angela; Laganà, Aldo; Capriotti, Anna Laura; Biagini, Giuseppe; Carbone, Luigi; Gigli, Giuseppe; Cannazza, Giuseppe
abstract

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to highresolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).


2020 - A proline derivative-enriched fraction from Sideroxylon obtusifolium protects against intracerebroventricular pilocarpine-induced injury associated with status epilepticus in mice [Articolo su rivista]
Everson Alexandre de Aquino, Pedro; Rabelo Bezerra, Jéssica; de Souza Nascimento, Tyciane; Tavares, Juliete; ROSAL LUSTOSA, Italo; José Maia Chaves Filho, Adriano; Mottin, Melina; Macêdo Gaspar, Danielle; Matos de Andrade, Geanne; Rose Tavares Neves, Kelly; Biagini, Giuseppe; Rocha Silveira, Edilberto; Socorro de Barros Viana, Glauce
abstract

The N-methyl-(2S,4R)-trans-4-hydroxy-L-proline-enriched fraction (NMP) from Sideroxylon obtusifolium was evaluated as a neuroprotective agent in the intracerebroventricular (icv) pilocarpine (Pilo) model. To this aim, male mice were subdivided into sham (SO, vehicle), Pilo (300 μg/1 μL icv, followed by the vehicle per os, po) and NMP-treated groups (Pilo 300 μg/1 μL icv, followed by 100 or 200 mg/kg po). The treatments started one day after the Pilo injection and continued for 15 days. The effects of NMP were assessed by characterizing the preservation of cognitive function in both the Y-maze and object recognition tests. The hippocampal cell viability was evaluated by Nissl staining. Additional markers of damage were studied—the glial fibrillary acidic protein (GFAP) and the ionized calcium-binding adaptor molecule 1 (Iba-1) expression using, respectively, immunofluorescence and western blot analyses. We also performed molecular docking experiments revealing that NMP binds to the γ-aminobutyric acid (GABA) transporter 1 (GAT1). GAT1 expression in the hippocampus was also characterized. Pilo induced cognitive deficits, cell damage, increased GFAP, Iba-1, and GAT1 expression in the hippocampus. These alterations were prevented, especially by the higher NMP dose. These data highlight NMP as a promising candidate for the protection of the hippocampus, as shown by the icv Pilo model.


2020 - Augmentation of endogenous neurosteroid synthesis alters experimental status epilepticus dynamics [Articolo su rivista]
Lucchi, C.; Costa, A. M.; Senn, L.; Messina, S.; Rustichelli, C.; Biagini, G.
abstract

Neurosteroids can modulate γ-aminobutyric acid type A receptor-mediated inhibitory currents. Recently, we discovered that the neurosteroids progesterone, 5α-dihydroprogesterone, allopregnanolone, and pregnanolone are reduced in the cerebrospinal fluid of patients with status epilepticus (SE). However, it is undetermined whether neurosteroids influence SE. For this reason, first we evaluated whether the inhibitor of adrenocortical steroid production trilostane (50 mg/kg) could modify the levels of neurosteroids in the hippocampus and neocortex, and we found a remarkable increase in pregnenolone, progesterone, 5α-dihydroprogesterone, and allopregnanolone levels using liquid chromatography tandem mass spectrometry. Second, we characterized the dynamics of SE in the presence of the varied neurosteroidal milieu by a single intraperitoneal kainic acid (KA; 15 mg/kg) injection in trilostane-treated rats and their controls. Convulsions started in advance in the trilostane group, already appearing 90 minutes after the KA injection. In contrast to controls, convulsions prevalently developed as generalized seizures with loss of posture in the trilostane group. However, this effect was transient, and convulsions waned 2 hours before the control group. Moreover, electrocorticographic traces of convulsions were shorter in trilostane-treated rats, especially at the 180-minute (P < .001) and 210-minute (P < .01) time points. These findings indicate that endogenous neurosteroids remarkably modulate SE dynamics.


2020 - BV-2 Microglial Cells Respond to Rotenone Toxic Insult by Modifying Pregnenolone, 5alpha-Dihydroprogesterone and Pregnanolone Level [Articolo su rivista]
Avallone, Rossella; Lucchi, Chiara; Puja, Giulia; Codeluppi, Alessandro; Filaferro, Monica; Vitale, Giovanni; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neuroinflammation, whose distinctive sign is the activation of microglia, is supposed to play a key role in the development and progression of neurodegenerative diseases. The aim of this investigation was to determine levels of neurosteroids produced by resting and injured BV-2 microglial cells. BV-2 cells were exposed to increasing concentrations of rotenone to progressively reduce their viability by increasing reactive oxygen species (ROS) production. BV-2 cell viability was significantly reduced 24, 48 and 72 h after rotenone (50–1000 nM) exposure. Concomitantly, rotenone (50–100 nM) determined a dose-independent augmentation of ROS production. Then, BV-2 cells were exposed to a single, threshold dose of rotenone (75 nM) to evaluate the overtime release of neurosteroids. In particular, pregnenolone, pregnenolone sulfate, progesterone, 5alpha-dihydroprogesterone (5-DHP), allopregnanolone, and pregnanolone, were quantified in the culture medium by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. BV-2 cells synthesized all the investigated neurosteroids and, after exposure to rotenone, 5DHP and pregnanolone production was remarkably increased. In conclusion, we found that BV-2 cells not only synthesize several neurosteroids, but further increase this production following oxidative damage. Pregnanolone and 5alpha-DHP may play a role in modifying the progression of neuroinflammation in neurodegenerative diseases.


2020 - GREASE II. A phase II randomized, 12-month, parallel-group, superiority study to evaluate the efficacy of a Modified Atkins Diet in Autosomal Dominant Polycystic Kidney Disease patients [Articolo su rivista]
Testa, Francesca; Marchiò, Maddalena; D’Amico, Roberto; Giovanella, Silvia; Ligabue, Giulia; Fontana, Francesco; Alfano, Gaetano; Cappelli, Gianni; Biagini, Giuseppe; Magistroni, Riccardo
abstract

Introduction Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a condition that causes progressive renal function decline. Preclinical data suggest the presence of a profound metabolic derangement in ADPKD. Cystic cells shift their energy metabolism from oxidative phosphorylation to aerobic glycolysis, show inhibition of fatty acid oxidation and become glutamine and arginine dependent. Recent preclinical experiences have suggested beneficial effect in terms of reduction of cystic size, interstitial fibrosis and disease progression, targeting these deregulated metabolic pathways by ketosis induction. The dietetic approach to ADPKD, because of low cost and absence of toxicity, represents an interesting therapeutic strategy. Methods and analysis The protocol describes a phase II clinical trial that will evaluate the effect on Total Kidney Volume, safety and tolerability of a ketogenic diet in a selected ADPKD population. The trial will have, as secondary objective, the evaluation of the ability of the ketogenic diet to slow down the renal function decline. This will be a 12-month randomized, parallel group, two arm, superiority trial with 1:1 allocation to evaluate the efficacy of a Modified Atkins Diet protocol compared to a balanced normocaloric diet on 90 ADPKD patients. Dissemination The study results will be released to the patients and the medical community.


2020 - Neurosteroids and focal epileptic disorders [Articolo su rivista]
Lévesque, Maxime; Biagini, Giuseppe; Avoli, Massimo
abstract

Neurosteroids are a family of compounds that are synthesized in principal excitatory neurons and glial cells, and derive from the transformation of cholesterol into pregnenolone. The most studied neurosteroids—allopregnanolone and allotetrahydrodeoxycorticosterone (THDOC)—are known to modulate GABAA receptor-mediated transmission, thus playing a role in controlling neuronal network excitability. Given the role of GABAA signaling in epileptic disorders, neurosteroids have profound eects on seizure generation and play a role in the development of chronic epileptic conditions (i.e., epileptogenesis). We review here studies showing the eects induced by neurosteroids on epileptiform synchronization in in vitro brain slices, on epileptic activity in in vivo models, i.e., in animals that were made epileptic with chemoconvulsant treatment, and in epileptic patients. These studies reveal that neurosteroids can modulate ictogenesis and the occurrence of pathological network activity such as interictal spikes and high-frequency oscillations (80–500 Hz). Moreover, they can delay the onset of spontaneous seizures in animal models of mesial temporal lobe epilepsy. Overall, this evidence suggests that neurosteroids represent a new target for the treatment of focal epileptic disorders.


2020 - Receptors and channels possibly mediating the effects of phytocannabinoids on seizures and epilepsy [Articolo su rivista]
Senn, Lara; Cannazza, Giuseppe; Biagini, Giuseppe
abstract

Epilepsy contributes to approximately 1% of the global disease burden. By affecting especially young children as well as older persons of all social and racial variety, epilepsy is a present disorder worldwide. Currently, only 65% of epileptic patients can be successfully treated with antiepileptic drugs. For this reason, alternative medicine receives more attention. Cannabis has been cultivated for over 6000 years to treat pain and insomnia and used since the 19th century to suppress epileptic seizures. The two best described phytocannabinoids, (−)-trans-Δ9- tetrahydrocannabinol (THC) and cannabidiol (CBD) are claimed to have positive effects on different neurological as well as neurodegenerative diseases, including epilepsy. There are different cannabinoids which act through different types of receptors and channels, including the cannabinoid receptor 1 and 2 (CB1, CB2), G protein-coupled receptor 55 (GPR55) and 18 (GPR18), opioid receptor μ and δ, transient receptor potential vanilloid type 1 (TRPV1) and 2 (TRPV2), type A γ-aminobutyric acid receptor (GABAAR) and voltage-gated sodium channels (VGSC). The mechanisms and importance of the interaction between phytocannabinoids and their different sites of action regarding epileptic seizures and their clinical value are described in this review.


2020 - Status epilepticus dynamics predicts latency to spontaneous seizures in the kainic acid model [Articolo su rivista]
COSTA, ANNA MARIA; LUCCHI, CHIARA; SIMONINI, CECILIA; ROSAL LUSTOSA, ITALO; BIAGINI, Giuseppe
abstract

(TLE), a common neurologic disorder characterized by spontaneous recurrent seizures (SRSs). However, the relationship between SE and TLE is still incompletely characterized. For this reason, in a model of TLE we evaluated the lesion extent and the onset of SRSs to determine if they were influenced by the SE dynamics. Methods: Sixty-two adult male Sprague-Dawley rats were implanted for video-electrocorticographic (v-ECoG) monitoring and intraperitoneally treated with saline or kainic acid (KA, 15 mg/kg) at 8 weeks of age. v-ECoG recordings were obtained during SE, in the following 9 weeks, and assessed by amplitude or power band spectrum. Rats were euthanized 3 or 64 days after SE to evaluate the lesion. Results: SE lasted about 10 h during which the mean duration of convulsive seizures (CSs) increased from 39 s, at 30 min, to 603 s at 4 h. The gamma power peaked 30 min after the SE onset and its peak was correlated (r²=0.13, p=0.042) with the overall SE duration. Subsequently, the gamma power was reduced under the baseline until the end of SE. The theta power increased at approximately 150% of basal levels 3 h after KA injection, but it went back to basal levels with the full development of CSs. Interestingly, the timing of the first SRS in chronic epilepsy was correlated with the latency to develop the first CS with loss of posture during SE (r²=0.60, p<0.001). Additionally, the overall duration of CSs observed during SE was related to the number of damaged brain regions (r²=0.60, p=0.005), but it did not influence the timing of the first SRS in chronic epilepsy. Conclusion: Overall, our results show that the onset of chronic epilepsy is modulated by SE dynamics, whereas brain damage is related to prolonged convulsions in SE.


2019 - A pilot study to evaluate Tolerability and Safety of a Modified Atkins Diet in ADPKD patients [Articolo su rivista]
Testa, Francesca; Marchiò, Maddalena; Belli, Michela; Giovanella, Silvia; Ligabue, Giulia; Cappelli, Gianni; Biagini, Giuseppe; Magistroni, Riccardo
abstract

Background Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the fourth cause of end stage renal disease (ESRD) and urgently requires the development of effective therapeutic treatments. The presence of a shift in energy metabolism from oxidative phosphorylation to aerobic glycolysis in ADPKD prompted us to explore the possibility to limit glucose availability by a nutritional approach. Methods We performed a single arm interventional pilot study to administer the modified Atkins diet to ADPKD patients, evaluating tolerability and safety by a questionnaire, along with glycemia and other biological parameters for 3 months. Results Satisfaction about the administered diet was high (median score of 4, IRQ 4–5). Compliance was also more than satisfactory (3, IRQ 2–5). Wellness reached the highest score (5, IRQ 4–5). Main side effect was the increase in cholesterol levels (mean net increase of 34 ± 13.1 mg/dl). Glycemia was reduced from the mean value of 105.8 ± 8.5 mg/dl (first week) to 92 ± 8.8 mg/dl (after 12ve weeks). Conclusions Our study showed the modified Atkins diet is able to reduce glucose availability and it, is well tolerated by adult patients with ADPKD.


2019 - Assessing caregiver informative materials on the ketogenic diet in Italy: A textual ethnographic approach [Articolo su rivista]
Cavalieri, Silvia; Marchio', Maddalena; Bondi, Marina; Biagini, Giuseppe
abstract

Caregiver informative materials are an important complement to verbal interaction in medical encounters enhancing caregivers’ health literacy and this is particularly true when dealing with treatments that are still little known, as is the ketogenic diet (KD) for pediatric refractory epilepsy in Italy. Their value is dependent upon whether they contain useful information from the viewpoint of the caregiver and are easily understood. The present study analyses informative booklets on the KD found in the Italian context, combining an ethnographic and a textual perspective, i.e. textual ethnography (Swales 1998) for assessing the quality of written caregiver information on the KD in Italy. We based our analysis on a two-fold methodology involving an Information Satisfaction Questionnaire (ISQ) and the application of a framework theory created by Clerehan et al. (2005), i.e. the Evaluative Linguistic Framework (ELF). Results show that together with the ethnographic assessment of informative materials obtained through the questionnaire, the analysis of key linguistic features gave important evidence to improve the quality of informational texts for caregivers.


2019 - Decreased ghrelin and des-acyl ghrelin plasma levels in patients affected by pharmacoresistant epilepsy and maintained on the ketogenic diet [Articolo su rivista]
Marchio', Maddalena; Roli, Laura; Giordano, Carmela; Trenti, Tommaso; Guerra, Azzurra; Biagini, Giuseppe
abstract

Background & aims. The gastric hormones ghrelin and des-acyl ghrelin have been found to be altered in patients treated with antiepileptic drugs. However, it is unknown if these hormones could be modified by other antiepileptic treatments, such as the ketogenic diet. Especially, a reduction in ghrelin levels could be relevant in view of the growth retardation observed under ketogenic diet treatment. For this reason we aimed to determine the changes in ghrelin and des-acyl ghrelin plasma levels in children affected by refractory epilepsy and treated with the ketogenic diet up to 90 days. Methods. Both peptides were measured by immunoassays in plasma obtained from 16 children. Results. Ghrelin plasma levels were progressively reduced by the ketogenic diet, reaching a minimum corresponding to 42% of basal levels after 90 days of ketogenic diet (P < 0.05, Duncan's test). Des-acyl ghrelin plasma levels were similarly affected, reaching minimal levels at 30 days (65% of basal levels), and maintaining a significant reduction until 90 days after the onset of ketogenic diet (P < 0.01 for both time intervals). No significant changes in growth were observed during the monitored period of ketogenic diet administration. Conclusions. Ghrelin and des-acyl ghrelin are downregulated by the ketogenic diet in children affected by refractory epilepsy. Although no significant changes in growth were observed during the short time period of our investigation, the reduction in ghrelin availability may explain the reported growth retardation found in children treated with the ketogenic diet in the long-term.


2019 - Ghrelin plasma levels after 1 year of ketogenic diet in children with refractory epilepsy [Articolo su rivista]
Marchiò, Maddalena; Roli, Laura; Lucchi, Chiara; Costa, ANNA MARIA; Borghi, Matteo; Iughetti, Lorenzo; Trenti, Tommaso; Guerra, Azzurra; Biagini, Giuseppe
abstract

The ketogenic diet (KD) is a high-fat, low carbohydrate nutritional treatment adopted in several countries for refractory epilepsy. However, the use of KD is limited by adverse events including growth retardation. In a previous investigation, we demonstrated that ghrelin is reduced in children maintained on KD for 3 months. As ghrelin regulates growth hormone (GH) secretion, it can be hypothesized that growth retardation depends on the reduced ghrelin availability. To assess this hypothesis, in this study we evaluate ghrelin and growth during 1 year of KD. We examined a small cohort of 6 children (2 males and 4 females, age range 3–10.4 years) affected by refractory epilepsy, who received the KD as add-on treatment. All patients were on drug polytherapy. Endpoints of the study were: (i) ghrelin plasma levels at 0, 15, 30, 90, and 365 days from KD onset, (ii) growth, and (iii) seizure control by ketogenesis. Ghrelin levels were −53 and −47% of basal levels, respectively, at 90 and 365 days (P &lt; 0.05 for both). Mean height index z scores were reduced, but not significantly, by comparing basal values with those at the end of observation. Instead, body mass index z scores slightly increased. Ketosis induced by the KD was within 2–5 mmol/L and satisfactorily reduced the seizure frequency (&gt;50%) in all patients. We show that ghrelin plasma levels are consistently reduced in children with refractory epilepsy and maintained on the KD. This change was associated with low growth indexes in the majority of patients.


2019 - Neuroplasticity in cholinergic projections from basal forebrain to the basolateral nucleus of amygdala in the kainic acid model of temporal lobe epilepsy [Articolo su rivista]
ROSAL LUSTOSA, ITALO; Soares, Joana I.; Biagini, Giuseppe; LUKOYANOV, NICOLAY VSEVOLODOVICH; Nikolay, V.
abstract

The amygdala is a cerebral region whose function is compromised in temporal lobe epilepsy (TLE). Patients with TLE present cognitive and emotional dysfunctions, of which impairments in recognizing facial expressions have been clearly attributed to amygdala damage. However, damage to the amygdala has been scarcely addressed, with the majority of studies focusing on the hippocampus. The aim of this study was to evaluate epilepsy-related plasticity of cholinergic projections to the basolateral nucleus (BL) of the amygdala. Adult rats received kainic acid (KA) injections and developed status epilepticus. Weeks later, they showed spontaneous recurrent seizures documented by behavioral observations. Changes in cholinergic innervation of the BL were investigated by using an antibody against the vesicular acetylcholine transporter (VAChT). In KA-treated rats, it was found that (i) the BL shrunk to 25% of its original size (p &lt; 0.01 vs. controls, Student’s t-test), (ii) the density of vesicular acetylcholine transporter-immunoreactive (VAChT-IR) varicosities was unchanged, (iii) the volumes of VAChT-IR cell bodies projecting to the BL from the horizontal limb of the diagonal band of Broca, ventral pallidum, and subcommissural part of the substantia innominata were significantly increased (p &lt; 0.05, Bonferroni correction). These results illustrate significant changes in the basal forebrain cholinergic cells projecting to the BL in the presence of spontaneous recurrent seizures.


2019 - miRNA-218 targets lipin-1 and glucose transporter type 4 genes in 3T3-L1 cells treated with lopinavir/ritonavir [Articolo su rivista]
Bresciani, Elena; Saletti, Cecilia; Squillace, Nicola; Rizzi, Laura; Molteni, Laura; Meanti, Ramona; Omeljaniuk, Robert J.; Biagini, Giuseppe; Gori, Andrea; Locatelli, Vittorio; Torsello, Antonio
abstract

Background: Metabolic complications represent a common and serious problem associated with HIV infection and combined Antiretroviral Therapy (cART). Alterations in body fat distribution are associated with significantly increased risks of (i) metabolic derangements, (ii) cardiovascular pathologies, and (iii) insulin resistance. A case control study showed that in subcutaneous adipose tissue from HIV-infected patients on cART presenting lipodystrophy (LS), the levels of miRNA-218 were upregulated and those of lipin-1, a putative target gene of miRNA-218, were downregulated compared with HIVnegative subjects. Lipin-1 is one of the most important factors linked to development of LS. Lipin-1, by controlling PPARg2, regulates the expression of specific genes, such as that of glucose transporter type 4 (GLUT-4), required for maturation and maintenance of adipocytes. Objectives: To determine whether lopinavir/ritonavir (LPV/RTV) can modulate lipogenesis in adipocytes affecting miRNA-218 and lipin-1 mRNA expression, and to investigate the functional link between miRNA-218 and GLUT-4 mRNA expression. Methods: Differentiated 3T3-L1 cells were treated with various combinations of LPV/RTV, followed by measurements of cell viability, lipid accumulation, lipin-1 and GLUT-4 mRNA and miRNA-218 levels. Transfection of anti-miR-218 or a miRNA-218 mimic were used to investigate the role of miRNA-218 in lipogenesis. Results: LPV/RTV treatment of 3T3-L1 cells did not affect the viability of differentiated 3T3-L1 cells, but caused (i) a significant decrease of lipid accumulation, (ii) an overexpression of miRNA-218, and (iii) a reduction of lipin-1 and GLUT-4 mRNA levels. The anti-miR-218 transfection of 3T3-L1 cells significantly ameliorated the adipogenic dysfunction and restored mRNA levels of lipin-1 and GLUT-4 consequent to LPV/RTV treatment. By contrast, 3T3-L1 cells transfected with a specific miRNA-218 mimic showed (i) an overexpression of miRNA-218, (ii) a reduced cellular lipid fraction, and (iii) decreased levels of mRNA for lipin-1 and GLUT-4. Conclusion: 3T3-L1 cells, treated with LPV/RTV, show altered lipid content due to increased miRNA-218 levels, which affects lipin-1 mRNA. Moreover, increased miRNA- 218 levels were inversely correlated with changes in GLUT-4 expression, which suggests a role for miRNA-218 in mediating the insulin resistance consequent to cART.


2018 - A hydroxypyrone-based inhibitor of metalloproteinase-12 displays neuroprotective properties in both status epilepticus and optic nerve crush animal models [Articolo su rivista]
Vinet, J.; Costa, A. M.; Salinas-Navarro, M.; Leo, G.; Moons, L.; Arkens, L.; Biagini, G.
abstract

Recently, we showed that matrix metalloproteinase-12 (MMP-12) is highly expressed in microglia and myeloid infiltrates, which are presumably involved in blood–brain barrier (BBB) leakage and subsequent neuronal cell death that follows status epilepticus (SE). Here, we assessed the effects of a hydroxypyrone-based inhibitor selective for MMP-12 in the pilocarpine-induced SE rat model to determine hippocampal cell survival. In the hippocampus of rats treated with pilocarpine, intra-hippocampal injections of the MMP-12 inhibitor protected Cornu Ammonis 3 (CA3) and hilus of dentate gyrus neurons against cell death and limited the development of the ischemic-like lesion that typically develops in the CA3 stratum lacunosum-moleculare of the hippocampus. Furthermore, we showed that MMP-12 inhibition limited immunoglobulin G and albumin extravasation after SE, suggesting a reduction in BBB leakage. Finally, to rule out any possible involvement of seizure modulation in the neuroprotective effects of MMP-12 inhibition, neuroprotection was also observed in the retina of treated animals after optic nerve crush. Overall, these results support the hypothesis that MMP-12 inhibition can directly counteract neuronal cell death and that the specific hydroxypyrone-based inhibitor used in this study could be a potential therapeutic agent against neurological diseases/disorders characterized by an important inflammatory response and/or neuronal cell loss.


2018 - High plasma levels of ghrelin and des-acyl ghrelin in responders to antiepileptic drugs [Articolo su rivista]
Marchiò, Maddalena; Roli, Laura; Giordano, Carmela; Caramaschi, Elisa; Guerra, Azzurra; Trenti, Tommaso; Biagini, Giuseppe
abstract

OBJECTIVE: To reconsider ghrelin and des-acyl ghrelin plasma levels in children with epilepsy in order to establish a possible relation with response to antiepileptic drugs (AEDs). METHODS: We designed an observational study in which 114 patients with epilepsy were classified as responders (77) or nonresponders (37) and compared to 59 controls. In these patients, we measured ghrelin and des-acyl ghrelin by immunoassays in blood samples obtained after overnight fast. RESULTS: Ghrelin plasma levels were higher (+94%; p < 0.001, Dunn test) in responders compared to controls. Des-acyl ghrelin plasma levels were also higher in the same group (+55%; p < 0.001). In addition, both hormones were unmodified in nonresponders compared to controls. By comparing responders to nonresponders, ghrelin and des-acyl ghrelin, respectively, were +126% (p < 0.001) and +29% (p < 0.001) in patients with a positive response to AEDs. CONCLUSIONS: These results indicate that ghrelin and des-acyl ghrelin plasma levels are especially high in patients with epilepsy who positively respond to AEDs. In view of the anticonvulsant properties of ghrelin and des-acyl ghrelin, we propose that their higher levels could play a role in modulating the response to AEDs. Moreover, these peptides could be promising markers of response to AEDs.


2018 - Low levels of progesterone and derivatives in cerebrospinal fluid of patients affected by status epilepticus [Articolo su rivista]
Meletti, Stefano; Lucchi, Chiara; Monti, Giulia; Giovannini, Giada; Bedin, Roberta; Trenti, Tommaso; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neurosteroids such as allopregnanolone may play a role in epilepsy as positive modulators of inhibitory currents mediated by γ-aminobutyric acid type A (GABAA ) receptor. Indeed, these molecules have been consistently shown to be anticonvulsants in animal models, but their role is still unclear in patients. For this reason, we investigated neurosteroids in the cerebrospinal fluid (CSF) of patients with status epilepticus (SE) by liquid chromatography tandem-mass spectrometry. Patients were retrospectively identified within subjects who received a lumbar puncture in the 2007-2017 period. Seventy-three patients (median age 65, ranging from 13 to 94 years; 67% women) with SE were evaluated. Controls (n = 52, median age 53, ranging from 16 to 93 years; 65% women) were patients presenting with symptoms for which a lumbar puncture was required by clinical guidelines, and who were negative at the end of the diagnostic work-up. Progesterone was 64% lower in patients with SE (p < 0.001). With respect to progesterone, upstream pregnenolone sulfate and pregnenolone did not change. Instead, downstream 5α-dihydroprogesterone, pregnanolone and allopregnanolone were respectively 49% (p < 0.001), 21% (p < 0.01) and 37% (p < 0.001) lower than in controls. Duration or type of SE, age and sex did not consistently affect CSF neurosteroid levels in the SE cohort. Instead, pregnenolone sulfate (Spearman's ρ = 0.4335, p < 0.01), allopregnanolone (ρ = 0.4121, p < 0.05), and pregnanolone (ρ = 0.592, p < 0.001) levels significantly increased by ageing in controls. We conclude that neurosteroidogenesis is defective in patients with SE. This article is protected by copyright. All rights reserved.


2018 - Ridotta concentrazione del progesterone e di altri neurosteroidi nel liquor di pazienti in stato di male epilettico [Abstract in Atti di Convegno]
Biagini, G.; Lucchi, C.; Monti, G.; Giovannini, G.; Bedin, R.; Rustichelli, C.; Meletti, S.
abstract

Lo stato di male epilettico (SE) costituisce una grave emergenza neurologica. In tale condizione, mediante cromatografia liquida e spettrometria di massa, abbiamo misurato e confrontato i livelli di pregnenolone solfato, pregnenolone, progesterone, 5α-diidroprogesterone, pregnanolone e allopregnanolone nel liquor di pazienti affetti da SE e in una coorte di controllo. Sono stati studiati 73 pazienti ricoverati per SE, con età di 65 anni (intervallo di variazione da 13 a 94 anni), dei quali il 67% donne. I controlli esaminati, pari a 52, avevano età compresa tra 16 e 93 anni (mediana pari a 53), dei quali il 65% donne. I risultati sono stati analizzati con il test di Mann-Whitney. Abbiamo anche correlato le concentrazioni dei neurosteroidi e l’età, il genere, la durata ed il tipo di SE. Il progesterone è risultato essere diminuito del 64% nel liquor dei pazienti con SE (p < 0.001). I neurosteroidi pregnenolone solfato e pregnenolone, precursori del progesterone, sono risultati a livelli analoghi a quelli dei controlli. I derivati del progesterone, al contrario, in particolare il 5α-diidroprogesterone (-49%, p < 0.001), il pregnanolone (-21%, p = 0.005) e l’allopregnanolone (-37%, p < 0.001), sono risultati essere inferiori ai controlli. Le variazioni osservate nei livelli liquorali di neurosteroidi non hanno travato spiegazioni nelle differenze di genere, età o tipo di SE. Inoltre, non sono stati riscontrati effetti dovuti alla durata del SE. In conclusione, questi risultati indicano che vari neurosteroidi anticonvulsivanti sono presenti a ridotte concentrazioni liquorali nei pazienti che presentano un’attività epilettica protratta.


2018 - STIM Proteins and Orai Ca2+ Channels Are Involved in the Intracellular Pathways Activated by TLQP-21 in RAW264.7 Macrophages [Articolo su rivista]
Molteni, L.; Rizzi, L.; Bresciani, E.; Meanti, R.; Fehrentz, J. -A.; Verdié, P.; Omeljaniuk, R. J.; Biagini, G.; Locatelli, Vittorio; Torsello, ANTONIO BIAGIO
abstract

TLQP-21 is a neuropeptide which has been implicated in regulation of nociception and other relevant physiologic functions. Although recent studies identified C3a and gC1q receptors as targets for TLQP-21, its intracellular molecular mechanisms of action remain largely unidentified. Our aim was (i) to explore the intracellular signaling pathway(s) activated by JMV5656, a novel derivative of TLQP-21, in RAW264.7 macrophages, and (ii) to assess linkages of these pathways with its purported receptors. JMV5656 stimulated, in a dose-dependent fashion, a rapid and transient increase in intracellular Ca2+ concentrations in RAW264.7 cells; repeated exposure to the peptide resulted in a lower response, suggesting a possible desensitization mechanism of the receptor. In particular, JMV5656 increased cytoplasmic Ca2+ levels by a PLC-dependent release of Ca2+ from the endoplasmic reticulum. STIM proteins and Orai Ca2+ channels were activated and played a crucial role. In fact, treatment of the cells with U73122 and thapsigargin modulated the increase of intracellular Ca2+ levels stimulated by JMV5656. Moreover, in RAW264.7 cells intracellular Ca2+ increases did not occur through the binding of JMV5656 to the C3a receptor, since the increase of intracellular Ca2+ levels induced by JMV5656 was not affected by specific siRNA against C3aR. In summary, our study provides new indications for the downstream effects of JMV5656 in macrophages, suggesting that it could activate receptors different from the C3aR.


2017 - Decreased allopregnanolone levels in cerebrospinal fluid obtained during status epilepticus [Articolo su rivista]
Meletti, Stefano; Lucchi, Chiara; Monti, Giulia; Giovannini, Giada; Bedin, Roberta; Trenti, Tommaso; Rustichelli, Cecilia; Biagini, Giuseppe
abstract

Neuroactive steroids are increasingly considered as relevant modulators of neuronal activity. Especially allopregnanolone (AP) and pregnenolone sulfate (PS) have been shown to possess, respectively, anticonvulsant or proconvulsant properties. In view of the potential role of these steroids, we aimed at evaluating AP and PS levels in cerebrospinal fluid (CSF) and blood samples obtained from patients with status epilepticus (SE). To this purpose, we enrolled 41 patients affected by SE and 41 subjects investigated for nonepileptic neurologic disorders. Liquid chromatographic procedures coupled with electrospray tandem mass spectrometry and routine laboratory investigations were performed. Significantly lower AP levels were found in the CSF of patients affected by SE (-30%; p &lt; 0.05, Mann-Whitney test). Notably, AP was not detectable in 28 of 41 patients affected by SE (p &lt; 0.01 vs. controls, Fisher's exact test). In serum, AP levels did not differ in the two considered groups. Conversely, PS was present at similar levels in the investigated groups. Finally, differences in AP levels could not be explained by a variation in CSF albumin content. These findings indicate that AP is defective in the CSF of patients affected by SE. This phenomenon was not dependent on carriers for steroids, such as albumin.


2017 - Electrographic changes accompanying recurrent seizures under ketogenic diet treatment. [Articolo su rivista]
Lucchi, Chiara; Marchio', Maddalena; Caramaschi, Elisa; Giordano, Carmela; Giordano, R.; Guerra, A.; Biagini, Giuseppe
abstract

The ketogenic diet (KD) is increasingly used to treat epilepsy refractory to antiepileptic drugs and other neurological disorders. In animal models, the KD was found to increase the threshold to seizures induced by different convulsive stimulations. However, in models in which suprathreshold stimuli were used, a paradoxical seizure worsening was consistently observed in KD-fed animals. To better define this phenomenon, we characterized the electrographic response to seizures induced in mice which were treated with the KD, and then corneally stimulated at 6-Hz in four different sessions. We also evaluated the electroencephalogram (EEG) in three patients in which the KD was associated with a paradoxical worsening of epileptic seizures. Although seizures were initially less severe, a remarkable prolongation of the electrographic response was observed in mice receiving the KD from the second session of 6-Hz corneal stimulation and onwards. The EEG was also markedly altered in the presence of progressive seizure aggravation observed in children treated with the KD, specifically one affected by Lennox–Gastaut syndrome and two by type I lissencephaly. These results suggest that when seizures are induced or recur because of resistance to therapeutic interventions, the KD may change the EEG by potentiating the electrographic epileptic activity.


2017 - FosB/ΔFosB and p-ERK1/2 expression respectively identified the lateral amygdala and CA1 as critical regions involved in the progressive seizure aggravation observed in mice exposed to repeated 6-Hz corneal stimulation [Abstract in Atti di Convegno]
Costa, Am; Giordano, C; Lucchi, C; Curia, G; Piat, M; Leo, G; Vinet, J; Biagini, G
abstract

FosB/ΔFosB and p-ERK1/2 expression respectively identified the lateral amygdala and CA1 as critical regions involved in the progressive seizure aggravation observed in mice exposed to repeated 6-Hz corneal stimulation


2017 - Involvement of PPARγ in the anticonvulsant activity of EP-80317, a ghrelin receptor antagonist [Articolo su rivista]
Lucchi, Chiara; Costa, ANNA MARIA; Giordano, Carmela; Curia, Giulia; Piat, Marika; Leo, Giuseppina; Vinet, Jonathan; Brunel, Luc; Fehrentz, Jean Alain; Martinez, Jean; Torsello, Antonio; Biagini, Giuseppe
abstract

Ghrelin, des-acyl ghrelin and other related peptides possess anticonvulsant activities. Although ghrelin and cognate peptides were shown to physiologically regulate only the ghrelin receptor, some of them were pharmacologically proved to activate the peroxisome proliferator-activated receptor gamma (PPARγ) through stimulation of the scavenger receptor CD36 in macrophages. In our study, we challenged the hypothesis that PPARγ could be involved in the anticonvulsant effects of EP-80317, a ghrelin receptor antagonist. For this purpose, we used the PPARγ antagonist GW9662 to evaluate the modulation of EP-80317 anticonvulsant properties in two different models. Firstly, the anticonvulsant effects of EP-80317 were studied in rats treated with pilocarpine to induce status epilepticus (SE). Secondly, the anticonvulsant activity of EP-80317 was ascertained in the repeated 6-Hz corneal stimulation model in mice. Behavioral and video electrocorticographic (ECoG) analyses were performed in both models. We also characterized levels of immunoreactivity for PPARγ in the hippocampus of 6-Hz corneally stimulated mice. EP-80317 predictably antagonized seizures in both models. Pre-treatment with GW9662 counteracted almost all EP-80317 effects both in mice and rats. Only the effects of EP-80317 on power spectra of ECoGs recorded during repeated 6-Hz corneal stimulation were practically unaffected by GW9662 administration. Moreover, GW9662 alone produced a decrease in the latency of tonic-clonic seizures and accelerated the onset of SE in rats. Finally, in the hippocampus of mice treated with EP-80317 we found increased levels of PPARγ immunoreactivity. Overall, these results support the hypothesis that PPARγ is able to modulate seizures and mediates the anticonvulsant effects of EP-80317.


2017 - JMV2894, a novel growth hormone secretagogue, accelerates body mass recovery in an experimental model of cachexia [Articolo su rivista]
Bresciani, Elena; Rizzi, Laura; Molteni, Laura; Ravelli, Monica; Liantonio, Antonella; Ben Haj Salah, Khoubaib; Fehrentz, Jean Alain; Martinez, Jean; Omeljaniuk, Robert J.; Biagini, Giuseppe; Locatelli, Vittorio; Torsello, Antonio
abstract

Oncologic patients subjected to chemotherapy frequently present aphagia, malnutrition, and cachexia. The purpose of this study was to investigate whether selected growth hormone secretagogues including hexarelin, JMV2894 and JMV2951 could antagonize body weight loss and wasting induced by cisplatin administration in rats. The three growth hormone secretagogues behaved as full agonists of the growth hormone secretagogues receptor both in terms of ability to stimulate calcium mobilization in Chinese hamster ovary cells and stimulation of growth hormone release in neonatal rats. Adult rats were (i) treated with vehicle throughout (controls), or (ii) treated with cisplatin (days 1–3) and a growth hormone secretagogues or vehicle, (days 1–12). Body weight and food consumption were measured daily. Although all growth hormone secretagogues caused initial transient acute increases in food intake, the total amount of food eaten by controls and growth hormone secretagogues treated groups over the 12 experimental days was not significantly different. All groups pre-treated with cisplatin lost up to 5–10 % body weight in the first 4 days; they subsequently gained weight at a rate comparable with controls. Interestingly, rats which received JMV2894 demonstrated a faster gain in body weight than any other growth hormone secretagogues treated group and at the end of the protocol reached a weight similar to that of controls. JMV2894 did not stimulate perirenal and epididymal fat accumulation but reduced MuRF mRNA levels in skeletal muscles. In conclusion, our findings demonstrate that JMV2894 antagonizes cisplatin induced weight loss in rats and may prove useful in antagonizing cachexia associated with cancer and chemotherapy in humans.


2017 - Pharmacological and biochemical characterization of TLQP-21 activation of a binding site on CHO cells [Articolo su rivista]
Molteni, L.; Rizzi, L.; Bresciani, E.; Possenti, R.; Petrocchi Passeri, P.; Ghè, C.; Muccioli, G.; Fehrentz, J. A.; Verdié, P.; Martinez, J.; Omeljaniuk, R. J.; Biagini, Giuseppe; Binda, A.; Rivolta, I.; Locatelli, V.; Torsello, A.
abstract

VGF is a propeptide of 617 amino acids expressed throughout the central and the peripheral nervous system. VGF and peptides derived from its processing have been found in dense core vesicles and are released from neuronal and neuroendocrine cells via the regulated secretory pathway. Among VGF-derived neuropeptides, TLQP-21 (VGF556-576) has raised a huge interest and is one of most studied. TLQP-21 is a multifunctional neuropeptide involved in the control of several physiological functions, potentially including energy homeostasis, pain modulation, stress responsiveness and reproduction. Although little information is available about its receptor and the intracellular mechanisms mediating its biological effects, recent reports suggest that TLQP-21 may bind to the complement receptors C3aR1 and/or gC1qR. The first aim of this study was to ascertain the existence and nature of TLQP-21 binding sites in CHO cells. Secondly, we endeavored to characterize the ligand binding to these sites by using a small panel of VGF-derived peptides. And finally, we investigated the influence of TLQP-21 on selected intracellular signaling pathways. We report that CHO cells express a single class of saturable and specific binding sites for TLQP-21 with an affinity and capacity of Kd = 0.55 ± 0.05 x 10-9 M and Bmax = 81.7 ± 3.9 fmol/mg protein, respectively. Among the many bioactive products derived from the C-terminal region of VGF that we tested, TLQP-21 was the most potent in stimulating intracellular calcium mobilization in CHO cells; this effect is primarily due to its C-terminal fragment (HFHH-10). TLQP-21 induced rapid and transient dephosphorylation of phospholipase Cγ1 and phospholipase A2. Generation of IP3 and diacylglycerol was crucial for TLQP‐21 bioactivity. In conclusion, our results suggest that the receptor stimulated by TLQP-21 belongs to the family of the Gq-coupled receptors, and its activation first increases membrane-lipid derived second messengers which thereby induce the mobilization of Ca2+ from the endoplasmic reticulum followed by a slower store-operated Ca2+ entry from outside the cell.


2017 - Reduced steroidogenesis in patients with PCDH19-female limited epilepsy [Articolo su rivista]
Trivisano, M.; Lucchi, Chiara; Rustichelli, Cecilia; Terracciano, A.; Cusmai, R.; Ubertini, G. M.; Giannone, G.; Bertini, E.; Vigevano, F.; Gecz, J.; Biagini, Giuseppe; Specchio, N.
abstract

Patients affected by protocadherin 19 (PCDH19)–female limited epilepsy (PCDH19- FE) present a remarkable reduction in allopregnanolone blood levels. However, no information is available on other neuroactive steroids and the steroidogenic response to hormonal stimulation. For this reason, we evaluated allopregnanolone, pregnanolone, and pregnenolone sulfate by liquid chromatographic procedures coupled with electrospray tandem mass spectrometry in 12 unrelated patients and 15 agematched controls. Wealso tested cortisol, estradiol, progesterone, and 17OH-progesterone using standard immunoassays. Apart from estradiol and progesterone, all the considered hormones were evaluated in basal condition and after stimulation with adrenocorticotropic hormone (ACTH). A generalized decrease in blood levels of almost all measured neuroactive steroids was found. When considering sexual development, cortisol and pregnenolone sulfate basal levels were significantly reduced in postpubertal girls affected by PCDH19-FE. Of interest, ACTH administration did not recover pregnenolone sulfate serum levels but restored cortisol to control levels. In prepubertal girls with PCDH19-FE, by challenging adrenal function with ACTH we disclosed defects in the production of cortisol, pregnenolone sulfate, and 17OH-progesterone, which were not apparent in basal condition. These findings point to multiple defects in peripheral steroidogenesis associated with and potentially relevant to PCDH19-FE. Some of these defects could be addressed by stimulating adrenocortical activity.


2016 - Allopregnanolone is reduced in patients with PCDH19-related epilepsy [Abstract in Rivista]
Trivisano, M; Lucchi, C; Terracciano, A; Rustichelli, C.; Cusmai, R; Ubertini, Gm; Giannone, G; Bertini, E; Vigevano, F; Gecz, J; Biagini, G; Specchio, N
abstract

Aim: The inherit base of PCDH19-related epilepsy suggests a hormonal involvement due to de-regulation of AKR1C1-3 genes, which encode for crucial steroid hormone-metabolizing enzymes. Both mRNA and protein levels of AKR1C3 have been reported to be decreased in a small number of PCDH19 mutated patients. Aim of this study is to verify allopregnanolone and other neuroactive steroids serum levels in PCDH19 mutated patients. Methods: We performed a prospective case-control study. We enrolled 12 patients affected by PCDH19-related epilepsy and 15 controls, age-and sex-matched. Controls were recruited among subjects evaluated for praecox puberty or hyperandrogenism. In both groups blood samples were taken at basal (T0) and 60 min after (ACTH) administration (T1). Laboratory methods: Quantitative analysis of neuroactive steroids in serum was performed by liquid chromatography-electrospray tandem mass spectrometry. Study population: median age of PCDH19-mutated patients was 8.3+5.7 years (range 2.5-18.9). Other than epilepsy, 6 patients had mental retardation. Controls were all females, age 9.2+4.0 years (range 6.1-17.9). All controls had a normal cognitive profile. Nine patients had a diagnosis of praecox puberty and 6 hyperandrogenism. Results: All neuroactive steroids resulted down produced in patients with PCDH19-related epilepsy rather than controls. At basal assessment (T0), allopregnanolone was 0.9+0.5 vs 0.18+0.9 ng/ml (p&lt;0.05); pregnanolone was 0.0+0.5 vs 0.3+0.5 ng/ml (p&gt;0.05), but pregnanolone solphate sulfate was 7.39+5.53 vs 75.41+52.69 ng/ml (p&lt;0.05). Also cortisol, progesterone and 17-OH progesterone resulted to be lowered in PCDH19-mutated patients compared with controls. After ACTH injection (T1), both neuroactive steroids and the other peripheral steroids, were confirmed to be lower compared with controls. ACTH test demonstrated a normal functioning of peripheral glands. Conclusion: We documented a down regulation of all steroidogenesis in PCDH19-related epilepsy. Particularly we found allopregnanolone and pregnaenolone solphate sulfate deficiency. Allopregnanolone is a GABA-A receptor modulators influencing the neuronal excitability, thus representing a realistic therapeutic target for PCDH19-related epilepsy.


2016 - Endogenous Neurosteroids Levels are Decreased in CSF During Status Epilepticus [Abstract in Rivista]
Monti, G; Lucchi, C; Rustichelli, C; Giovannini, G; Meletti, S; Biagini, G
abstract

Purpose: Neuroactive steroids, such as allopregnanolone (AP), are positive allosteric modulators of GABA-A receptors enhancing both synaptic and extrasynaptic GABA-A mediated inhibition and might be effective in the treatment of benzodiazepine-resistant status epilepticus (SE). No data are available on AP levels in central nervous system (CNS) of patients suffering from SE. This lack of information hampers the possibility to correctly design a rational therapeutic approach to SE. We designed a study to evaluate AP in serum and cerebrospinal fluid (CSF) of patients affected by SE. Method: We retrospectively (2007–2015) evaluated blood and CSF samples from 41 adult patients with diagnosis of SE who received lumbar puncture at SE onset (median of 4 days) for clinical purposes and for whom a CNS infection was finally excluded. 41 subjects (matched for age and sex) that had negative results after lumbar puncture for suspected idiopathic intracranial hypertension, CNS infection, or inflammatory disease served as control group. Quantitative analysis of neurosteroids was performed by liquid chromatography-electrospray tandem mass spectrometry. Results: Serum AP levels did not revealed significant differences between controls and SE patients. However, CSF AP levels were decreased by approximately 30% (p &lt; 0.05, Mann-Whitney test) in patients compared with controls. Interestingly, linear regression did not reveal a relationship between serum and CSF levels for AP in controls (R2 = 0.06, p = 0.27). On the contrary, a significant relationship (R2 = 0.57, p = 0.003) was present in patients affected by SE. Conclusion: We demonstrated that endogenous allopregnenolone is significantly reduced during SE in CSF. Administration of AP to patients suffering of SE could be useful to reestablish AP level in the CNS. In addition, exogenously administered AP may result to have a privileged access to brain tissue by virtue of the higher permeability of the blood- brain barrier during SE.


2016 - MicroRNA-101 Regulates Multiple Developmental Programs to Constrain Excitation in Adult Neural Networks [Articolo su rivista]
Lippi, Giordano; Fernandes, Catarina C; Ewell, Laura A; John, Danielle; Romoli, Benedetto; Curia, Giulia; Taylor, Seth R; Frady, E.  Paxon; Jensen, Anne B; Liu, Jerry C; Chaabane, Melanie M; Belal, Cherine; Nathanson, Jason l; Zoli, Michele; Leutgeb, Jill K; Biagini, Giuseppe; Yeo, Gene W; Berg, Darwin K
abstract

A critical feature of neural networks is that they balance excitation and inhibition to prevent pathological dysfunction. How this is achieved is largely unknown, although deficits in the balance contribute to many neurological disorders. We show here that a microRNA (miR-101) is a key orchestrator of this essential feature, shaping the developing network to constrain excitation in the adult. Transient early blockade of miR-101 induces long-lasting hyper-excitability and persistent memory deficits. Using target site blockers in vivo, we identify multiple developmental programs regulated in parallel by miR-101 to achieve balanced networks. Repression of one target, NKCC1, initiates the switch in γ-aminobutyric acid (GABA) signaling, limits early spontaneous activity, and constrains dendritic growth. Kif1a and Ank2 are targeted to prevent excessive synapse formation. Simultaneous de-repression of these three targets completely phenocopies major dysfunctions produced by miR-101 blockade. Our results provide new mechanistic insight into brain development and suggest novel candidates for therapeutic intervention.


2016 - Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus [Articolo su rivista]
Vinet, Jonathan; Vainchtein, Ilia D.; Spano, Maria Carlotta; Giordano, Carmela; Bordini, Domenico; Curia, Giulia; Dominici, Massimo; Boddeke, Hendrikus W. G. M.; Eggen, Bart J. L.; Biagini, Giuseppe
abstract

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4pos T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile.


2016 - Progressive Seizure Aggravation in the Repeated 6-Hz Corneal Stimulation Model Is Accompanied by Marked Increase in Hippocampal p-ERK1/2 Immunoreactivity in Neurons [Articolo su rivista]
Giordano, Carmela; Costa, ANNA MARIA; Lucchi, Chiara; Leo, Giuseppina; Brunel, Luc; Fehrentz, Jean Alain; Martinez, Jean; Torsello, Antonio; Biagini, Giuseppe
abstract

The 6-Hz corneal stimulation test is used to screen novel antiepileptic molecules to overcome the problem of drug refractoriness. Although recognized as a standard test, it has been evaluated only recently in the attempt to characterize the putative neuronal networks involved in seizures caused by corneal stimulation. In particular, by recording from the CA1 region we previously established that the hippocampus participates to propagation of seizure activity. However, these findings were not corroborated by using markers of neuronal activation such as FosB/ΔFosB antigens. In view of this discrepancy, we performed new experiments to characterize the changes in levels of phosphorylated extracellular signal-regulated kinases1/2 (p-ERK1/2), which are also used as markers of neuronal activation. To this aim, mice underwent corneal stimulation up to three different times, in three sessions separated by an interval of 3 days. To characterize a group in which seizures could be prevented by pharmacological treatment, we also considered pretreatment with the ghrelin receptor antagonist EP-80317 (330 μg/kg). Control mice were sham-treated. Video electrocorticographic (ECoG) recordings were obtained from mice belonging to each group of treatment. Animals were finally used to characterize the immunoreactivity for FosB/ΔFosB and p-ERK1/2 in the hippocampus. As previously shown, FosB/ΔFosB levels were highly increased throughout the hippocampus by the first induced seizure but, in spite of the progressively increased seizure severity, they were restored to control levels after the third stimulation. At variance, corneal stimulation caused a progressive increase in p-ERK1/2 immunoreactivity all over the hippocampus, especially in CA1, peaking in the third session. Predictably, EP-80317 administration reduced both duration and severity of seizures, prevented the increase in FosB/ΔFosB levels in the first session, and partially counteracted the increase in p-ERK1/2 levels in the third session. The vast majority of p-ERK1/2 immunopositive cells were co-labeled with FosB/ΔFosB antibodies, suggesting the existence of a relationship between the investigated markers in a subpopulation of neurons activated by seizures. These findings suggest that p-ERK1/2 are useful markers to define the aggravation of seizures and the response to anticonvulsant treatments. In particular, p-ERK1/2 expression clearly identified the involvement of hippocampal regions during seizure aggravation in the 6-Hz model.


2015 - Activity-dependent changes in excitability of perirhinal cortex networks in vitro [Articolo su rivista]
Biagini, Giuseppe; D’Antuono, Margherita; Inaba, Yuji; Kano, Toshiyuki; Ragsdale, David; Avoli, Massimo
abstract

Rat brain slices comprising the perirhinal cortex (PC) and a portion of the lateral nucleus of the amygdala (LA), in standard medium, can generate synchronous oscillatory activity that is associated with action potential discharge and reflects the activation of glutamatergic and GABAergic receptors. We report here that similar synchronous oscillatory events are recorded in the PC in response to single-shock, electrical stimuli delivered in LA. In addition, we found that the latency of these responses progressively increased when the stimulus interval was varied from 10 to 1 s; for example, the response latency during stimuli delivered at 1 Hz was more than twofold longer than that seen during stimulation at 0.1 Hz. This prolongation in latency occurred after approximately 5 stimuli, attained a steady value after 24-35 stimuli, and recovered to control values 30 s after stimulation arrest. These frequency-dependent changes in latency continued to occur during NMDA receptor antagonism but weakened following application of GABAA and/or GABAB receptor blockers. Our findings identify a new type of short-term plasticity that is mediated by GABA receptor function and may play a role in decreasing neuronal network synchronization during repeated activation. We propose that this frequency-dependent adaptive mechanism influences the excitability of limbic networks, thus potentially controlling epileptiform synchronization.


2015 - Blockade of in vitro ictogenesis by low-frequency stimulation coincides with increased epileptiform response latency [Articolo su rivista]
Kano, Toshiyuki; Inaba, Yuji; D'Antuono, Margherita; Biagini, Giuseppe; Lévesque, Maxime; Avoli, Massimo
abstract

Low-frequency stimulation, delivered through transcranial magnetic or deep-brain electrical procedures, reduces seizures in patients with pharmacoresistant epilepsy. A similar control of ictallike discharges is exerted by low-frequency electrical stimulation in rodent brain slices maintained in vitro during convulsant treatment. By employing field and “sharp” intracellular recordings, we analyzed here the effects of stimuli delivered at 0.1 or 1 Hz in the lateral nucleus of the amygdala on ictallike epileptiform discharges induced by the K+ channel blocker 4-aminopyridine in the perirhinal cortex, in a rat brain slice preparation. We found that 1) ictal events were nominally abolished when the stimulus rate was brought from 0.1 to 1 Hz; 2) this effect was associated with an increased latency of the epileptiform responses recorded in perirhinal cortex following each stimulus; and 3) both changes recovered to control values following arrest of the 1-Hz stimulation protocol. The control of ictal activity by 1-Hz stimulation and the concomitant latency increase were significantly reduced by GABAB receptor antagonism. We propose that this frequency-dependent increase in latency represents a short-lasting, GABAB receptor-dependent adaptive mechanism that contributes to decrease epileptiform synchronization, thus blocking seizures in epileptic patients and animal models.


2015 - Ischemic-hypoxic mechanisms leading to hippocampal dysfunction as a consequence of status epilepticus [Articolo su rivista]
Lucchi, Chiara; Vinet, Jonathan; Meletti, Stefano; Biagini, Giuseppe
abstract

Status epilepticus (SE) is one of the recognized primary precipitating events that can lead to temporal lobe epilepsy (TLE) associated with hippocampal sclerosis. This type of epilepsy is characterized by poor response to drug treatment, often requiring surgical intervention to remove the mesial temporal regions involved in the seizure onset. However, even neurosurgery may not be completely successful. Thus, the prevention of hippocampal damage and epileptogenesis is currently evaluated as a possible alternative therapeutic approach to prevent the development of pharmacoresistant TLE. Lines of evidence suggest that ischemic-hypoxic lesions might occur in different brain regions, including the hippocampus, during SE. Especially in the hippocampal CA3 region, an ischemic-like lesion develops in the stratum lacunosum-moleculare and is mainly characterized by a loss of astrocytes and neuronal processes and increased immunostaining of pimonidazole which probes areas exposed to hypoxia. Interestingly, these mechanisms can contribute to neuronal cell loss and may be counteracted by drugs that can afford vascular protection, as in the case of ligands of the ghrelin receptor. Notably, some of the ghrelin receptor ligands possess a double edge effect, since they are anticonvulsant and vascular-protective, thus, potentially representing new tools to counteract the consequences of SE. This article is part of a Special Issue entitled Status Epilepticus.


2015 - Neurosteroids as Endogenous Modulators of Epileptic Seizures [Abstract in Atti di Convegno]
Biagini, Giuseppe; Rustichelli, Cecilia; Lucchi, Chiara; Meletti, Stefano
abstract

Neurosteroids are a family of steroidal compounds which produce a variety of biological effects, mainly by interacting with the γ-aminobutyric acid receptor A (GABAA). Among neurosteroids, allopregnanolone and tetrahydrodeoxycorticosterone are positive modulators of GABAA receptors that were shown to counteract seizures in animal models. However, it is still to be defined the role of neurosteroids both in patients affected by epilepsy and in animal models based on spontaneously recurrent seizures. By using finasteride, an irreversible inhibitor of the enzyme 5α-reductase, we found that allopregnanolone cerebral levels were significantly decreased in rats treated with the convulsant pilocarpine. Interestingly, this decrease was followed by enhanced epileptogenesis. We also described a case of antiepileptic drug resistance in a patient that was maintained on finasteride to treat a dermatological disorder: seizures ceased by withdrawing finasteride. These findings point to a modulatory role of allopregnanalone and other neurosteroids on epileptic seizures.


2015 - Repeated 6-Hz Corneal Stimulation Progressively Increases FosB/ΔFosB Levels in the Lateral Amygdala and Induces Seizure Generalization to the Hippocampus [Articolo su rivista]
Giordano, Carmela; Vinet, Jonathan; Curia, Giulia; Biagini, Giuseppe
abstract

Exposure to repetitive seizures is known to promote convulsions which depend on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by a modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures always appeared in frontal cortex, but only at the fourth stimulation they involved the hippocampus, suggesting the establishment of an epileptogenic process. Duration of seizure non-motor component progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session, most likely due to activity of principal cells. These findings indicate a predominant role of amygdala in promoting progressively more severe convulsions as well as the late recruitment of the hippocampus in the seizure spread. We propose that the repeated 6-Hz corneal stimulation model may be used to investigate some mechanisms of epileptogenesis and to test putative antiepileptogenic drugs.


2015 - Repeated 6-Hz corneal stimulation progressively increases FosB/∆FosB levels in the lateral amygdala and induces seizure generalization to the hippocampus. [Abstract in Atti di Convegno]
Giordano, C; Vinet, Jonathan; Curia, Giulia; Biagini, Giuseppe
abstract

Exposure to repetitive seizures is known to promote convulsions which depends on specific patterns of network activity. We aimed at evaluating the changes in seizure phenotype and neuronal network activation caused by the modified 6-Hz corneal stimulation model of psychomotor seizures. Mice received up to 4 sessions of 6-Hz corneal stimulation with fixed current amplitude of 32 mA and an inter-stimulation interval of 72 h. Video-electroencephalography showed that evoked seizures were characterized by a motor component and a non-motor component. Seizures appeared always in frontal cortex, but only at the fourth stimulation they involved the hippocampus. Duration of non-motor seizures progressively decreased after the second session, whereas convulsive seizures remained unchanged. In addition, a more severe seizure phenotype, consisting of tonic-clonic generalized convulsions, was predominant after the second session. Immunohistochemistry and double immunofluorescence experiments revealed a significant increase in neuronal activity occurring in the lateral amygdala after the fourth session most likely due to activity of principal cells. These findings suggest a predominant role of amygdala in promoting progressively more severe convulsions.


2015 - The phenotypes of microglia and macrophages during epileptogenesis [Abstract in Rivista]
Vinet, Jonathan; Vainchtein, I. D.; Spano, Maria Carlotta; Giordano, Carmela; Bordini, D.; Dominici, Massimo; Eggen, B. J. L.; Biagini, Giuseppe
abstract

Question: A growing body of evidence is now supporting a relationship between inflammation and epilepsy. Indeed, activated microglia, reactive astrocytes, local expression of pro-inflammatory cytokines, blood brain barrier leakage and peripheral immune cell infiltration have all been observed in temporal lobe epilepsy (TLE) animal models as well as in humans. Accordingly, inflammatory mechanisms are thought to play a central role in the initiation and maintenance of seizures, starting in the acute phase represented by status epilepticus (SE) induction. Microglia activation has been correlated with the expression of several pro-inflammatory cytokines which are thought to contribute to the neuronal cell death occurring after SE. Data point towards a pro-inflammatory phenotype of microglia that precedes neuronal injury and cell death. Because of this, microglia are generally considered to play a pro-epileptogenic role. However, infiltration of peripheral immune cells during epileptogenesis such as leukocytes, granulocytes and monocytes/macrophages might also contribute to the development of chronic epilepsy and recurrent seizures. Uncertainty on the role of these POSTER ABSTRACTS E355 GLIA different inflammatory cells depended on technical limitations in the discrimination of microglia from macrophages. For this reason, it is possible that the detrimental function that is currently attributed to microglia might be incorrect and should be ascribed to infiltrating macrophages. Methods: Both microglia and macrophages were acutely isolated from the hippocampi of control and pilocarpine-treated CD1 mice (24h and 96h after SE) and FACS sorted. Microglia were defined as CD11b+ CD45int Ly-6Cneg and infiltrated macrophages as CD11b+ CD45hi Ly-6Cpos. After sorting, qPCR and flow cytometry analysis were performed. Results: During epileptogenesis, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecule CD40 and pro-inflammatory gene IL-1. In contrast, infiltrated macrophages were strongly immune activated. Both cell types expressed high levels of the phagocytosis marker AXL. Conclusions: These data suggest that macrophages might be more detrimental than microglia during epileptogenesis.


2014 - Electrocorticography demonstrates beneficial effects of EP-80317 in a model of status epilepticus [Abstract in Atti di Convegno]
Lucchi, C; Curia, Giulia; Torsello, A; Biagini, Giuseppe
abstract

Status epilepticus (SE) is a serious life-threatening condition. Treatment of SE is based on a stage approach, going from benzodiazepines in the early stage to intensive care unit and general anesthesia in severe stages. Treatment of SE is frequently unsuccessful, and high levels of mortality and morbidity are reported. Several risk factors for SE occurrence and recurrence have been identified, making prevention of this condition a primary objective. EP-80317, a ghrelin receptor antagonist, displays anticonvulsant properties on behavioral convulsive episodes. In this study, we have characterized the ability of EP-80317 to prevent progression from non-convulsive to convulsive seizures and to SE in the pilocarpine model by video-electrocorticography. SE developed in 100% of rats pretreated with saline, but only in 50% of those pretreated with EP-80317. Seizures were similarly evoked in both groups, but different rates of convulsive vs. non-convulsive episodes were observed. In particular, the rate of convulsive seizures was 15% in EP-80317-pretreated rats that did not experience SE (EP-80317/Non-SE), whereas it was about 75% in saline-pretreated group and in EP-80317-pretreated rats that experienced SE (EP-80317/SE). Moreover, after pilocarpine administration, saline-pretreated and EP-80317/SE rats showed a worsening in behavioral manifestation and seizure duration, and a progression of the power spectrum of convulsive seizures. This progressive worsening was not observed in EP-80317/Non-SE rats. We report for the first time that EP-80317 prevents the progression from non-convulsive to convulsive seizures and to SE in pilocarpine-treated rats.


2014 - Electrographic and behavioral characterization of EP-80317 anticonvulsant effects in pilocarpine-treated rats. [Abstract in Rivista]
Curia, Giulia; Lucchi, Chiara; Torsello, A; Biagini, Giuseppe
abstract

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2014 - In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed. [Articolo su rivista]
Vainchtein, Id; Vinet, Jonathan; Brouwer, N; Brendecke, S; Biagini, Giuseppe; Biber, K; Boddeke, Hw; Eggen, Bj
abstract

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS) characterized by loss of myelin accompanied by infiltration of T-lymphocytes and monocytes. Although it has been shown that these infiltrates are important for the progression of MS, the role of microglia, the resident macrophages of the CNS, remains ambiguous. Therefore, we have compared the phenotypes of microglia and macrophages in a mouse model for MS, experimental autoimmune encephalomyelitis (EAE). In order to properly discriminate between these two cell types, microglia were defined as CD11bpos CD45int Ly-6Cneg, and infiltrated macrophages as CD11bpos CD45high Ly-6Cpos. During clinical EAE, microglia displayed a weakly immune-activated phenotype, based on the expression of MHCII, co-stimulatory molecules (CD80, CD86, and CD40) and proinflammatory genes [interleukin-1b (IL-1b) and tumour necrosis factor-a (TNF-a)]. In contrast, CD11bpos CD45high Ly-6Cpos infiltrated macrophages were strongly activated and could be divided into two populations Ly-6Cint and Ly-6Chigh, respectively. Ly-6Chigh macrophages contained less myelin than Ly-6Cint macrophages and expression levels of the proinflammatory cytokines IL-1b and TNF-a were higher in Ly-6Cint macrophages. Together, our data show that during clinical EAE, microglia are only weakly activated whereas infiltrated macrophages are highly immune reactive.


2014 - Neuroactive Peptides as Putative Mediators of Antiepileptic Ketogenic Diets [Articolo su rivista]
GIORDANO, CARMELA; Maddalena, Marchiò; Elena, Timofeeva; BIAGINI, Giuseppe
abstract

Various ketogenic diet (KD) therapies, including classic KD, medium chain triglyceride administration, low glycemic index treatment, and a modified Atkins diet, have been suggested as useful in patients affected by pharmacoresistant epilepsy. A common goal of these approaches is to achieve an adequate decrease in the plasma glucose level combined with ketogenesis, in order to mimic the metabolic state of fasting. Although several metabolic hypotheses have been advanced to explain the anticonvulsant effect of KDs, including changes in the plasma levels of ketone bodies, polyunsaturated fatty acids, and brain pH, direct modulation of neurotransmitter release, especially purinergic (i.e., adenosine) and γ-aminobutyric acidergic neurotransmission, was also postulated. Neuropeptides and peptide hormones are potent modulators of synaptic activity, and their levels are regulated by metabolic states. This is the case for neuroactive peptides such as neuropeptide Y, galanin, cholecystokinin and peptide hormones such as leptin, adiponectin, and growth hormone-releasing peptides (GHRPs). In particular, the GHRP ghrelin and its related peptide des-acyl ghrelin are well-known controllers of energy homeostasis, food intake, and lipid metabolism. Notably, ghrelin has also been shown to regulate the neuronal excitability and epileptic activation of neuronal networks. Several lines of evidence suggest that GHRPs are upregulated in response to starvation and, particularly, in patients affected by anorexia and cachexia, all conditions in which also ketone bodies are upregulated. Moreover, starvation and anorexia nervosa are accompanied by changes in other peptide hormones such as adiponectin, which has received less attention. Adipocytokines such as adiponectin have also been involved in modulating epileptic activity. Thus, neuroactive peptides whose plasma levels and activity change in the presence of ketogenesis might be potential candidates for elucidating the neurohormonal mechanisms involved in the beneficial effects of KDs. In this review, we summarize the current evidence for altered regulation of the synthesis of neuropeptides and peripheral hormones in response to KDs, and we try to define a possible role for specific neuroactive peptides in mediating the antiepileptic properties of diet-induced ketogenesis.


2014 - Pathophysiogenesis of Mesial Temporal Lobe Epilepsy: Is Prevention of Damage Antiepileptogenic? [Articolo su rivista]
Curia, Giulia; Lucchi, Chiara; Vinet, Jonathan; Gualtieri, F; Marinelli, C; Torsello, A; Costantino, Luca; Biagini, Giuseppe
abstract

Temporal lobe epilepsy (TLE) is frequently associated with hippocampal sclerosis, possibly caused by a primary brain injury that occurred a long time before the appearance of neurological symptoms. This type of epilepsy is characterized by refractoriness to drug treatment, so to require surgical resection of mesial temporal regions involved in seizure onset. Even this last therapeutic approach may fail in giving relief to patients. Although prevention of hippocampal damage and epileptogenesis after a primary event could be a key innovative approach to TLE, the lack of clear data on the pathophysiological mechanisms leading to TLE does not allow any rational therapy. Here we address the current knowledge on mechanisms supposed to be involved in epileptogenesis, as well as on the possible innovative treatments that may lead to a preventive approach. Besides loss of principal neurons and of specific interneurons, network rearrangement caused by axonal sprouting and neurogenesis are well known phenomena that are integrated by changes in receptor and channel functioning and modifications in other cellular components. In particular, a growing body of evidence from the study of animal models suggests that disruption of vascular and astrocytic components of the blood-brain barrier takes place in injured brain regions such as the hippocampus and piriform cortex. These events may be counteracted by drugs able to prevent damage to the vascular component, as in the case of the growth hormone secretagogue ghrelin and its analogues. A thoroughly investigation on these new pharmacological tools may lead to design effective preventive therapies.


2014 - Proprietà anticonvulsivanti di ligandi del recettore GHS-R1a. [Abstract in Atti di Convegno]
Biagini, Giuseppe; Giordano, Carmela; Lucchi, Chiara; Marchio', Maddalena; Bresciani, E; Torsello, A; Curia, Giulia
abstract

Status epilepticus (SE) is a serious life-threatening condition. Treatment of SE is based on a stage approach, going from benzodiazepines in the early stage to intensive care unit and general anesthesia in severe stages. Treatment of SE is frequently unsuccessful, and high levels of mortality and morbidity are reported. Several risk factors for SE occurrence and recurrence have been identified, making prevention of this condition a primary objective. EP-80317, a ghrelin receptor antagonist, displays anticonvulsant properties on behavioral convulsive episodes. In this study, we have characterized the ability of EP-80317 to prevent progression from non-convulsive to convulsive seizures and to SE in the pilocarpine model by video-electrocorticography. SE developed in 100% of rats pretreated with saline, but only in 50% of those pretreated with EP-80317. Seizures were similarly evoked in both groups, but different rates of convulsive vs. non-convulsive episodes were observed. In particular, the rate of convulsive seizures was 15% in EP-80317-pretreated rats that did not experience SE (EP-80317/Non-SE), whereas it was about 75% in saline-pretreated group and in EP-80317-pretreated rats that experienced SE (EP-80317/SE). Moreover, after pilocarpine administration, saline-pretreated and EP-80317/SE rats showed a worsening in behavioral manifestation and seizure duration, and a progression of the power spectrum of convulsive seizures. This progressive worsening was not observed in EP-80317/Non-SE rats. We report for the first time that EP-80317 prevents the progression from non-convulsive to convulsive seizures and to SE in pilocarpine-treated rats.


2013 - Hypoxia markers are expressed in interneurons exposed to recurrent seizures. [Articolo su rivista]
Gualtieri, Fabio; C., Marinelli; Longo, Daniela; Pugnaghi, Matteo; Nichelli, Paolo Frigio; Meletti, Stefano; Biagini, Giuseppe
abstract

An early but transient decrease in oxygen availability occurs during experimentally induced seizures. Using pimonidazole, which probes hypoxic insults, we found that by increasing the duration of pilocarpine-induced status epilepticus (SE) from 30 min to 120 min, counts of pimonidazole-immunoreactive neurons also increased (P < 0.01, 120 vs 60 and 30 min). All the animals exposed to SE were immunopositive to pimonidazole, but a different scenario emerged during epileptogenesis when a decrease in pimonidazole immunostained cells occurred from 7 to 14 days, so that only 1 out of 4 rats presented with pimonidazole-immunopositive cells. Pimonidazole-immunoreactive cells robustly reappeared at 21 days post-SE induction when all animals (7 out of 7) had developed spontaneous recurrent seizures. Specific neuronal markers revealed that immunopositivity to pimonidazole was present in cells identified by neuropeptide Y (NPY) or somatostatin antibodies. At variance, neurons immunopositive to parvalbumin or cholecystokinin were not immunopositive to pimonidazole. Pimonidazoleimmunopositive neurons expressed remarkable immunoreactivity to hypoxia-inducible factor 1α (HIF-1α). Interestingly, surgical samples obtained from pharmacoresistant patients showed neurons co-labeled by HIF-1α and NPY antibodies. These interneurons, along with parvalbumin-positive interneurons that were negative to HIF-1α, showed immunopositivity to markers of cell damage, such as high-mobility group box 1 in the cytoplasm and cleaved caspase-3 in the nucleus. These findings suggest that interneurons are continuously endangered in rodent and human epileptogenic tissue. The presence of hypoxia and cell damage markers in NPY interneurons of rats and patients presenting with recurrent seizures indicates a mechanism of selective vulnerability in a specific neuronal subpopulation.


2013 - Neurosteroids And Epileptogenesis [Articolo su rivista]
Biagini, Giuseppe; Rustichelli, Cecilia; Curia, Giulia; Vinet, Jonathan; Lucchi, Chiara; Pugnaghi, Matteo; Meletti, Stefano
abstract

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its upregulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus (SE) induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.


2013 - Neurosteroids and epileptogenesis. [Abstract in Atti di Convegno]
Biagini, Giuseppe; Rustichelli, Cecilia; Curia, Giulia; Lucchi, Chiara; Pugnaghi, Matteo; Meletti, Stefano; Avoli, M.
abstract

Epileptogenesis is defined as the latent period at the end of which spontaneous recurrent seizures occur. This concept has been recently re-evaluated to include exacerbation of clinically-manifested epilepsy. Thus, in patients affected by pharmacoresistant seizures, the progression toward a worse condition may be viewed as the result of a durable epileptogenic process. However, the mechanism potentially responsible for this progression remains unclear. Neuroinflammation has been consistently detected both in the latent period and in the chronic phase of epilepsy, especially when brain damage is present. This phenomenon is accompanied by glial cell reaction, leading to gliosis. We have previously described rats presenting an increased expression of the cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, during the latent period, in glial cells of the hippocampus. The P450scc enzyme is critically involved in the synthesis of neurosteroids and its up-regulation is associated with a delayed appearance of spontaneous recurrent seizures in rats that experienced status epilepticus induced by pilocarpine. Moreover, by decreasing the synthesis of neurosteroids able to promote inhibition, such as allopregnanolone, through the administration of the 5α-reductase blocker finasteride, it is possible to terminate the latent period in pilocarpine-treated rats. Finasteride was also found to promote seizures in the chronic period of epileptic rats, suggesting that neurosteroids are continuously produced to counteract seizures. In humans, exacerbation of epilepsy has been also described in patients occasionally exposed to finasteride. Overall, these findings suggest a major role of neurosteroids in the progression of epilepsy and a possible antiepileptogenic role of allopregnanolone and cognate molecules.


2013 - Perirhinal cortex and temporal lobe epilepsy. [Articolo su rivista]
Biagini, Giuseppe; D'Antuono, M; Benini, R; de Guzman, P; Longo, Daniela; Avoli, M.
abstract

The perirhinal cortex – which is interconnected with several limbic structures and is intimately involved in learning and memory - plays major roles in pathological processes such as the kindling phenomenon of epileptogenesis and the spread of limbic seizures. Both features may be relevant to the pathophysiology of mesial temporal lobe epilepsy that represents the most refractory adult form of epilepsy with up to 30% of patients not achieving adequate seizure control. Compared to other limbic structures such as the hippocampus or the entorhinal cortex, the perirhinal area remains understudied and, in particular, detailed information on its dysfunctional characteristics remains scarce; this lack of information may be due to the fact that the perirhinal cortex is not grossly damaged in mesial temporal lobe epilepsy and in models mimicking this epileptic disorder. However, we have recently identified in pilocarpine-treated epileptic rats the presence of selective losses of interneuron subtypes along with increased synaptic excitability. In this review we: (i) highlight the fundamental electrophysiological properties of perirhinal cortex neurons; (ii) briefly stress the mechanisms underlying epileptiform synchronization in perirhinal cortex networks following epileptogenic pharmacological manipulations; and (iii) focus on the changes in neuronal excitability and cytoarchitecture of the perirhinal cortex occurring in the pilocarpine model of mesial temporal lobe epilepsy. Overall, these data indicate that perirhinal cortex networks are hyperexcitable in an animal model of temporal lobe epilepsy, and that this condition is associated with a selective cellular damage that is characterized by an age-dependent sensitivity of interneurons to precipitating injuries, such as status epilepticus.


2013 - Protective but Not Anticonvulsant Effects of Ghrelin and JMV-1843 in the Pilocarpine Model of Status epilepticus. [Articolo su rivista]
Lucchi, Chiara; Curia, Giulia; Vinet, Jonathan; Gualtieri, Fabio; Bresciani, E; Locatelli, V; Torsello, A; Biagini, Giuseppe
abstract

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45±0.07 mm2 in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P&lt;0.05) and JMV-1843 (P&lt;0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P&lt;0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P&lt;0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.


2013 - Protective but not anticonvulsive effects of ghrelin and JMV-1843 in the pilocarpine model of status epilepticus. [Abstract in Atti di Convegno]
Lucchi, Chiara; Curia, Giulia; Vinet, Jonathan; Gualtieri, F; Torsello, A; Biagini, Giuseppe
abstract

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45 ± 0.07 mm(2) in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P&lt;0.05) and JMV-1843 (P&lt;0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P&lt;0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P&lt;0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.


2013 - Resilience to audiogenic seizures is associated with p-ERK1/2 dephosphorylation in the subiculum of Fmr1 knockout mice [Articolo su rivista]
Curia, Giulia; Gualtieri, Fabio; Bartolomeo, Regina; Riccardo, Vezzali; Biagini, Giuseppe
abstract

Young, but not adult, Fmr1 knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P&lt;0.01 vs WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P&lt;0.05 vs WT) and CA3 (P&lt;0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P&lt;0.05 vs WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P&lt;0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice.


2013 - Resilience to audiogenic seizures is associated with p-ERK1/2 dephosphorylation in the subiculum of a mice model of Fragile X syndrome. [Abstract in Atti di Convegno]
Curia, Giulia; Gualtieri, F; Bartolomeo, Regina; Vezzali, R; Biagini, Giuseppe
abstract

Young, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P &lt; 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P &lt; 0.05 vs. WT) and CA3 (P &lt; 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P &lt; 0.05 vs. WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P &lt; 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice


2013 - Temporal lobe epilepsy exacerbation during pharmacological inhibition of endogenous neurosteroid synthesis [Articolo su rivista]
Pugnaghi, Matteo; Monti, Giulia; Biagini, Giuseppe; Meletti, Stefano
abstract

We report the case of a woman who presented cryptogenic temporal lobe seizures from the age of 43 years. Antiepileptic drug (AED) treatment with carbamazepine was able to control seizures for 1 year, but seizures relapsed and an add-on treatment with lamotrigine was started without achieving seizures control. The patient's medical history was unremarkable except for a mild hirsutism for which she was taking finasteride since 45 years of age. In view of the possible relationship between finasteride, a known inhibitor of neurosteroids synthesis, and patient's seizures exacerbation, we stopped finasteride resulting in prompt recovery of seizures control. It is know that 5α-dihydrosteroids are precursors of powerful positive modulators of γ-aminobutyric acid-A inhibitory currents and exert antiseizure effects in animal epilepsy models. This case supports the hypothesis that endogenous neurosteroids can modulate seizure susceptibility and response to AEDs also in humans, suggesting their possible use as a new therapeutic option.


2012 - Ghrelin anticonvulsive properties: is it matter of desacylation? [Articolo su rivista]
Biagini, Giuseppe; V., Locatelli; A., Torsello
abstract

We have read the article by Portelli et al. (2012) that thoroughly reviews the still scarce literature existing on the role of ghrelin and its receptor (GHSR1a) in epilepsy. As the authors recognize, clinical studies on the relationship between ghrelin and epilepsy are largely contradictory, since some of them show increased ghrelin levels, other no changes or even a decrease in patients affected by epilepsy: so, factors causing such variability need to be carefully addressed in future investigations. On the other hand, Portelli and coworkers look at data coming from animal studies as clearly demonstrating a role for ghrelin as anticonvulsant. Supporting this view, they cite two different studies showing that ghrelin is able to counteract the convulsive effects of pentylenetetrazole (Obay et al., 2007) or kainic acid (Lee et al., 2010), and a third one that reports negative findings both in pilocarpine and kainate models (Biagini et al, 2001). In our opinion, these discrepancies are only apparent, since Lee and coworkers (2010) reported beneficial effects not on seizure prevention, but on seizure severity, whereas we evaluated just seizure induction. More intriguingly, Obay and coworkers (2007) found that ghrelin dose-dependently increased latency to (but did not prevent) myoclonic jerks, tonic generalized extension and generalized clonic seizures. Duration of tonic generalized extension was also decreased by ghrelin, but duration of the initial myoclonic jerk was increased as well as the duration of clonic generalized seizures. Furthermore, a small effect on overall seizure duration was obtained only with the highest ghrelin dose. Alternatively, we have reported that GHSR1a ligands other than ghrelin prevent seizures in pilocarpine-treated rats, and that desacyl ghrelin increases the latency to first generalized seizures in the kainate model.A key observation to understand these discrepancies is that “…In human plasma, acylated ghrelin was found to disappear more quickly than total ghrelin, with elimination half-lives of 9-13 and 27-31 min, respectively (Akamizu et al., 2004)”, as cited by Portelli and coauthors (page 586 of the review). In spite that ghrelin was always administered 30 min before the tested convulsant (Obay et al., 2007; Lee et al., 2010), in no one of the mentioned studies circulating levels of ghrelin and desacyl ghrelin were assessed. In view of the probable conversion of ghrelin to desacyl ghrelin by butyrylcholinesterase (De Vriese et al., 2004) during latency to convulsions, it cannot be excluded that the anticonvulsive effects attributed to ghrelin could be due to desacyl ghrelin. Accordingly, the highly effective GHSR1a agonist JMV-1843 (also known as EP01572 or ARD-07), which has a better kinetics than ghrelin, in our hands failed to counteract the pilocarpine effects. We believe that the present evidence is still too poor to suggest a role for GHSR1a agonists in seizure prevention, at least in animal models. The same questions apply to clinical investigations, in which the balance between ghrelin and desacyl ghrelin is not clearly addressed.


2012 - Glia-neuron interactions: neurosteroids and epileptogenesis. [Capitolo/Saggio]
Biagini, Giuseppe; C., Marinelli; G., Panuccio; Puja, Giulia; M., Avoli
abstract

Glia can influence the outcome of an epileptogenic insult by controlling the recovery of neuronalnetworks and functions. In particular, glia may facilitate the establishment of epilepsy by impairedremoval of glutamate from synapses or by releasing inflammatory cytokines and excitatoryneurotransmitters, such as interleukin-1β or, respectively, glutamate, aspartate and D-serine.Opposed to these pro-excitatory/pro-epileptogenic mediators, glia can also release molecules thatrestrain neuronal excitability such as neurosteroids, which are potent modulators of inhibitorycurrents dependent on γ-aminobutyric acid (GABA) type A receptors. In normal conditions,neurosteroids are mainly synthesized in neurons by conversion of cholesterol to pregnenolone, a stepcatalyzed by the cytochrome P450 cholesterol-side chain cleavage enzyme (P450scc). Following anepileptogenic insult, astrocytes transform into reactive cells and express high levels of P450scc, thusbecoming major players in neurosteroid synthesis. In this context, we found that the degree ofP450scc expression in astrocytes dictates the duration of the latent period. In line with this view,inhibition of neurosteroid synthesis anticipates the establishment of chronic epilepsy only when theP450scc induction is intense and long lasting. Thus, we hypothesize that reactive astrocytes maydampen neuronal excitability in the course of epileptogenesis through neurosteroid-mediatedmechanisms that likely enhance GABAergic neurotransmission.


2012 - Increased perivascular laminin predicts damage to astrocytes in CA3 and piriform cortex following chemoconvulsive treatments. [Articolo su rivista]
Gualtieri, Fabio; Curia, Giulia; C., Marinelli; Biagini, Giuseppe
abstract

Status epilepticus (SE) induced by pilocarpine or kainate is associated with yet notsystemically investigated astrocytic and vascular injuries. To investigate their possibleassociation with neuronal damage, the changes in glial fibrillary acidic protein (GFAP),laminin and neuron-specific nuclear protein (NeuN) immunoreactivities were analyzed in ratstreated with pilocarpine (380 mg/kg) or kainate (15 mg/kg), and receiving diazepam (20mg/kg) after 10 min of SE. A different group of rats was injected with endothelin-1 (ET-1) inthe caudate putamen to reproduce the changes in GFAP and laminin immunoreactivitiesassociated with ischemia. Focal loss of GFAP immunostaining was accompanied byincreased laminin immunoreactivity in blood vessels, in all the examined groups. Regressionanalysis revealed a significant (P &lt; 0.01) relationship between astrocytic lesion andincreased laminin immunoreactivity in the piriform cortex (Pir) of both pilocarpine (R2 = 0.88)and kainate (R2 = 0.94) groups of treatment. A significant relationship (P &lt; 0.01; R2 = 0.81)was also present in the CA3 hippocampal region of pilocarpine-treated rats. At variance,neuronal and glial lesions were significantly related (P &lt; 0.05, R2 = 0.74) only in thesubstantia nigra of pilocarpine-treated rats. The ratio between areas of GFAP and lamininchanges of immunoreactivity in the ET-1 group was similar to those found in pilocarpineandkainate-treated rats in specific brain regions, such as the hippocampal CA3 subfield, Pirand the anterior olfactory nucleus. The amygdala and submedius thalamic nucleus in thepilocarpine group, and the perirhinal and entorhinal cortices in the kainate group, alsopresented ischemic-like changes. These results indicate that laminin immunoreactivity isupregulated in the basal lamina of blood vessels after SE induced by pilocarpine or kainate.This phenomenon is significantly associated with lesions involving more glial than neuronalcells, in specific cerebral regions.


2011 - Audiogenic seizures selectively activate hippocampal neurons in young mice affected by fragile X syndrome [Abstract in Atti di Convegno]
Gualtieri, F; Bartolomeo, Regina; Vezzali, R; Curia, Giulia; Biagini, Giuseppe
abstract

Young, but not adult, fragile X mental retardation gene (Fmr1) knockout (KO) mice display audiogenic seizures (AGS) that can be prevented by inhibiting extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation. In order to identify the cerebral regions involved in these phenomena, we characterized the response to AGS in Fmr1 KO mice and wild type (WT) controls at postnatal day (P) 45 and P90. To characterize the diverse response to AGS in various cerebral regions, we evaluated the activity markers FosB/ΔFosB and phosphorylated ERK1/2 (p-ERK1/2). Wild running (100% of tested mice) followed by clonic/tonic seizures (30%) were observed in P45 Fmr1 KO mice, but not in WT mice. In P90 Fmr1 KO mice, wild running was only present in 25% of tested animals. Basal FosB/ΔFosB immunoreactivity was higher (P < 0.01 vs. WT) in the CA1 and subiculum of P45 Fmr1 KO mice. Following the AGS test, FosB/ΔFosB expression consistently increased in most of the analyzed regions in both groups at P45, but not at P90. Interestingly, FosB/ΔFosB immunoreactivity was significantly higher in P45 Fmr1 KO mice in the medial geniculate body (P < 0.05 vs. WT) and CA3 (P < 0.01). Neurons presenting with immunopositivity to p-ERK1/2 were more abundant in the subiculum of Fmr1 KO mice in control condition (P < 0.05 vs. WT, in both age groups). In this region, p-ERK1/2-immunopositive cells significantly decreased (-75%, P < 0.01) in P90 Fmr1 KO mice exposed to the AGS test, but no changes were found in P45 mice or in other brain regions. In both age groups of WT mice, p-ERK1/2-immunopositive cells increased in the subiculum after exposure to the acoustic test. Our findings illustrate that FosB/ΔFosB markers are overexpressed in the medial geniculate body and CA3 in Fmr1 KO mice experiencing AGS, and that p-ERK1/2 is markedly decreased in the subiculum of Fmr1 KO mice resistant to AGS induction. These findings suggest that resilience to AGS is associated with dephosphorylation of p-ERK1/2 in the subiculum of mature Fmr1 KO mice.


2011 - Beneficial effects of desacyl-ghrelin, hexarelin and EP-80317 in models of status epilepticus. [Articolo su rivista]
Biagini, Giuseppe; A., Torsello; C., Marinelli; Gualtieri, Fabio; R., Vezzali; S., Coco; E., Bresciani; V., Locatelli
abstract

It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this study we aimed to characterize the anticonvulsive activity of ghrelin and other growth hormone secretagogue receptor 1a (GHSR1a) ligands in rats exposed to status epilepticus induced by pilocarpine or kainate. Firstly, in three independent experiments, before receiving pilocarpine (380 mg/kg, i.p.), rats were pretreated with one among ghrelin (1.5 mg/kg), desacyl-ghrelin (1.5 mg/kg), hexarelin (330 μg/kg), EP-80317 (330 μg/kg), JMV-1843 (330 μg/kg), JMV-2959 (330 μg/kg) or saline. Secondly, in the fourth experiment, rats were pretreated with i.p. ghrelin, desacyl-ghrelin, hexarelin, EP-80317 or saline, followed by kainate (15 mg/kg, i.p.). We evaluated: induction of generalized seizures, latency to generalized seizures, status epilepticus, latency to status epilepticus (the time lag between the first tonic–clonic convulsion and the switch to continuous seizures) and mortality. In the pilocarpine model, 60% of rats pretreated with EP-80317 (P < 0.05) showed no seizure. Hexarelin and EP-80317 were both able to prevent progression to status epilepticus in pilocarpine-treated rats (P < 0.05). When status epilepticus was induced by kainate, seizures developed with few exceptions. However, latency to status epilepticus was significantly (P < 0.01) longer in rats pretreated with desacyl-ghrelin, whereas hexarelin and EP-80317 did not display any effect. Almost all GHSR1a ligands prevented pilocarpine-induced mortality, which was observed only in rats pretreated with saline or JMV-2959. After kainate administration, all rats survived to status epilepticus. These findings demonstrate that desacyl-ghrelin, hexarelin and EP-80317 but not other GHSR1a ligands display relevant anticonvulsive properties in models of limbic seizures.


2011 - Effetti anticonvulsivanti di desacyl-ghrelin in un modello d'epilessia del lobo temporale. [Relazione in Atti di Convegno]
Gualtieri, Fabio; A., Torsello; C., Marinelli; E., Bresciani; R., Vezzali; V., Locatelli; Biagini, Giuseppe
abstract

We investigated the effects of the growth hormone secretagogue ghrelin and its precursor/metabolitedesacyl-ghrelin in rats exposed to pilocarpine-induced seizures. Pilocarpine (380 mg/kg) was administered toSprague-Dawley rats pretreated with scopolamine, followed by saline (control group) or the tested hormone(ghrelin and desacyl-ghrelin at 1.5 mg/kg). After induction of status epilepticus (SE), seizures were quelled byinjecting diazepam (20 mg/kg). Four days after SE, rats were sacrificed to evaluate lesions in hippocampus.Pilocarpine did not induce seizures in 20% and SE was prevented in 60% of desacyl-ghrelin treated rats. In ghrelintreated group, all rats developed seizures and SE. Interestingly, both ghrelin and desacyl-ghrelin preventedpost-SE mortality. Hippocampal lesions were larger in control rats than in ghrelin or desacyl-ghrelin treated rats(p < 0.05 unpaired Student’s t-test). We thereby conclude that desacyl-ghrelin possesses antiepileptic propertiesin the pilocarpine model of temporal lobe epilepsy.


2011 - Perirhinal cortex hyperexcitability in pilocarpine-treated rats [Articolo su rivista]
R., Benini; Longo, Daniela; Biagini, Giuseppe; M., Avoli
abstract

The perirhinal cortex (PC), which is heavily connected with several epileptogenic regions of the limbic system such as the entorhinal cortex and amygdala, is involved in the generation and spread of seizures. However, the functional alterations occurring within an epileptic PC network are unknown. Here, we analyzed this issue by employing in vitro electrophysiology and immunohistochemistry in brain tissue obtained from pilocarpine-treated epileptic rats and age-matched, non-epileptic controls (NECs). Neurons recorded intracellularly from the PC deep layers in the two experimental groups had similar intrinsic and firing properties, and generated spontaneous depolarizing and hyperpolarizing postsynaptic potentials with comparable duration and amplitude. However, spontaneous and stimulus-induced epileptiform discharges were seen with field potential recordings in over one fifth of pilocarpine-treated slices but never in NEC tissue. These network events were reduced in duration by antagonizing NMDA receptors and abolished by NMDA+non-NMDA glutamatergic receptor antagonists. Pharmacologically isolated IPSPs had reversal potentials for the early GABAA receptor-mediated component that were significantly more depolarized in pilocarpine-treated cells. Experiments with a potassium-chloride co-transporter 2 antibody identified in pilocarpine-treated PC a significant immunostaining decrease that could not be explained by neuronal loss. However, interneurons expressing parvalbumin and neuropeptide Y were found to be decreased throughout the PC, whereas cholecystokinin-positive cells were diminished in superficial layers. These findings demonstrate synaptic hyperexcitability that is contributed by attenuated inhibition in the PC of pilocarpine-treated epileptic rats, and underscore the role of PC networks in temporal lobe epilepsy.


2010 - A novel pharmacological approach and identification of peripheral cellular biomarkers in Niemann-Pick Type C disease patients [Relazione in Atti di Convegno]
C., Frank; S., Rufini; D., Grossi; G., De Chiara; C., Dionisi Vici; Biagini, Giuseppe; V., Tancredi; D., Merlo; G., D’Arcangelo
abstract

In our electrophysiological results input-output curve, BST and PPF didn’t show significantdifferences in CA1 pyramidal layer of hippocampal formation both in WT and NPC1 -/- miceslices. On the contrary, synaptic plasticity was affected: in fact induction and maintenance ofNMDA-dependent LTP in the CA1 region of NPC1 -/- hippocampal slices were significantlyreduced, suggesting that NMDA receptor activity is impaired. In line with these observations,we found a significantly reduction in NMDA-induced calcium influx in NPC culturedhippocampal neurons as compared with WT neurons. To evaluate if glutamate receptors displayfunctional changes in NPC1 -/- mice slices, we studied synaptic transmission duringpharmacological stimulation by adding to the bath solution KA and AMPA (2M). While theperfusion of KA depressed synaptic transmission at CA3-CA1 synapses in both groups ofanimals, followed by a recovery during washout only in NPC1 -/- mice slices, AMPAapplication induced a disappearance of synaptic transmission only in NPC1 -/- mice slices; theseresults indicate that AMPA-Kainate receptors present quantitative and/or a qualitativedifferences probably linked to lipid rafts disruption. Moreover electrophysiological results aresupported by data outcoming from cell culture experiments on excitatory aminoacid-inducedintracellular calcium increase. Indeed, application of KA and AMPA in WT and NPC1 -/-cultured cells induced different calcium influxes, which were increased in homozygote cells.However, Western Blot analysis of synaptosomal membrane fractions from NPC and WT micedid not reveal any significant difference in AMPA (GluR1), KA (GluR6/7, KA2) or NMDA(R2) receptor expression in the two groups of animals. We further investigated whether thiscould result in compromised intracellular signal transduction pathways in NPC -/- slicesfollowing kainic acid perfusion.


2010 - Antiepileptic drugs abolish ictal but not interictal epileptiform discharges in vitro [Articolo su rivista]
M., D’Antuono M; R., Kohling; S., Ricalzone; J., Gotman; Biagini, Giuseppe; M., Avoli
abstract

Purpose: We established the effects of the antiepilepticdrugs (AEDs) carbamazepine (CBZ), topiramate(TPM), and valproic acid (VPA) on theepileptiform activity induced by 4-aminopyridine(4AP) in the rat entorhinal cortex (EC) in anin vitro brain slice preparation.Methods: Brain slices were obtained from Sprague-Dawley rats (200–250 g). Field and intracellularrecordings were made from the EC during bathapplication of 4AP (50 microM). AEDs, and in someexperiments, picrotoxin were bath applied concomitantly.Results: Prolonged (>3 s), ictal-like epileptiformevents were abolished by CBZ (50 microM), TPM(50 microM), and VPA (1 mM), whereas shorter (<3 s)interictal-like discharges continued to occur, evenat concentrations up to 4-fold as high. gamma-Aminobutyricacid (GABA)A–receptor antagonism changedthe 4AP-induced activity into recurrent interictallikeevents that were not affected by CBZ or TPM,even at the highest concentrations. To establishwhether these findings reflected the temporal featuresof the epileptiform discharges, we testedCBZ and TPM on 4AP-induced epileptiform activitydriven by stimuli delivered at 100-, 10-, and 5-sintervals; these AEDs reduced ictal-like responsesto stimuli at 100-s intervals at nearly therapeuticconcentrations, but did not influence shorter interictal-like events elicited by stimuli deliveredevery 10 or 5 s.Conclusions: We conclude that the AED ability tocontrol epileptiform synchronization in vitrodepends mainly on activity-dependent characteristicssuch as discharge duration. Our data are inkeeping with clinical evidence indicating that interictalactivity is unaffected by AED levels that areeffective to stop seizures.


2010 - Convulsive status epilepticus duration as determinant for epileptogenesis and interictal discharge generation in the rat limbic system. [Articolo su rivista]
A., Bortel; M., Lévesque; Biagini, Giuseppe; J., Gotman; M., Avoli
abstract

We analyzed with EEG-video monitoring the epileptic activity recorded during the latent and chronic periods in rats undergoing 30 or 120 min pilocarpine-induced convulsive status epilepticus (SE). Interictal discharges frequency in the entorhinal cortex (EC) of animals exposed to 120 min SE was significantly higher in the chronic than in the latent period. Following seizure appearance, interictal spikes diminished in duration in the CA3 of the 120 min SE group, and occurred at higher rates in the amygdala in all animals. Rats exposed to 120 min SE generated shorter seizures but presented twice as many non-convulsive seizures per day as the 30 min group. Finally, seizures most frequently initiated in CA3 in the 120 min SE group but had similar onset in CA3 and EC in the 30 min group. These findings indicate that convulsive SE duration influences the development of interictal and ictal activity, and that interictal discharges undergo structure-specific changes after seizure appearance.


2010 - Glia-neuron interactions: neurosteroids. [Articolo su rivista]
Biagini, Giuseppe; M., Avoli
abstract

Glial cells have both beneficial and detrimentaleffects on recovery from brain damage and playa role in epileptogenesis. Herein, we summarizeevidence indicating that the ability of astrocytesto produce neurosteroids that reinforce c-aminobutyricacid receptor A (GABAA) functionsimpinge on epileptogenesis, as shown in temporallobe epilepsy models. For an expanded treatmentof this topic see Jasper’s Basic Mechanisms of theEpilepsies, Fourth Edition (Noebels JL, Avoli M, RogawskiMA, Olsen RW, Delgado-Escueta AV, eds)published by Oxford University Press (available onthe NCBI Bookshelf).


2010 - Homocysteine potentiates seizures and cell loss induced by pilocarpine treatment [Articolo su rivista]
Baldelli, Enrica; Leo, Giuseppina; Andreoli, Nicola; K., Fuxe; Biagini, Giuseppe; Agnati, Luigi Francesco
abstract

Patients affected by recurrent seizures frequently present increased homocysteine plasma levels in consequence of treatment with antiepileptic drugs. Homocysteine is proconvulsant and can affect the response to antiepileptic drugs. In addition, high homocysteine plasma levels represent a risk factor for cardiovascular and neurodegenerative diseases. To better define the role of increased homocysteine in epilepsy, we analyzed the effects of homocysteine pretreatment in the pilocarpine model of status epilepticus (SE), which is used to mimic temporal lobe epilepsy (TLE) in rodents. Precisely, we investigated whether a moderate hyperhomocysteinemia, unable to cause seizures, could sensitize rats to pilocarpine and cooperate in inducing brain lesions. We found that a subthreshold dose of pilocarpine (200 mg/kg) is sufficient to induce SE in the majority (approximately 90%) of rats pretreated with homocysteine for 2 weeks, whereas only 40% of saline-treated controls developed SE following the same pilocarpine dose. Furthermore, homocysteine pretreatment led to a significant increase in neuronal cell loss evaluated by counting toluidine blue-stained or Fluoro-Jade-positive cells in hippocampal and parahippocampal regions. Pilocarpine augmented amyloid beta expression in both animal groups. However, pretreatment with homocysteine favored the intraneuronal fibrillar conformation of amyloid beta, thus promoting neurodegeneration. These findings indicate that increased homocysteine levels enhance seizure activity and neurodegeneration in pilocarpine-treated rats and suggest that similar detrimental effects may occur in patients affected by TLE.


2010 - In vitro ictogenesis and parahippocampal networks in a rodent model of temporal lobe epilepsy [Articolo su rivista]
G., Panuccio; M., D’Antuono; P., de Guzman; L., Delannoy; Biagini, Giuseppe; M., Avoli
abstract

Temporal lobe epilepsy (TLE) is a chronic epileptic disorder involving the hippocampal formation. Details onthe interactions between the hippocampus proper and parahippocampal networks during ictogenesisremain, however, unclear. In addition, recent findings have shown that epileptic limbic networks maintainedin vitro are paradoxically less responsive than non-epileptic control (NEC) tissue to application of theconvulsant drug 4-aminopyridine (4AP). Field potential recordings allowed us to establish here the effects of4AP in brain slices obtained from NEC and pilocarpine-treated epileptic rats; these slices included thehippocampus and parahippocampal areas such as entorhinal and perirhinal cortices and the amygdala. First,we found that both types of tissue generate epileptiform discharges with similar electrographiccharacteristics. Further investigation showed that generation of robust ictal-like discharges in the epilepticrat tissue is (i) favored by decreased hippocampal output (ii) reinforced by EC–subiculum interactions and(iii) predominantly driven by amygdala networks. We propose that a functional switch to alternativesynaptic routes may promote network hyperexcitability in the epileptic limbic system.


2010 - Neurosteroids and epilepsy [Articolo su rivista]
Biagini, Giuseppe; G., Panuccio; M., Avoli
abstract

Purpose of reviewNeurosteroids are a family of compounds synthesized directly in the brain bytransforming cholesterol into pregnenolone, which is then converted to compoundssuch as allopregnanolone and allotetrahydrodeoxycorticosterone. In view of their abilityto modulate neurotransmission, neurosteroids may influence the clinical course ofepileptic disorders. In this review, we highlight two emerging properties ofneurosteroids, that is, their anticonvulsant and antiepileptogenic activities.Recent findingsIt has been shown that fluctuations in neurosteroid synthesis, such as those seen inresponse to stress or during the ovarian cycle, determine an increase in seizurethreshold. Moreover, increased neurosteroid synthesis, presumably occurring in glialcells during epileptogenesis, delays the appearance of recurrent spontaneous seizuresin an animal model of temporal lobe epilepsy; such an effect may be due to augmentedtonic g-aminobutyric acid type A receptor-mediated inhibition. Finally, clinical trials withganaxolone, an allopregnanolone analogue, have demonstrated beneficial effects inpharmacoresistant epileptic patients, whereas finasteride – which interferes withneurosteroid synthesis – facilitates seizures in catamenial epilepsy.SummaryThe overall evidence suggests that neurosteroids may represent a novel therapeuticstrategy in epileptic disorders and a future perspective to control epileptogenicity.


2010 - Selective changes in inhibition as determinants for limited hyperexcitability in the insular cortex of epileptic rats [Articolo su rivista]
A., Bortel; Longo, Daniela; P., de Guzman; F., Dubeau; Biagini, Giuseppe; M., Avoli
abstract

The insular cortex (IC) is involved in the generalization of epileptic discharges in temporal lobe epilepsy (TLE), while seizures originating in IC can mimic the epileptic phenotype seen in some TLE patients. Few studies have however addressed the changes occurring in the IC in TLE animal models. Here, we analyzed the immunohistochemical and electrophysiological properties of IC networks in non-epileptic control and pilocarpine-treated epileptic rats. Neurons identified with a neuron-specific nuclear protein antibody showed similar counts in the two types of tissue but parvalbumin- and neuropeptide Y-positive interneurons were significantly decreased (parvalbumin, approx. -35%; neuropeptide Y, approx. -38%; P<0.01) in the epileptic IC. Non-adapting neurons were more frequently seen in the epileptic IC during intracellular injection of depolarizing current pulses. In addition, single-shock electrical stimuli elicited network-driven epileptiform responses in 87% of epileptic and in 22% of non-epileptic control neurons (P<0.01) but spontaneous postsynaptic potentials had similar amplitude, duration and intervals of occurrence in the two groups. Finally, pharmacologically isolated, GABAA receptor-mediated inhibitory postsynaptic potentials had more negative reversal potential (P<0.01) and higher peak conductance (P<0.05) in epileptic tissue. These data reveal moderate increased network excitability in the IC of pilocarpine-treated epileptic rats. We propose that such limited degree of hyperexcitability originates from loss of parvalbumin- and neuropeptide Y-positive interneurons that is compensated by an increased drive for GABAA receptor-mediated inhibition.


2010 - Uso di agonisti e antagonisti dei recettori per i growth hormone secretagogues per la prevenzione e il trattamento di convulsioni ed epilessia [Brevetto]
Biagini, Giuseppe
abstract

L’invenzione riguarda l’uso di agonisti e antagonisti dei recettori per i Growth Hormone Secretagogues utili nella prevenzione e nel trattamento di convulsioni, ad esempio acute, ricorrenti, a grappolo, associate o non associate ad epilessia, e di stati epilettici, come ad esempio le crisi motorie associate ad epilessia del lobo temporale. Tali composti possono essere somministrati da soli o in associazione con uno o più composti anticonvulsivanti o antiepilettici per la prevenzione e il trattamento delle convulsioni e dell’epilessia.


2010 - Voltage-gated sodium channels as therapeutic targets in epilepsy and other neurological disorders [Articolo su rivista]
Mantegazza, M.; Curia, Giulia; Biagini, Giuseppe; Ragsdale, D.; Avoli, M.
abstract

Voltage-gated sodium channels (VGSCs) are key mediators of intrinsic neuronal and muscle excitability. AbnormalVGSC activity is central to the pathophysiology of epileptic seizures, and many of the most widely used antiepilepticdrugs, including phenytoin, carbamazepine, and lamotrigine, are in hibitors of VGSC function. These antiepilepticdrugs might also be effi cacious in the treatment of other nervous system disorders, such as migraine, multiplesclerosis, neurodegenerative diseases, and neuropathic pain. In this Review, we summarise the structure and functionof VGSCs and their involvement in the pathophysiology of several neurological disorders. We also describe thebiophysical and molecular bases for the mechanisms of action of antiepileptic VGSC blockers and discuss the effi cacyof these drugs in the treat ment of epileptic and non-epileptic disorders. Overall, clinical and experimental data indicatethat these drugs are effi cacious for a range of diseases, and that the development of drugs with enhanced selectivity forspecifi c VGSC isoforms might be an eff ective and novel approach for the treatment of several neurological diseases.


2009 - Diminished presynaptic GABA(B) receptor function in the neocortex of a genetic model of absence epilepsy [Articolo su rivista]
Y., Inaba; M., D'Antuono; G., Bertazzoni; Biagini, Giuseppe; M., Avoli
abstract

Changes in GABA(B) receptor subunit expression have been recently reported in the neocortexof epileptic WAG/Rij rats that are genetically prone to experience absence seizures.These alterations may lead to hyperexcitability by downregulating the function of presynapticGABA(B) receptors in neocortical networks as suggested by a reduction in paired-pulsedepression. Here, we tested further this hypothesis by analyzing the effects induced by theGABA(B) receptor agonist baclofen (0.1-10 μM) on the inhibitory events recorded in vitro fromneocortical slices obtained from epileptic (>180 day-old) WAG/Rij and age-matched, nonepilepticcontrol (NEC) rats. We found that higher doses of baclofen were required todepress pharmacologically isolated, stimulus-induced IPSPs generated by WAG/Rij neuronsas compared to NEC. We also obtained similar evidence by comparing the effects ofbaclofen on the rate of occurrence of synchronous GABAergic events recorded by WAG/Rijand NEC neocortical slices treated with 4-aminopyridine+glutamatergic receptor antagonists.In conclusion, these data highlight a decreased function of presynaptic GABA(B) receptorsin the WAG/Rij rat neocortex. We propose that this alteration may contribute toneocortical hyperexcitability and thus to absence seizures.


2009 - Evaluation of reference models for [11C]ABP688 targeting the metabotropic glutamate receptor 5 in rats—application to an epilepsy model [Abstract in Rivista]
Elmenhorst, ; Biagini, Giuseppe; L., Minuzzi; A., Aliaga; G., Massarweh; M., Diksic; M., Avoli; A., Bauer; P., Rosa Neto
abstract

The cerebellum is a suitable referenceregion for the quantification of mGluR5 availabilitywith [11C]ABP688. Blood sample based quantificationof BPnd correlates well with reference regionbased analyses. Scan duration of 50 mins is required.The observed increase in receptor density in humans(hippocampal specimens) suffering from temporallobe epilepsy was not observed in the pilocarpinemodel, but the stastical power has to be increased toconfirm these results. The present results in thepilocarpine model contrast with the immunohistochemicalstudies in humans but are in agreementwith receptor binding studies conducted by ourgroup.


2009 - Farmacoresistenza e sclerosi ippocampale in un modello di epilessia del lobo temporale: evidenze a favore di una relazione con la lesione dell’area CA3Pharmacoresistance and hippocampal sclerosis in a model of temporal lobe epilepsy: evidence for a relationship with lesion of the CA3 region [Relazione in Atti di Convegno]
Longo, Daniela; Baldelli, Enrica; Manca, Lidia; G., Gatti; E., Perucca; M., Avoli; Biagini, Giuseppe
abstract

Temporal lobe epilepsy (TLE) with hippocampal sclerosis is the most common type of pharmacoresistantepilepsy in adults.We investigated whether hippocampal damage could influence the response to antiepilepticdrugs in the pilocarpine model of TLE. Sprague-Dawley rats were injected with intraperitoneal (i.p.) pilocarpine(380 mg/kg), and the provoked status epilepticus (SE) was quelled after 30 or 120 minutes with i.p.diazepam (20 mg/kg). After 3 weeks, when all the animals developed spontaneous recurrent seizures, weimplanted osmotic minipumps to assure a constant release of carbamazepine (CBZ, 4 mg/kg/h) or vehicle(epileptic controls). After one week, during which we monitored seizure frequency, we sacrificed the animalsto assess the hippocampal damage.We found a highly predictable ischemic-hemorrhagic lesion in the CA3stratum lacunosum-molecolare of rats exposed to 120 min SE. Although ablating the perforant path terminalfield, this lesion was significantly (p < 0.05) less pronounced in animals with a SE of 30 minutes.All the rats wereresistant to CBZ.Moreover, rats exposed to 120 minutes of SE showed a 6-fold increase in frequency of spontaneousseizures during CBZ administration. These data suggest that the loss of direct inputs from the entorhinalcortex to CA3 can worsen the response to CBZ treatment in a model of TLE.


2009 - Glia-derived steroids modulate epileptogenesis in a model of temporal lobe epilepsy [Abstract in Rivista]
Longo, Daniela; Baldelli, Enrica; G., Bertazzoni; M., Avoli; Biagini, Giuseppe
abstract

Purpose: The conversion of cholesterol into pregnenolone by cytochromeP450 cholesterol side-chain cleavage enzyme (P450 scc) is therate-limiting step in the steroid synthesis. Glial cells and neurons bothexpress P450 scc and synthesize neurosteroids, which are positive modulatorsof GABAergic transmission. Astrocytes become activated followingstatus epilepticus (SE), but it is presently unclear whether thisactivation leads to enhanced neurosteroidogenesis.Method: We studied the time course of P450 scc immunoreactivitychanges after pilocarpine-induced SE in adult (8 week-old) and young (3weeks-old) rats. To evaluate the role of P450 scc upregulation, we usedthe 5a-reductase inhibitor finasteride (100/kg s.c. for 3 weeks) to suppressthe synthesis of neurosteroids.Results: We demonstrated that P450 scc is upregulated in the CA3 hippocampalregion. Moreover the extent of P450 scc induction was directlyrelated to the onset of spontaneous recurrent seizures in adult (8-weekold)rats. In 3-week-old rats compared with adults, higher P450 scc levelsand a longer latent period were found. Interestingly, adult epileptic rats,treated with finasteride, compared with a group of vehicle-treated rats,presented anticipated generalized seizures (p<0.01). In young rats, finasterideanticipates seizures in approximately 50% of the animals.Conclusion: These findings suggest that neurosteroids can modulateepileptogenesis in the pilocarpine model of temporal lobe epilepsy.


2009 - Lacosamide: a new approach to target voltage-gated sodium currents in epileptic disorders [Articolo su rivista]
Curia, Giulia; Biagini, Giuseppe; E., Perucca; M., Avoli
abstract

The mechanism of action of several antiepileptic drugs (AEDs) rests on their ability tomodulate the activity of voltage-gated sodium currents that are responsible for fast action potentialgeneration. Recent data indicate that lacosamide - a compound with analgesic and anticonvulsanteffects in animal models - shares a similar mechanism. When compared with other AEDs, lacosamidehas the unique ability to interact with sodium channel slow inactivation without affecting fastinactivation. This article reviews these findings and discusses their relevance within the context ofneuronal activity seen during epileptiform discharges generated by limbic neuronal networks in thepresence of chemical convulsants. These seizure-like events are characterized by sustained dischargesof sodium-dependent action potentials supported by robust depolarizations thus providingsynchronization within neuronal networks. Generally, AEDs such as phenytoin, carbamazepine andlamotrigine block sodium channels when activated. By contrasts, lacosamide facilitates slowinactivation of sodium channels both in term of kinetics and voltage-dependency. This effect may berelatively selective for repeatedly depolarized neurons such as those participating in seizure activity inwhich the persistence of sodium currents is more pronounced and promotes neuronal excitation. Theclinical effectiveness of lacosamide has been demonstrated in randomized placebo-controlled doubleblindparallel-group, adjunctive-therapy trials in patients with refractory partial seizures. Furtherstudies should determine whether lacosamide effects in animal models and in clinical settings are fullyexplained by its selective action on sodium current slow inactivation or whether other effects (e.g.,interactions with the collapsin-response mediator protein 2) play a contributory role.


2009 - Network interactions in the limbic system and epileptiform synchronization [Capitolo/Saggio]
M., Avoli; Biagini, Giuseppe; L., Uva; M., de Curtis
abstract

Neuronal networks in the hippocampal and parahippocampal regions are damaged in patients affected by temporallobe epilepsy (TLE). The consequences of the lesions are still poorly understood, but evidence suggests thatinteractions between the entorhinal cortex (EC) and hippocampus are changed from polysynaptic to monosynaptic,bypassing the CA3 region. In addition, the perirhinal/postrhinal cortex (PC), which normally filters the signalsentering the hippocampal formation, may lose its capacity to inhibit the paroxysmal neuronal activity directed to orleaving the EC. Finally, emphasis has recently been placed on the epileptogenic role of the subiculum that isdisinhibited both in epileptic animals and in TLE patients.


2009 - Neuroprotective effects of neurosteroids in pilocarpine-treated rats [Abstract in Rivista]
LONGO, Daniela; SILVESTRI, Chiara; M. A., Rogawski; M., Avoli; BIAGINI, Giuseppe
abstract

The pilocarpine model is based on status epilepticus (SE) induction in rodents to mimictemporal lobe epilepsy as observed in humans. Depending on SE duration, widespreaddamage is observed in hippocampal or extrahippocampal regions leading to gliosis andreorganization of neuronal circuits [1]. Glial cells can play a role in modulating the lesionoutcome through the release of neurotrophic factors or mediators of the inflammatoryresponse. Moreover, glial cells are able to synthesize neurosteroids that can interact withGABAA receptors. In a previous study [2], we have reported an increased expression of therate-limiting enzyme cholesterol side-chain cleavage cytochrome P450 (P450scc), after SEin the hippocampal formation. P450scc converts cholesterol into pregnenolone. In addition,we found that the upregulation of P450scc was more pronounced in rats exposed to longerintervals of SE, and that these animals had a delayed onset of spontaneous recurrentseizures (SRSs). Finally, we have shown that when treated with the 5α-reductase inhibitorfinasteride, a drug that blocks the synthesis of allopregnanolone, the appearance of SRSswas anticipated. We have recently identified a highly predictable lesion in the CA3 stratumlacunosum-moleculare of pilocarpine-treated adult rats [3]. This lesion enlarged withlonger SE intervals and was partially reduced or, in some cases, prevented by treatinganimals with diazepam. In addition, the CA3 lesion was uncommon in young (3-weekold)rats. A characteristic of the CA3 lesion was its association with an increasedpropensity to develop SRSs.Here, we investigated whether the higher expression of P450scc associated with glialreactivity could influence the development of the CA3 lesion as well as the frequency ofoccurrence of SRSs in both young and adult rats. To this aim, we exposed 8 and 3-weekoldrats to 1 h of SE, characterizing both the consequent brain damage and epileptogenesis.Adult animals presented a well defined lesion in CA3, consisting of loss of glial cells (Fig.1), nerve fibers and neurons. Brains in young rats, instead, presented a pronounced, longlastingglial reaction in the same area in which the CA3 lesion was localized in adultanimals. As expected, young rats presented with P450scc induction that was larger than inadults. In addition, spontaneous seizures were significantly delayed (p&lt;0.01) in younganimals exposed to 1 h SE compared with adults. To further evaluate the role ofneurosteroids on the onset of the CA3 lesion we treated the adult group (which presentsthis type of damage) with ganaxolone (60 mg/kg p.o.), a synthetic analogue ofallopregnanolone. Ganaxolone was able to significantly prevent the CA3 lesion in 3/13,whereas all untreated rats (n=10) were damaged. However, no differences were found inthe onset of SRSs in both groups of treatment (Fig. 2). Taken together, these resultssuggest that high levels of neurosteroids protect the CA3 hippocampal region from damageoccurring during SE. However, this effect was particularly evident in young rats. Theallopregnanolone analogue ganaxolone only modestly prevented the lesion in adult rats;such a lesion is known to be prevented by diazepam administration (3). These findingssuggest that endogenous neurosteroids are more effective than those administered exogenously. We propose that the development of more effective neurosteroid mimeticdrugs could provide promising tools for the treatment of temporal lobeepilepsy.


2009 - Neurosteroids and epileptogenesis in the pilocarpine model: evidence for a relationship between P450scc induction and length of the latent period [Articolo su rivista]
Biagini, Giuseppe; Longo, Daniela; Baldelli, Enrica; Zoli, Michele; M. A., Rogawski; G., Bertazzoni; M., Avoli
abstract

Purpose: Cytochrome P450 cholesterol side-chaincleavage enzyme (P450scc) catalyzes the initialstep in the biosynthesis of neurosteroids withinthe brain. We sought to determine which cellsexpress P450cc and whether neurosteroids play arole in the regulation of epileptogenesis followingpilocarpine-induced status epilepticus (SE).Methods: Rats experienced uninterrupted SE orSE terminated with diazepam at 60, 120, and180 min. P450scc induction in CA3 hippocampuswas determined by double immunolabeling withP450scc antiserum and monoclonal antibodiesagainst GFAP (astrocytes), RIP (oligodendrocytes),or heme oxygenase-1 (microglia).Results: SE was associated with P450scc inductionin many astrocytes and a small number of microgliaand oligodendrocytes in the hippocampal CA3strata radiatum and lacunosum-moleculare. Theextent of P450scc induction increased withincreasing SE duration. Paradoxically, increasedP450scc induction in rats experiencing SE for180 min or more was associated with the delayedonset of spontaneous recurrent seizures. Treatmentwith the 5a-reductase inhibitor finasteride(100 mg/kg/day for 25 days), which inhibits thesynthesis of c-aminobutyric acid (GABA)A receptormodulating neurosteroids such as allopregnanolone,was associated with a significantreduction in time to the onset of spontaneous seizuresin rats exposed to 180-min but not 90-minSE.Discussion: P450scc is induced by SE in a diversepopulation of hippocampal glia. Induction ofP450scc is associated with the delayed onsetof spontaneous seizures. Conversely, inhibitionof neurosteroid synthesis accelerated the onset ofspontaneous seizures, but only in animals exhibitingsignificant increases in P450scc. These findingssuggest that induction of neurosteroid synthesis inreactive glial cells is associated with delayed onsetof spontaneously recurrent seizures.


2009 - Thalamocortical synchronization and absence epilepsy [Capitolo/Saggio]
M., Avoli; Biagini, Giuseppe
abstract

Generalized spike-and-wave (SW) discharges are often associated with periods of impaired consciousness (i.e., withabsence seizures) and reflect thalamocortical oscillations similar to those involved in sleep spindles, but at a lowerfrequency. Both cortical and thalamic neuronal networks contribute to SWdischarge. Moreover, according to recentevidence obtained from rodent models, discrete cortical networks initiate these seizures by producing enhancedcorticothalamic output activity that initiates the following sequence: (i) corticothalamic activity makes thalamicreticular γ-aminobutyric acid (GABA)-ergic cells fire intense action potential bursts; (ii) these bursts cause extended inhibitory postsynaptic potentials (IPSPs) in thalamic relaycells, which results in (iii) a slowing of relay cell pacing frequency and (iv) a recruitment of a larger number of corticaland thalamic neurons – which leads to SW activity. The changes that cause the initiating cortical hyperexcitabilityinclude both intrinsic and synaptic mechanisms.


2008 - Characterizationof a focal vascular lesionaffecting entorhinalcortex-CA3connections after statusepilepticus [Abstract in Rivista]
Biagini, Giuseppe; Longo, Daniela; Baldelli, Enrica; Contri, Miranda; Nichelli, Paolo Frigio; U., Guerrini; L., Sironi; P., Gelosa; G., Bertazzoni; M., Avoli
abstract

Pilocarpine mimics temporal lobeepilepsy by inducing SE associatedwith damage in hippocampal andextrahippocampal areas. We havecharacterized a vascular lesion thatdestroys the perforant path (PP) inCA3 after SE. This lesion (white arrows,Figure 1) was evident withmagnetic resonance imaging 1 day aftera SE lasting for 120 min and its appearancewas delayed by limiting SEto 60 or 30 min. Rats exposed to SEfor 30 min developed the lesion unlesssubsequently treated with diazepam(20 mg/kg s.c. for 3 days), whichprotected 50% of the animals. Antibodiesfor astrocytes (GFAP), dendrites(MAP2) and PPnerve terminals(mGluR2/3), showed areas of immunoreactivityloss in which we localizedincreased staining for laminin, inthe basement membrane of vessels.This lesion was uncommon in young(3-week-old) rats. The latent periodfor seizure appearance was similar inadult rats exposed to SE only or to SEfollowed by neuroprotection with diazepam;however, the frequency ofspontaneous seizures was significantlylower (p < 0.01) in the neuroprotectedgroup. Moreover, spontaneousseizures were delayed in youngrats (p < 0.01) exposed to 60 min SEcompared to adults experiencing asimilar SE. To investigate the role ofdamaged CA3 in seizure activity, were-induced SE in adult and young epilepticrats. Using FosB/FosB markers,we found induction of FosB/FosB immunopositivity in CA3neurons of young but not in adult rats.These experiments reveal that SE canproduce a focal lesion in the PP, whichaffects the role of the hippocampus inepileptic rats.


2008 - Glia-derived neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy [Abstract in Rivista]
Longo, Daniela; Baldelli, Enrica; G., Bertazzoni; M., Avoli M; Biagini, Giuseppe
abstract

The conversion of cholesterol intopregnenolone by the enzyme cholesterolside-chain cleavage cytochromeP450 (P450scc) is the rate-limitingstep in the steroid synthesis. Glialcells and neurons both expressP450scc and synthesize neurosteroidsthat act as positive modulators ofGABAergic transmission. Astrocytesbecome activated following statusepilepticus (SE), but it is presently unclearwhether this activation leads toenhanced neurosteroid synthesis. Westudied the time course of P450sccimmunoreactivity changes afterpilocarpine-induced SE and its relationshipwith epileptogenesis. Wefound that P450scc is upregulated inthe CA3 hippocampal region, afterSE. Induction of P450scc was mainlyobserved in astrocytes identified by aGFAP antibody, although hemeoxygenase-1-positive microglial cells,RIP-positive oligodendrocytes andNeuN-neurons were also stained forP450scc (Fig. 1). The extent ofP450scc induction was directly relatedwith the onset of spontaneouslyrecurrent seizures in adult (8-weekold)rats. In addition, in young (3-week-old) rats the induction ofP450scc and the latent period durationwere much larger than in adultrats. To further evaluate the role ofP450scc upregulation, we used the5-reductase inhibitor finasteride(100 mg/kg/day for approximately 3weeks) to suppress the synthesis ofGABA-modulating neurosteroidssuch as allopregnanolone. Interestingly,adult epileptic rats, comparedwith a group of vehicle-treated ratsthat experienced a similar SE, presentedwith significantly (p<0.01) anticipatedgeneralized seizures whentreated with finasteride. In young rats,we observed early generalized seizuresin approximately 50% of the animals.These findings suggest thatneurosteroids can modulate epileptogenesisin the pilocarpine modelof temporal lobe epilepsy.


2008 - Network hyperexcitability within the deep layers of pilocarpine-treated entorhinal cortex. [Articolo su rivista]
P., De Guzman; Y., Inaba; Baldelli, Enrica; M., De Curtis; Biagini, Giuseppe; M., Avoli
abstract

In this study we report that in the presence of normal buffer, epileptiform dischargesoccur spontaneously (duration=2.60±0.49 s) or can be induced by electrical stimuli(duration=2.50±0.62 s) in the entorhinal cortex (EC) of brain slices obtained frompilocarpine-treated rats but not in those from age-matched, nonepileptic control (NEC)animals. These network-driven epileptiform events consist of field oscillatory sequences atfrequencies greater than 200 Hz that most often initiate in the lateral EC and propagateto the medial EC with 4–63 ms delays. The NMDA receptor antagonist CPP depressesthe rate of occurrence (P <0.01) of these spontaneous epileptiform discharges but failsin blocking them. Paradoxically, stimulus-induced epileptiform responses are enhanced induration during CPP application.However, concomitant application ofNMDAand non-NMDAglutamatergic antagonists abolishes spontaneous and stimulus-induced epileptiform events.Intracellular recordings from lateral EC layer V cells indicate a lower frequency of spontaneoushyperpolarizing postsynaptic potentials in pilocarpine-treated tissue than in NEC (P <0.002)both under control conditions and with glutamatergic receptor blockade; the reversal potentialof pharmacologically isolated GABAA receptor-mediated inhibitory postsynaptic potentialshas similar values in the two types of tissue. Finally, immunohistochemical analysis showsthat parvalbumin-positive interneurons are selectively reduced in number in EC deep layers.Collectively, these results indicate that reduced inhibition within the pilocarpine-treated EClayer V may promote network epileptic hyperexcitability.


2008 - Proepileptic influence of a focal vascular lesion affecting entorhinal cortex-CA3 connections after status epilepticus. [Articolo su rivista]
Biagini, Giuseppe; Baldelli, Enrica; Longo, Daniela; Contri, Miranda; U., Guerrini; L., Sironi; P., Gelosa; Zini, Isabella; M., Avoli
abstract

In limbic seizures, neuronal excitation is conveyed from the entorhinalcortex directly to CA1 and subicular regions. This phenomenonis associated with a reduced ability of CA3 to respond toentorhinal cortex inputs. Here, we describe a lesion that destroys theperforant path in CA3 after status epilepticus (SE) induced bypilocarpine injection in 8-week-old rats. Using magnetic resonanceimaging, immunohistochemical, and ultrastructural analyses, wedetermined that this lesion develops after 30 minutes of SE and ischaracterized by microhemorrhages and ischemia. After a longerperiod of SE, the lesion invariably involves the upper blade ofthe dentate gyrus. Adult rats treated with subcutaneous diazepam(20 mg kgj1 for 3 days) did not develop the dentate gyrus lesionand had less frequent spontaneous recurrent seizures (p G 0.01).Young (3-week-old) rats rarely (20%) developed the CA3 lesion,and their spontaneous seizures were delayed (p G 0.01). To investigatethe role of the damaged CA3 in seizure activity, wereinduced SE in adult and young epileptic rats. Using FosB/$FosBmarkers, we found induction of FosB/$FosB immunopositivity inCA3 neurons of young but not in adult rats. These experimentsindicate that SE can produce a focal lesion in the perforant path thatmay affect the roles of the hippocampus in epileptic rats.


2008 - The pilocarpine model of temporal lobe epilepsy [Articolo su rivista]
Curia, Giulia; Longo, Daniela; Biagini, Giuseppe; R., Jones; M., Avoli
abstract

Understanding the pathophysiogenesis of temporal lobe epilepsy (TLE) largely rests on the use of modelsof status epilepticus (SE), as in the case of the pilocarpine model. The main features of TLE are: (i) epilepticfoci in the limbic system; (ii) an “initial precipitating injury”; (iii) the so-called “latent period”; and (iv)the presence of hippocampal sclerosis leading to reorganization of neuronal networks. Many of thesecharacteristics can be reproduced in rodents by systemic injection of pilocarpine; in this animal model, SEis followed by a latent period and later by the appearance of spontaneous recurrent seizures (SRSs). Theseprocesses are, however, influenced by experimental conditions such as rodent species, strain, gender, age,doses and routes of pilocarpine administration, as well as combinations with other drugs administeredbefore and/or after SE. In the attempt to limit these sources of variability,we evaluated themethodologicalprocedures used by several investigators in the pilocarpine model; in particular, we have focused on thebehavioural, electrophysiological and histopathological findings obtained with different protocols. Weaddressed the various experimental approaches published to date, by comparing mortality rates, onset ofSRSs, neuronal damage, and network reorganization. Based on the evidence reviewed here, we proposethat the pilocarpine model can be a valuable tool to investigate the mechanisms involved in TLE, and evenmore so when standardized to reduce mortality at the time of pilocarpine injection, differences in latentperiod duration, variability in the lesion extent, and SRS frequency.


2007 - Antiepileptic drugs and muscarinic receptor-dependent excitation in the rat subiculum. [Articolo su rivista]
M., D'Antuono; H., Kawasaki; C., Palmieri; Curia, Giulia; Biagini, Giuseppe; M., Avoli
abstract

Field and intracellular recordings were made in an in vitro slice preparation to establish whether the antiepileptic drugs topiramate andlamotrigine modulate cholinergic excitation in the rat subiculum. Bath application of carbachol (CCh, 70e100 mM) induced: (i) spontaneousand synchronous field oscillations (duration ¼ up to 7 s) that were mirrored by intracellular depolarizations with rhythmic action potentialbursts; and (ii) depolarizing plateau potentials (DPPs, duration ¼ up to 2.5 s) associated with action potential discharge in response to brief(50e100 ms) intracellular depolarizing current pulses. Ionotropic glutamatergic receptor antagonists abolished the field oscillations withoutinfluencing DPPs, while atropine (1 mM) markedly reduced both types of activity. Topiramate (10e100 mM, n ¼ 8e13 slices) or lamotrigine(50e400 mM, n ¼ 3e12) decreased in a dose-dependent manner, and eventually abolished, CCh-induced field oscillations. During topiramateapplication, these effects were accompanied by marked DPP reduction. When these antiepileptic drugs were tested on DPPs recorded in thepresence of CCh þ ionotropic glutamatergic and GABA receptor antagonists, only topiramate reduced DPPs (n ¼ 5e19/dose; IC50 ¼ 18 mM,n ¼ 48). Similar effects were induced by topiramate during metabotropic glutamate receptor antagonism (n ¼ 5), which did not influenceDPPs. Thus, topiramate and lamotrigine reduce CCh-induced epileptiform synchronization in the rat subiculum but only topiramate is effectivein controlling DPPs. We propose that muscarinic receptor-mediated excitation represents a target for the action of some antiepileptic drugs suchas topiramate.


2006 - Does interictal activity sustain seizures and epileptogenesis? [Articolo su rivista]
M., Avoli; Biagini, Giuseppe; M., De Curtis
abstract

Interictal spiking is seen in the EEG of epileptic patients betweenseizures. To date, the roles played by interictal events in seizureoccurrence and in epileptogenesis remain elusive. While interictalspikes may herald the onset of electrographic seizures, experimentaldata indicate that hippocampus-driven interictal events preventseizure precipitation. Even less clear than the role of interictalevents in seizure occurrence is whether and how interictal spikescontribute to epileptogenesis. Thus, while plastic changes withinlimbic neuronal networks may result from ongoing interictal activity,experimental evidence supports the view that epileptogenesisis accompanied by a decrease in hippocampus-driven interictalactivity.


2006 - Endogenous neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy [Articolo su rivista]
Biagini, Giuseppe; Baldelli, Enrica; Longo, Daniela; L., Pradelli; Zini, Isabella; M. A., Rogawski; M., Avoli
abstract

Neurosteroids modulate seizure susceptibility, but their role in the regulation of epileptogenesis is unknown. Status epilepticus (SE) induces temporal lobe epileptogenesis following a latent period in which glial cells are activated. Here, we found that P450scc, the rate-limiting enzyme in steroid synthesis.. is upregulated in hippocampal glia during the latent period after pilocarpine-induced SE in rats. More prolonged SE was associated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retards epileptogenesis. The 5 alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that glia-derived neurosteroids may be antiepileptogenic. (c) 2006 Elsevier Inc. All rights reserved.


2006 - Epileptiform hyperexcitability in the rat neocortex: modulation by the H current blocker ZD7288 [Abstract in Rivista]
Y., Inaba; Biagini, Giuseppe; M., Avoli
abstract

Objectives: Neurons in the CNS respond to intracellular injection of hyperpolarizing current pulses by generating depolarizing sags contributed by a cation current termed Ih. Ih modulates neuron excitability and rhythmicity. However, whether the net effect of Ih on cortical networks results in facilitation or depression of epileptiform activity remains debatable. Here, we addressed this issue by studying the effects of the Ih blocker ZD7288 on the epileptiform discharges.Methods: We studied with field and intracellular recordings the effects of the Ih blocker ZD7288 on the epileptiform discharges (duration=2.54±0.33s, mean±SEM; interval of occurrence=34.2±3.3s, n=30) induced in rat neocortical slices by bath applying 4- aminopyridine+picrotoxin+CGP55845.Results: ZD7288 (10–100µM; n=18) abolished the depolarizing sags seen during injection of intracellular hyperpolarizing current pulses while increasing both resting membrane potential and apparent input resistance. These effects were fully established with 10µM ZD7288 and were accompanied by a dosedependent decrease in the occurrence of spontaneous epileptiform events and a reduction in their duration (this last effect becoming apparent with concentrations >20µM). ZD7288 (10–100µM; n=17) also caused a dose-dependent decrease of background postsynaptic potentials. Finally, 10, 50 or 100µM ZD7288 (n=6, 5 and 8, respectively) depressed the epileptiform activity during application of Cs+, which is known to reduce Ih.Conclusion: This evidence indicates that ZD7288 depresses neocortical epileptiform synchronization. However, most of this action may reflect the ability of ZD7288 to interfer with synaptic transmission.


2006 - Neurosteroidi ed epilettogenesi: evidenze a favore di una relazione tra induzione dell’enzima P450scc e durata della fase di latenza nel modello della pilocarpina. [Relazione in Atti di Convegno]
Baldelli, Enrica; Longo, Daniela; Zini, Isabella; Zoli, Michele; M., Avoli; Biagini, Giuseppe
abstract

Neurosteroids are GABAA receptor agonists able to modulate seizure susceptibility. They are mainly synthesizedin astrocytes, a type of glial cells that are activated by neuronal damage. Interestingly, status epilepticus (SE)induces neuronal damage and a chronic epileptic condition that becomes evident following a “latent” period,in which glial cells are highly activated. Investigating glial cell activation in the pilocarpine model, wefound that P450scc, the rate-limiting enzyme in neurosteroid synthesis, is upregulated in hippocampal astrocytesduring the latent period. The induction of P450scc immunoreactivity was stronger with prolonged statusepilepticus and was associated with longer latencies to the development of seizures, suggesting thatenhanced neurosteroid synthesis retards epileptogenesis. Accordingly, the 5α-reductase inhibitor finasteride,which blocks neurosteroid synthesis, reduced the latent period, thus indicating that glia-derived neurosteroidsmay be antiepileptogenic.


2006 - Neurosteroids modulate epileptogenesis in a model of temporal lobe epilepsy [Abstract in Rivista]
Longo, Daniela; Baldelli, Enrica; L., Pradelli; Zini, Isabella; M. A., Rogawski; M., Avoli; Biagini, Giuseppe
abstract

Neurosteroids modulate seizure susceptibility, but their role in the regulation of epileptogenesis is unknown. Status epilepticus inducestemporal lobe epileptogenesis following a latent period in which glial cells are activated. Here, we found that P450scc, the rate-limiting enzyme insteroid synthesis, is upregulated in hippocampal glia during the latent period after pilocarpine-induced SE in rats. More prolonged SE wasassociated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retardsepileptogenesis. The 5alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that glia-derivedneurosteroids may be antiepileptogenic.


2006 - Subiculum network excitability is increased in a rodent model of temporal lobe epilepsy [Articolo su rivista]
P., de Guzman; Y., Inaba; Biagini, Giuseppe; Baldelli, Enrica; C., Mollinari; D., Merlo; M., Avoli
abstract

In this study, we used in vitro electrophysiology along with immunohistochemistry and molecular techniques to study the subiculum-a limbic structure that gates the information flow from and to the hippocampus-in pilocarpine-treated epileptic rats. Comparative data were obtained from age-matched nonepileptic controls (NEC). Subicular neurons in hippocampal-entorhinal cortex (EC) slices of epileptic rats were: (i) hyperexcitable when activated by CA1 or EC inputs; and (ii) generated spontaneous postsynaptic potentials at higher frequencies than NEC cells. Analysis of pharmacologically isolated, GABA(A) receptor-mediated inhibitory postsynaptic potentials revealed more positive reversal potentials in epileptic tissue (-67.8 +/- 6.3 mV, n = 16 vs. -74.8 +/- 3.6 mV in NEC, n = 13; P < 0.001) combined with a reduction in peak conductance (17.6 +/- 11.3 nS vs. 41.1 +/- 26.7 nS in NEC; P < 0.003). These electrophysiological data correlated in the epileptic subiculum with (i) reduced levels of mRNA expression and immunoreactivity of the neuron-specific potassium-chloride cotransporter 2; (ii) decreased number of parvalbumin-positive cells; and (iii) increased synaptophysin (a putative marker of sprouting) immunoreactivity. These findings identify an increase in network excitability within the subiculum of pilocarpine-treated, epileptic rats and point at a reduction in inhibition as an underlying mechanism. (c) 2006 Wiley-Liss, Inc.


2006 - Synaptic hyperexcitability of deep layer neocortical cells in a genetic model of absence seizures [Articolo su rivista]
M., D'Antuono; Y., Inaba; Biagini, Giuseppe; G., D'Arcangelo; V., Tancredi; M., Avoli
abstract

We used sharp-electrode, intracellular recordings in an in vitro brain slice preparation to study the excitability of neocortical neurons located in the deep layers (> 900 mu m from the pia) of epileptic (180-210-days old) Wistar Albino Glaxo/Rijswijk (WAG/Rij) and age-matched, non-epileptic control (NEC) rats. Wistar Albino Glaxo/Rijswijk rats represent a genetic model of absence seizures associated with generalized spike and wave (SW) discharges in vivo. When filled with neurobiotin, these neurons had a typical pyramidal shape with extensive apical and basal dendritic trees; moreover, WAG/Rij and NEC cells had similar fundamental electrophysiological and repetitive firing properties. Sequences of excitatory postsynaptic potentials (EPSPs) and hyperpolarizing inhibitory postsynaptic potentials (IPSPs) were induced in both the strains by electrical stimuli delivered to the underlying white matter or within the neocortex; however, in 24 of 55 regularly firing WAG/Rij cells but only in 2 of 25 NEC neurons, we identified a late EPSP that (1) led to action potential discharge and (2) was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (20 mu M; n = 8/8 WAG/Rij cells). Finally, we found that the fast and slow components of the stimulus-induced IPSPs recorded during the application of glutamatergic receptor antagonists had similar reversal potentials in the two strains, while the peak conductance of the fast IPSP was significantly reduced in WAG/Rij cells. These findings document an increase in synaptic excitability that is mediated by NMDA receptors, in epileptic WAG/Rij rat neurons located in neocortical deep layers. We propose that this mechanism may be instrumental for initiating and maintaining generalized SW discharges in vivo.


2006 - The H current blocker ZD7288 decreases epileptiform hyperexcitability in the rat neocortex by depressing synaptic transmission [Articolo su rivista]
Y., Inaba; Biagini, Giuseppe; M., Avoli
abstract

Neurons respond to intracellular injection of hyperpolarizing current pulses by generating depolarizing sags contributed by a cation current termed I-h. I-h modulates neuron excitability and rhythmicity. It is, however, unclear whether the net effect of changing Ih leads to facilitation or depression of cortical epileptiform activity. Here, we addressed this issue by using field and intracellular recordings to study the effects of ZD7288 (10-100 mu M), a bradycardic agent known to abolish I-h, on the epileptiform discharges (duration = 2.5 +/- 0.3 s, mean +/- SEM; interval of occurrence = 34.2 +/- 3.3 s, n = 30 slices) induced in rat neocortical slices by 4-aminopyridine and GABA receptor antagonists. ZD7288 abolished the depolarizing sags seen during injection of intracellular hyperpolarizing current pulses while increasing resting membrane potential and apparent input resistance. These effects, which were fully established with 10 mu M ZD7288, were associated with a dose-dependent decrease in the occurrence of spontaneous epileptiform events and a reduction in their duration (the latter change occurring at doses > 20 mu M). ZD7288 also caused a dose-dependent decrease of background postsynaptic potentials. Finally, ZD7288 could depress epileptiform activity during Cs+ pretreatment, a procedure known to block Ih. These data indicate that ZD7288 hampers neocortical epileptiform synchronization, but also suggest that most of this action reflects the ability of ZD7288 to decrease synaptic transmission. (c) 2006 Elsevier Ltd. All rights reserved.


2005 - Entorhinal cortex-subiculum interactions in an experimental model of mesial temporal lobe epilepsy [Abstract in Rivista]
M., Avoli; Biagini, Giuseppe; G., D'Arcangelo; M., D'Antuono; V., Tancredi
abstract

Mesial temporal lobe epilepsy (MTLE) patients present with seizuresinvolving the limbic system and with a pattern of brain damage characterizedby neuronal loss in CA1/CA3 areas, dentate hilus, and entorhinalcortex (EC), layer III (Houser CR. Adv Neurol 1999;79:743–61). Similarfindings are seen in laboratory animals following pilocarpine injection(Turski WA, et al. Behav Brain Res 1983;9:315–35). This procedure inducesan initial convulsive response, which is followed within 2–3 weeksby recurrent seizures. Limbic network hyperexcitability in MTLE andin animal models results from seizure-induced brain damage leading to(a) synaptic reorganization (Cavazos JE, et al. J Neurosci 1991;11:2795–803; Houser CR. Adv Neurol 1999;79:743–61) and (b) changes inGABAreceptor–mediated inhibition (Buhl EH, et al. Science 1996;271:369–7;Doherty J, Dingledine R. J Neurosci 2001;21:2048–57. However, it isunclear how these changes lead to a chronic epileptic condition.CA3-driven interictal activity induced in normal brain tissue by epileptogenicstimuli inhibits the EC from generating ictal discharges (BarbarosieM, Avoli M. J Neurosci 1997;17:9308–14), suggesting that CA3damage causes a decrease of hippocampal output activity that wouldrelease EC ictogenesis and establish a chronic epileptic condition. Accordingly,slices obtained from pilocarpine-treated epileptic mice respondto 4-aminopyridine (4AP) application by generating (a) CA3-driven interictal activity that is less frequent than in nonepileptic control(NEC) tissue, and (b) ictal discharges that do not disappear overtime and propagate to the CA1-subiculum via the temporoammonic path(D’Antuono M, et al. J Neurophysiol 2002;87:634–9). From these findings,we predicted that limbic seizures result from EC–subiculum interactions.Using brain slices obtained from pilocarpine-treated, epilepticrats, we found that decreased CA3 output function, along with reverberationbetween EC and subiculum networks, lead to in vitro epileptogenesis.First, intense activation of EC and subiculum was identifiedwith intrinsic optical signal (IOS) recordings in pilocarpine-treated, butnot in NEC slices. Second, using field potential recordings during 4APapplication, we established that CA3-driven interictal activity occursat lower frequency in pilocarpine-treated slices and that disconnectionof the EC from the subiculum attenuates 4AP-induced ictal dischargesin pilocarpine-treated, but not in NEC slices. Third, the distributionof FosB/FosB-related proteins in epileptic tissue demonstrated distinctpatterns overlapping those seen with IOS recordings, with the highestintensity in layer III of the lateral EC.In conclusion, our data show that hippocampal damage in epilepticrats, and perhaps in MTLE patients, hampers the ability of CA3 outputactivity to control ictogenesis in the EC. Such a process is reinforced byinteractions between subiculum and EC networks.


2005 - Impaired activation of CA3 pyramidal neurons in the epileptic hippocampus [Articolo su rivista]
Biagini, Giuseppe; G., D'Arcangelo; Baldelli, Enrica; M., D'Antuono; V., Tancredi; M., Avoli
abstract

We employed in vitro and ex vivo imaging tools to characterize the function of limbic neuron networks in pilocarpine-treated and age-matched, nonepileptic control (NEC) rats. Pilocarpine-treated animals represent an established model of mesial temporal lobe epilepsy. Intrinsic optical signal (IOS) analysis of hippocampal-entorhinal cortex (EC) slices obtained from epileptic rats 3 wk after pilocarpine-induced status epilepticus (SE) revealed hyperexcitability in many limbic areas, but not in CA3 and medial EC layer III. By visualizing immunopositivity for FosB/Delta FosB-related proteins-which accumulate in the nuclei of neurons activated by seizures-we found that: (1) 24 h after SE, FosB/Delta FosB immunoreactivity was absent in medial EC layer III, but abundant in dentate gyrus, hippocampus proper (including CA3) and subiculum; (2) FosB/Delta FosB levels progressively diminished 3 and 7 d after SE, whereas remaining elevated (p < 0.01) in subiculum; (3) FosB/Delta FosB levels sharply increased 2 wk after SE (and remained elevated up to 3 wk) in dentate gyrus and in most of the other areas but not in CA3. A conspicuous neuronal damage was noticed in medial EC layer III, whereas hippocampus was more preserved. IOS analysis of the stimulus-induced responses in slices 3 wk after SE demonstrated that IOSs in CA3 were lower (p < 0.05) than in NEC slices following dentate gyrus stimulation, but not when stimuli were delivered in CA3. These findings indicate that CA3 networks are hypoactive in comparison with other epileptic limbic areas. We propose that this feature may affect the ability of hippocampal outputs to control epileptiform synchronization in EC.


2005 - Protein kinase C epsilon mediates angiotensin II-induced activation of beta(1)-integrins in cardiac fibroblasts [Articolo su rivista]
P., Stawowy; C., Margeta; F., Blaschke; C., Lindschau; C., Spencer Hansch; M., Leitges; Biagini, Giuseppe; E., Fleck; K., Graf
abstract

Objective: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. All and other growth factors augment beta(1)-integrin-dependent adhesion and spreading by inside-out signaling without affecting the total number of integrin receptors. Inside-out signaling involves specific signaling pathways, including protein kinase C (PKC), leading to activation of beta(1)-integrins. In the present study we investigated the mechanisms involved in All-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague-Dawley rats, to collagen I mediated by beta(1)-integrin. Methods and results: Treatment of CFBs with All induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, p < 0.01) within 3-6 h of treatment. This effect was mediated by beta 1-integrin via the angiotensin AT(1) receptor and was significantly reduced by protein kinase C inhibition. All significantly induced phosphorylation of PKC epsilon (PKC epsilon), which is involved in beta(1)-integrin-dependent adhesion and motility and phosphorylation of the cytoplasmatic tail of beta(1)-integrin at threonine 788/789, required for adhesion. Phosphorylation of beta(1)-integrin and an increase in adhesion was also induced by L-alpha-phosphatidylinositol-3,4,5-triphosphate (L-alpha-PIP3) an activator of endogeneous PKCe. All failed to increase adhesion in myofibroblasts obtained from PKCe (-/-) mice, but not in cells obtained from control mice. Co-immunoprecipitation and double immunofluorescence demonstrated that All induced a close association of PKC epsilon with beta(1)-integrin in CFBs. Conclusion: The present study demonstrates that All increased beta(1)-integfin-dependent adhesion to collagen I in cardiac fibroblasts by inside-out signaling via PKC epsilon and phosphorylation of the cytoplasmatic tail of the beta(1)-integrin.


2004 - Hypoactivity of CA3 pyramidal neurons in the pilocarpine model of mesial temporal lobe epilepsy [Articolo su rivista]
Baldelli, E.; Zini, I.; Biagini, G.; D'Arcangelo, G.; Tancredi, V.; D'Antuono, M.; Avoli, M.
abstract

Reduced CA3-driven interictal activity in hippocampal-entorhinal cortex (EC) slices obtained from pilocarpine-treated rodents is accompanied by the persistence of ictal discharges originating from EC. Therefore, loss of CA3 pyramidal cells could be critical for unveiling limbic seizures in mesial temporal lobe epilepsy (MTLE). We analyzed here this issue by means of in vitro and ex vivo imaging along with histopathology in pilocarpine-treated and age-matched, non-epileptic control (NEC) rats. By visualizing immunopositivity for FosB/ΔFosB-related proteins - which accumulate in the nuclei of seizing neurons - we found that activated neurons can be found in many hippocampal and parahippocampal regions but not in CA3. Intrinsic optical signal (IOS) analysis of the stimulus-induced responses in slices obtained 3 weeks after SE revealed that IOSs in CA3 were lower (p &lt; 0.05) than in NEC slices following dentate gyrus stimulation, while comparable responses were elicited by stimuli delivered in the CA3 pyramidal layer. We conclude that in epileptic rats, and perhaps in MTLE patients, mechanisms other than neuronal loss hamper CA3 network synchronization and thus the ability of hippocampal outputs to control EC ictogenesis.


2004 - Ipoattività dei neuroni piramidali dell’area CA3 in un modello animale di epilessia del lobo temporale. [Articolo su rivista]
Baldelli, Enrica; Zini, Isabella; Biagini, Giuseppe; G., D'Arcangelo; V., Tancredi; M., D'Antuono; M., Avoli
abstract

Rational and Aims - Reduced CA3-driven interictal activity has been reported in hippocampal-entorhinal cortex (EC)-slices obtained from pilocarpine-treated rodents during 4-aminopyridine (4AP) treatment. In addition, ictal discharges initiating in EC persist throughout the experiment in these slices, while they disappear within the first 2 hours of 4AP superfusion in non-epileptic control (NEC) tissue. This evidence has led us to hypothesize that CA3 cell damage, which is known to occur in several models of mesial temporal lobe epilepsy (MTLE), may release EC ictogenesis. However, this in vitro data could also be related to the artificial condition set to record seizure activity in the slice. We addressed this issue by determining in vivo the extent of hippocampal network activation by using a marker sensitive to repetitive seizure activity: ΔFosB. Methods – We induced status epilepticus (SE) in Sprague-Dawley male rat by injecting pilocarpine i.p. (380 mg/kg). The animals where then sacrificed at 1, 3, 7, 14, 21 days and processed for ΔFosB immunodetection and other histopathological studies; alternatively, at 21 days they were used for in vitro analysis of the hippocampal-entorhinal cortex slice by intrinsic optical signal detection after electrical stimulation.Results – Immediately after SE, ΔFosB immunoreactivity was widespread in all the hippocampal regions, while no positive cell nuclei were found in NEC. In the following days, a general decrease in ΔFosB levels was observed in all the positive areas, reaching the lowest levels 7 days after. However, the subiculum presented a smoother decrease and ΔFosB levels were significantly higher than in the other areas. A sharp increase in ΔFosB immunoreactivity occurred in the dentate gyrus 14 days after SE, when positive cell nuclei reappeared also in other regions. Twenty-one days after SE, ΔFosB levels were increased in all the hippocampal regions apart CA3 and the medial entorhinal cortex. The histopathological analysis demonstrated a large damage in layer III of the medial entorhinal cortex, while CA was generally preserved. By counting pyramidal neurons in CA3, only a 10% decrease in cell number was found (p<0.05 vs NEC). Hence, we postulated that CA3 could be hypoactive in pilocarpine-treated spontaneously epileptic animals. This hypothesis was confirmed by analyzing the intensity of intrinsic optical signals detected in CA3 after maximal stimulation of the dentate gyrus. Again, by directly stimulating the CA3 pyramidal layer we were able to record IOSs as intense as in NEC, proving that this region is spared from damage.Conclusions - These findings indicate that in epileptic rats, and perhaps in MTLE patients, mechanisms other than neuronal loss hamper CA3 network synchronization and thus the ability of hippocampal output ability to control ictogenesis in the EC.


2003 - Histomorphometric, biochemical and ultrastructural changes in the aorta of salt-loaded stroke-prone spontaneously hypertensive rats fed a Japanese-style diet [Articolo su rivista]
Contri, Miranda; Taparelli, Francesca; M., Miselli; B., Bacchelli; Biagini, Giuseppe
abstract

Background and Aim: It is demonstrated that dietary habits play a role in cardiovascular diseases. In stroke-prone spontaneously hypertensive rats ( SHRsp), concomitant salt loading and a Japanese-style diet greatly accelerate hypertension and the appearance of cerebrovascular lesions by directly damaging arterial vessels. A number of studies have characterised medium and small vessel lesions in SHRsp, but little attention has been paid to the changes in the wall structure of large arteries induced by exposure to a salt-enriched diet. The aim of this study was to investigate the effects of a Japanese-style diet and salt loading on the thoracic aorta. Methods and Results: Two-month-old SHRsp were kept on a Japanese-style diet with 1% sodium chloride solution replacing tap water. Two months later, they were sacrificed and compared with age-matched or two-month-old control SHRsp kept on a standard diet and tap water in terms of the histomorphometry; ultrastructure and biochemical composition of the thoracic aorta. The vessel was consistently thicker in the four-month-old SHRsp (+20%, p<0.05 vs two-month-old rats) regardless of diet The salt-loaded SHRsp showed a significant reduction in elastic fibre density (-20%, p<0.05 vs two-month-old rats) and art increase it? the other matrix components (+50%), whereas the four-month-old controls showed preserved elastic fibres and a significant increase in the other matrix components (+65%, p<0.05 vs two-month-old rats). There was a considerable increase in the amounts of 4-OH-proline (+147%), 5-OH-lysine (+174%) and desmosines (+360%) in the four-month-old controls vs their two-month-old counterparts (p<0.01), but not in the salt-loaded animals. Ultrastructural analysis revealed clear damage and accelerated aging in the thoracic aorta of the salt-loaded SHRsp. Conclusions: Salt loading and a Japanese-style diet destabilise thoracic aorta architecture in SHRsp after two months of treatment.


2002 - Corticosterone administration to rat pups, but not maternal separation, affects sexual maturation and glucocorticoid receptor immunoreactivity in the testis [Articolo su rivista]
Biagini, Giuseppe; E., Merlo Pich
abstract

Prenatal stress strongly affects sexual dimorphism of male rats. Much less information is instead available on the effects of postnatal stress on sexual maturation during the so-called stress hyporesponsive period (SHRP). For this reason, we compared corticosterone-treated (CS; 10 mg/kg sc, suspended in sesame oil) or maternally separated pups (MS; 5 h/day in the first week of life) with control rats. Control and MS PUPS also received sesame oil injections. The effects of these procedures on physical development (body weight and eye opening), sexual maturation [anogenital distance, testis weight, 3beta-hydroxysteroid dehydrogenase/(Delta5-4) (3betaHSD) isomerase activity and time to testis descent] and glucocorticoid receptor (GR) immunoreactivity in the testis were examined. Corticosterone treatment significantly (P<.05) advanced testis descent and increased testis weight and 3betaHSD activity at puberty. In addition, adult CS rats presented higher levels of GR inummoreactivity in testicular tubules when compared to control and MS rats. No differences were found between control and MS rats. On this basis, we propose that the silencing of adrenocortical function during the SHRP could be finalized to preserve sexual maturation from the influence of glucocorticoid effects. As SHRP is unique to rodents, this phenomenon could be related to their successful reproductive strategy. (C) 2002 Elsevier Science Inc. All rights reserved.


2002 - Dose-Dependent Prevention of Fibrosis in Aorta of Salt-Loaded Stroke-Prone Spontaneously Hypertensive Rats by Combined Delapril and Indapamide treatment. [Articolo su rivista]
Contri, Miranda; Taparelli, Francesca; M., Miselli; G., Pedrazzi; B., Bacchelli; Biagini, Giuseppe
abstract

Combined treatment with the angiotensin-converting enzyme (ACE) inhibitor delapril and the diuretic indapamide prevented vascular damage in vital organs of salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp). Whether the changes occurring after long-term hypertension could also be modulated in large arteries was investigated. Two-month-old SHRsp were salt loaded and treated with the drug regimen until they reached 50 +/- 10 mortality or around midlife. In a first experiment, delapril (12 mg/kg) and indapamide (1 mg/kg) were administered daily separately or in combination. In the second dose-finding experiment, delapril (6, 3, 1.5 mg/kg) and indapamide (0.5, 0.25, 0.125 mg/kg) in decreasing dose combinations were analyzed. Ultrastructural, histomorphometric, and biochemical studies were performed on the thoracic aorta. When compared with delapril (12 mg/kg) or indapamide (1 mg/kg) administered individually for 5 months, the combination 12 + 1 mg/kg was able to prevent the increase in extracellular matrix deposition observed in other treatment groups, as assessed by histomorphometry or 4-OH-proline biochemical determination. In the second experiment, a half-dose (delapril 6 mg/kg + indapamide 0.5 mg/kg) combination was similarly effective in counteracting fibrosis, but the other doses progressively failed. In the first experiment, the combination had a stabilizing effect on hypertension and stimulated diuresis. In the second experiment, arterial blood pressure values and sodium balance were not consistently affected by the treatments that antagonized fibrosis (i.e., delapril 6 mg/kg + indapamide 0.5 mg/kg and, less efficiently, delapril 3 mg/kg + indapamide 0.25 mg/kg). These results suggest that indapamide interacts with ACE inhibitors to limit aortic fibrosis independent of any well-established mechanism.


2002 - Limbic network interactions leading to hyperexcitability in a model of temporal lobe epilepsy [Articolo su rivista]
M., D'Antuono; R., Benini; BIAGINI, Giuseppe; G., D'Arcangelo; M., Barbarosie; V., Tancredi; M., Avoli
abstract

In mouse brain slices that contain reciprocally connected hippocampus and entorhinal cortex (EC) networks, CA3 outputs control the EC propensity to generate experimentally induced ictal-like discharges resembling electrographic seizures. Neuronal damage in limbic areas, such as CA3 and dentate hilus, occurs in patients with temporal lobe epilepsy and in animal models (e.g., pilocarpine- or kainate-treated rodents) mimicking this epileptic disorder. Hence, hippocampal damage in epileptic mice may lead to decreased CA3 output function that in turn would allow EC networks to generate ictal-like events. Here we tested this hypothesis and found that CA3-driven interictal discharges induced by 4-aminopyridine (4AP, 50 muM) in hippocampus-EC slices from mice injected with pilocarpine 13-22 days earlier have a lower frequency than in age-matched control slices. Moreover, EC-driven ictal-like discharges in pilocarpine- treated slices occur throughout the experiment (less than or equal to6 h) and spread to the CA1/subicular area via the temporoammonic path; in contrast, they disappear in control slices within 2 h of 4AP application and propagate via the trisynaptic hippocampal circuit. Thus, different network interactions within the hippocampus-EC loop characterize control and pilocarpine-treated slices maintained in vitro. We propose that these functional changes, which are presumably caused by seizure-induced cell damage, lead to seizures in vivo. This process is facilitated by a decreased control of EC excitability by hippocampal outputs and possibly sustained by the reverberant activity between EC and CA1/subiculum networks that are excited via the temporoammonic path.


2002 - Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro [Articolo su rivista]
M., Avoli; M., D'Antuono; J., Louvel; R., Kohling; Biagini, Giuseppe; R., Pumain; G., D'Arcangelo; V., Tancredi
abstract

Seizures in patients presenting with mesial temporal lobe epilepsy result from the interaction among neuronal networks in limbic structures such as the hippocampus, amygdala and entorhinal cortex. Mesial temporal lobe epilepsy, one of the most common forms of partial epilepsy in adulthood, is generally accompanied by a pattern of brain damage known as mesial temporal sclerosis. Limbic seizures can be mimicked in vitro using preparations of combined hippocampus-entorhinal cortex slices perfused with artificial cerebrospinal fluid containing convulsants or nominally zero Mg2+, in order to produce epileptiform synchronization. Here, we summarize experimental evidence obtained in such slices from rodents. These data indicate that in control animals: (i) prolonged, NMDA receptor-dependent epileptiform discharges, resembling electrographic limbic seizures, originate in the entorhinal cortex from where they propagate to the hippocampus via the perforant path-dentate gyrus route; (ii) the initiation and maintenance of these ictal discharges is paradoxically contributed by GABA (mainly type A) receptor-mediated mechanisms; and (iii) CA3 outputs, which relay a continuous pattern of interictal discharge at approximately 1 Hz, control rather than sustain ictal discharge generation in entorhinal cortex. Recent work indicates that such a control is weakened in the pilocarpine model of epilepsy (presumably as a result of CA3 cell damage). In addition, in these experiments electrographic seizure activity spreads directly to the CA1-subiculum regions through the temporoammonic pathway. Studies reviewed here indicate that these changes in network interactions, along with other mechanisms of synaptic plasticity (e.g. axonal sprouting, decreased activation of interneurons, upregulation of bursting neurons) can confer to the epileptic, damaged limbic system, the ability to produce recurrent limbic seizures as seen in patients with mesial temporal lobe epilepsy. (C) 2002 Elsevier Science Ltd. All rights reserved.


2002 - Nicotine and neurodegeneration in ageing [Articolo su rivista]
Zanardi, Alessio; Leo, Giuseppina; Biagini, Giuseppe; Zoli, Michele
abstract

Impairment in cholinergic systems is a highly consistent finding in human dementia. Among cholinergic markers, marked decreases in nicotine binding have been most consistently observed in the telencephalic regions of demented patients and are thought to contribute to the cognitive deficits associated with ageing and age-related neurodegenerative diseases. New evidence that the cholinergic system has a specific pathogenic role in the neurodegenerative alterations of aged and, especially, demented patients is fast accumulating. Both in vivo and in culture, nicotine protects striatal, hippocampal and cortical neurons against the neurotoxicity induced by excitotoxic amino acids as well as the toxicity caused by beta-amyloid, the major component of senile plaques. Further support for the implication of nicotinic receptors in brain ageing is come from recent studies on transgenic animals lacking nicotinic receptor subtypes, which shed light on the mechanisms of nicotine neuroprotection and neurotoxicity. (C) 2002 Published by Elsevier Science Ireland Ltd.


2002 - Thalamocortical oscillations in a genetic model of absence seizures [Articolo su rivista]
D'Arcangelo, G; D'Antuono, M; Biagini, Giuseppe; Warren, R; Tancredi, V; Avoli, M.
abstract

We used field potential recordings in an in vitro thalamocortical slice preparation to compare the rhythmic oscillations generated by reciprocally connected networks of the thalamus and cerebral cortex obtained from epileptic (>160 days old) WAG/Rij and age-matched, nonepileptic control (NEC) rats. To increase neuronal excitability, and thus to elicit spontaneous field potential activity in vitro , we applied medium containing: (i) zero [Mg2+]; (ii) high [K+] (8.25 mm); or (iii) low concentrations of the K+ channel blocker 4-aminopyridine (4AP, 0.5-1 mum). Of these procedures, only the last was effective in triggering oscillatory activity that depended on the type of tissue. Thus, during 4AP application: (i) sequences of fast (intraburst frequency 9.5-16.1 Hz) and slower (5-8.9 Hz) field potential oscillations (FPOs) were recorded in WAG/Rij slices (n = 23), but (ii) only fast FPOs were seen in NEC slices (n = 7). Slower FPOs in WAG/Rij slices reflected a larger degree of thalamocortical synchronization than fast FPOs, and disappeared after surgical separation of cortex and thalamus (n = 5); under these conditions fast FPOs continued to occur in thalamus only. In addition, fast and slower FPOs disappeared in all areas of the WAG/Rij slice during thalamic application of the excitatory amino acid receptor antagonist kynurenic acid (n = 3), while fast FPOs continued to occur in thalamus when kynurenic acid was applied to the cortex (n = 4). Bath application of the N -methyl-d-aspartic acid (NMDA) receptor antagonist 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (CPP) abolished slower FPOs in WAG/Rij cortex and thalamus (n = 6) without infuencing fast FPOs recorded in WAG/Rij (n = 6) or NEC slices (n = 4). Moreover, cortical application of CPP (n = 6) abated slower FPOs although they persisted following CPP application to the thalamus (n = 7). Our data demonstrate that highly synchronized, slower FPOs can occur during 4AP application in WAG/Rij but not in NEC slices. This activity, which may represent an in vitro hallmark of thalamocortical epileptogenicity, requires the function of reciprocally connected thalamic and cortical networks and depends on cortical NMDA receptor-mediated mechanisms.


2001 - Brain-derived neurotrophic factor superinduction parallels anti-epileptic-neuroprotective treatment in the pilocarpine epilepsy model [Articolo su rivista]
Biagini, Giuseppe; M., Avoli; J., Marcinkiewicz; M., Marcinkiewicz
abstract

Antiepileptic drugs provide neuroprotection in several animal models of brain damage, including those induced by status epilepticus (SE). The mechanisms involved in this action are unknown, but neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may play a role. In this study we investigated the changes in BDNF levels in rats in which SE had been induced by pilocarpine injection (400 mg/kg i.p.) and continued for several hours (unprotected group). In other animals (protected groups), SE was suppressed after 30 min by intraperitoneal injection of either diazepam (10 mg/kg) + pentobarbital (30 mg/kg) or paraldehyde (0.3 mg/kg). In diazepam + pentobarbital-treated rats the hippocampal damage caused by SE was significantly lower (p < 0.05) than in unprotected animals. In addition, 2 and 24 h after pilocarpine injection, the levels of BDNF mRNA were moderately increased in the unprotected group, but 'super-induced' in protected animals, especially in the neocortex and hippocampus. A time-dependent increase in BDNF immunoreactivity was also found by western blot analysis in rats treated with diazepam + pentobarbital. In contrast, a decrease of BDNF immunoreactivity occurred in the unprotected group. In conclusion, these results show that neuroprotection induced by anti-epileptic drugs in pilocarpine-treated rats is accompanied by strong potentiation of BDNF synthesis in brain regions involved in SE.


2001 - Decreased fibrosis in aorta of salt loaded stroke-prone spontaneously hypertensive rats by combining delapril and indapamide treatments: a dose response analysis. [Abstract in Rivista]
Contri, Miranda; Taparelli, Francesca; Pedrazzi, Giada; Biagini, Giuseppe
abstract

The protective effects of the angiotensin-converting enzyme (ACE) inhibitor delapril and the diuretic indapamide were investigated in stroke-prone spontaneously hypertensive rats (SHRsp) by studying vascular wall fibrosis in the thoracic aorta.


2001 - Electrophysiology of regular firing cells in the rat perirhinal cortex [Articolo su rivista]
M., D'Antuono; Biagini, Giuseppe; V., Tancredi; M., Avoli
abstract

The electrophysiological properties of neurons in the rat perirhinal cortex were analyzed with intracellular recordings in an in vitro slice preparation. Cells included in this study (n = 59) had resting membrane potential (RMP) = -73.9 +/- 8.5 mV (mean +/- SD), action potential amplitude = 95.5 +/- 10.4 mV, input resistance = 36.1 +/- v 15.7 M Omega, and time constant = 13.9 +/- 3.4 ms. When filled with neurobiotin (n = 27) they displayed a pyramidal shape with an apical dendrite and extensive basal dendritic tree. injection of intracellular current pulses revealed: 1) tetrodotoxin (TTX, 1 muM)-sensitive, inward rectification in the depolarizing direction (n = 6), and 2) a time- and voltage-dependent hyperpolarizing sag that was blocked by extracellular Cs+ (3 mM, n = 5) application. Prolonged (up to 3 s) depolarizing pulses made perirhinal cells discharge regular firing of fast action potentials that diminished over time in frequency and reached a steady level (i.e., adapted). Repetitive firing was followed by an afterhyperpolarization that was decreased, along with firing adaptation, by the Ca2+-channel blocker Co2+ (2 mM, n = 6). Action potential broadening became evident during repetitive firing. This behavior, which was more pronounced when larger pulses of depolarizing current were injected (and thus when repetitive firing attained higher rates), was markedly decreased by Co2+ application. Subthreshold membrane oscillations at 5-12 Hz became apparent when cells were depolarized by 10-20 mV from RMP, and action potential clusters appeared with further depolarization. Application of glutamatergic and GABA(A) receptor antagonists (n = 4), Co2+ (n = 6), or Cs+ (n = 5) did not prevent the occurrence of these oscillations that were abolished by TTX (n = 6). Our results show that pyramidal-like neurons in the perirhinal cortex are regular firing cells with electrophysiological features resembling those of other cortical pyramidal elements. The ability to generate subthreshold membrane oscillations may play a role in synaptic plasticity and thus in the mnemonic processes typical of this limbic structure. Hippocampus 2001;11:662-672.


2001 - Regional and subunit-specific downregulation of acid-sensing ion channels in the pilocarpine model of epilepsy [Articolo su rivista]
Biagini, Giuseppe; K., Babinski; M., Avoli; M., Marcinkiewicz; P., Seguela
abstract

Acid-sensing ion channels (ASICs) constitute a recently discovered family of excitatory cation channels, structurally related to the superfamily of degenerin/epithelial sodium channels. ASIC1b and ASIC3 are highly expressed in primary sensory neurons and are thought to play a role in pain transmission related to acidosis. ASIC1a, ASIC2a, and ASIC2b are also distributed in the central nervous system where their function remains unclear. We investigated here the regulation of their expression during status epilepticus (SE), a condition in which neuronal overexcitation leads to acidosis. In animals treated with pilocarpine (380 mg/kg) to induce SE, we observed a marked decrease of ASlC2b mRNA levels in all hippocampal areas and of ASIC1a mRNA levels in the CA1-2 fields. These changes were also observed after protective treatment from neuronal cell death with diazepam (10 mg/kg) and pentobarbital (30 mg/kg). These findings suggest a key role of channels containing ASIC1a and ASIC2b subunits in both normal and pathological activity of hippocampus.


2001 - Thalamocortical connectivity in a rat brain slice preparation: participation of the ventrobasal complex to synchronous activities. [Articolo su rivista]
Biagini, Giuseppe; D`antuono, M.; Tancredi, V.; Motalli, R.; Louvel, J.; D`arcangelo, G.; Pumain, R.; Warren, R.; Avoli, M.
abstract

We studied the synchronous cortical and thalamic activities induced by low (0.5–1 mM) and high (50–100 mM) concentrations of theK+ channel blocker 4-aminopyridine (4AP) in a rat thalamocortical preparation. The presence of reciprocal thalamocortical connectivitywas documented by diffusion of the fluorescent tracer Di-IC18 between the somatosensory cortex and the ventrobasal complex (VB) of thethalamus in vitro. Functional reciprocal connectivity was also demonstrated by stimulating the cortical middle-deep layers (which elicitedorthodromic responses in VB) or the VB (which induced orthodromic and antidromic responses in the cortex). Spontaneous field potentialswere not recorded in either the thalamus or cortex in control medium. Low concentrations of 4AP produced local spindle-like rhythmicoscillations in cortex and VB (duration = 0.4–3.5 s; frequency = 9–16 Hz). In contrast, high concentrations of 4AP induced widespreadictal-like epileptiform discharges (duration = 8–45 s) characterised by a ‘tonic’ component followed by a period of ‘clonic’ discharges inboth cortex and VB. Spindle-like activity was abolished in cortex and thalamus by applying the excitatory amino acid receptor antagonistkynurenic acid in VB. In contrast, the same procedure exacerbated ictal-like discharges, while depressing VB activity. Our results indicatethat thalamus and cortex can produce synchronous activities in this in vitro thalamocortical network: spindle-like rhythmic oscillationsare generated at the thalamic level and imposed upon the cortical network whereas ictal-like discharges have a cortical origin and aremodulated by the thalamic network activity. In addition, we have shown that it is possible to preserve reciprocal projections between cortexand thalamus in an in vitro rat slice preparation that could be a valuable tool to study epileptic-prone rat strains.


2000 - Degenerazione e rigenerazione nel sistema nervoso centrale: rilevanza dei fattori trofici e della trasmissione neuronale colinergica nel trofismo del sistema nervoso centrale. [Articolo su rivista]
Biagini, Giuseppe; Zoli, Michele; Zanardi, Alessio; Zini, Isabella; Agnati, Luigi Francesco
abstract

Contrary to a diffuse belief lasting until the end of the past century, recent data demonstrate that even the mammalian central nervous system possesses regenerative capabilities. The recovery that follows neuronal lesions could be morphological and/or functional. In both cases, the recovery is dependent on chemical signals (i.e., trophic factors and neurotransmitters) that influence neuronal cell survival and capability of producing structural changes. The synthesis and release of these chemical signals could be modulated by drugs acting on glial and neuronal systems. We showed that it is possible to maintain, at least in part, the anatomo-functional integrity of damaged brain areas by modulating polyamine synthesis. These molecules are synthesized in neurons belonging to damaged areas and can stimulate astroglial reactivity upon their release, leading to astrocyte hypertrophy and hyperplasia and neurotrophic factor synthesis. Trophic factor synthesis can be modulated also by specific drugs acting on glial and/or neuronal metabolism. We found that selegilin is able to induce basic fibroblast growth factor (bFGF), and that certain antiepileptic drugs can induce brain-derived neurotrophic factor (BDNF) by acting, respectively, on glial and neuronal cells.


2000 - Mammalian ASIC2a and ASIC3 subunits co-assemble into heteromeric proton-gated channels sensitive to Gd3+ [Articolo su rivista]
K., Babinski; S., Catarsi; Biagini, Giuseppe; P., Seguela
abstract

Proton receptors of the acid-sensing ion channel (ASIC) family are expressed in sensory neurons and thus could play a critical role in the detection of noxious acidosis. To investigate the subunit composition of native ASICs in peripheral and central neurons, we coinjected human as well as rodent ASIC2a and ASIC3 subunits in Xenopus oocytes, The amplitudes of acid-induced biphasic responses mediated by co-expressed ASIC2a and ASIC3 subunits were much larger (as much as 20-fold) than the currents mediated by the respective homomers, clearly indicating functional association. The reversal potential of the ASIC2a+3 current (greater than or equal to+20 mV) reflected a cationic current mainly selective for sodium, The sensitivity to pH or amiloride of single versus co-expressed ASIC subunits was not significantly different; however, gadolinium ions inhibited ASICS and ASIC2a+3 responses with much higher potency (IC50 similar to 40 mu M) than the ASIC2a response (IC50 greater than or equal to 1 mM). Biochemical interaction between ASIC2a and ASIC3 subunits was demonstrated by co-purification from transfected human embryonic kidney (HEK293) cells and Xenopus oocytes. Our in situ hybridization data showed that rat ASIC2a and ASIC3 transcripts are colocalized centrally, whereas reverse transcription-polymerase chain reaction data led us to detect co-expression of human ASIC2a and ASIC3 subunits in trigeminal sensory ganglia, brain, and testis where they might coassemble into a novel subtype of proton-gated channels sensitive to gadolinium.


2000 - Protective effects of delapril combined with indapamide or hydrochlorothiazide in spontaneously hypertensive stroke-prone rats: a comparative dose-response analysis [Articolo su rivista]
S., Boschi; G., Vantaggiato; C., Torri; Zini, Isabella; Agnati, Luigi Francesco; Zoli, Michele; Biagini, Giuseppe
abstract

In previous articles, we have shown that the combination of the angiotensin-converting enzyme (ACE) inhibitor delapril (12 mg/kg/day) and the diuretic indapamide (1 mg/kg/day) was able to prolong the life span significantly in salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp). Because this finding was partly dependent on the antagonism of salt-loading effects by pharmacologic induction of diuresis, which prevented any increase in blood pressure values, we decided to evaluate whether lower doses of the combination could be equally protective without changing the progression of hypertension. Thus, we studied several treatments with progressively lower doses of delapril (6, 3, or 1.5 mg/kg/day) combined with indapamide (0.5, 0.25, or 0.125 mg/kg/day) or hydrochlorothiazide (2.5, 1.25, or 0.625 mg/kg/day) in salt-loaded SHRsp. Salt-loaded untreated animals were considered to be the control group. In agreement with previous experiments, control rats reached 50% mortality similar to 7 weeks after the beginning of salt loading. The combination of delapril and hydrochlorothiazide at the two lowest doses was nor able to delay animal death significantly, whereas treatment with april and indapamide at the lowest dose was effective (50% survival rate, 15 weeks). The groups treated with the highest dose of delapril and hydrochlorothiazide or with the intermediate or highest dose of delapril and indapamide did not reach 50% mortality by the end of the experiment, at 44 weeks of treatment (i.e., when animals reached age 1 year). Only the highest delapril and indapamide doses were able to increase diuresis, but for a relatively short period. None of the treatments was able to lower or control blood pressure levels adequately. Therefore, blood pressure levels by themselves were not predictive of rat mortality. In contrast, the maximal value of proteinuria in the weeks preceding death was inversely correlated with the survival time. In conclusion, this study shows that low doses of an ACE inhibitor in combination with a diuretic can be effectively protective in a model of severe hypertension, independent of any change in blood pressure levels.


2000 - Spindle-like thalamocortical synchronization in a rat brain slice preparation [Articolo su rivista]
V., Tancredi; Biagini, Giuseppe; M., D' Antuono; J., Louvel; R., Pumain; M., Avoli
abstract

We obtained rat brain slices (550-650 mm) that contained part of the frontoparietal cortex along with a portion of the thalamic ventrobasal complex (VB) and of the reticular nucleus (RTN). Maintained reciprocal thalamocortical connectivity was demonstrated by VB stimulation, which elicited orthodromic and antidromic responses in the cortex, along with re-entry of thalamocortical firing originating in VB neurons excited by cortical output activity. In addition, orthodromic responses were recorded in VB and RTN following stimuli delivered in the cortex. Spontaneous and stimulus-induced coherent rhythmic oscillations (duration = 0.4-3.5 s; frequency = 9-16 Hz) occurred in cortex, VB, and RTN during application of medium containing low concentrations of the K+ channel blocker 4-aminopyridine (0.5-1 mu M). This activity, which resembled electroencephalograph (EEG) spindles recorded in vivo, disappeared in both cortex and thalamus during application of the excitatory amino acid receptor antagonist kynurenic acid in VB (n = 6). By contrast, cortical application of kynurenic acid (n = 4) abolished spindle-like oscillations at this site, but not those recorded in VB, where their frequency was higher than under control conditions. Our findings demonstrate the preservation of reciprocally interconnected cortical and thalamic neuron networks that generate thalamocortical spindle-like oscillations in an in vitro rat brain slice. As shown in intact animals, these oscillations originate in the thalamus where they are presumably caused by interactions between RTN and VB neurons. We propose that this preparation may help to analyze thalamocortical synchronization and to understand the physiopathogenesis of absence attacks.


1999 - Biochemical and morphometric studies of the aortic extracellular matrix in long term hypertension: Effects of diet and anti-hypertensive drugs [Abstract in Rivista]
Contri, Miranda; Taparelli, Francesca; Miselli, M; Tollari, J; Biagini, Giuseppe
abstract

The investigation was aimed to characterize long term changes in the aortic wall composition related to hypertension and drug admnistration.


1998 - Brain oxidative damage following acute immobilization and mild emotional stress [Articolo su rivista]
Marzatico, F; Bertorelli, L; Pansarasa, O; Guallini, P; Torri, C; Biagini, Giuseppe
abstract

We studied the role of free radicals on brain oxidative damage in rats after acute immobilization stress (restraint) and mild emotional stress (handling). To investigate brain oxidative damage, CuZn and Mn dependent superoxide dismutase (CuZn SOD, Mn SOD) activities, lipid peroxidation (TBARs), Na+K+ ATPase activity, protein carbonyl (PrC), and reduced and oxidized glutathione (GSH GSSG) levels were measured in the cerebral cortex (CTX), hippocampus (HIP), and striatum (ST) of the animals after the two different stress stimuli. Because stress produces abnormalities in the hypothalamic-p pituitary-adrenal axis, the intensity of the two stress conditions were measured by plasmatic corticosteroid (COR) levels: particularly, COR levels doubled in handled rats and increased 15-fold in restrained animals. The SOD activities increased in CTX and decreased in HIP of the handled rats, while in ST a significant decrease in handled animals but an increase in restrained animals occurred. TBARs, GSH, and GSSG levels remained unchanged while an index of glutathione redox decreased significantly in ST of handled animals and in CTX of restrained ones. Na+K+ ATPase activity increased significantly in the HIP and ST of both groups of stressed rats. The stress induced a remarkable increase in PrC levels in all studied cerebral areas. These findings provide evidence to support the idea that stress produces oxidants but that the oxidative damage in stress differs in cerebral areas and could contribute to the degenerative mechanism of aging.


1998 - Postnatal maternal separation during the stress hyporesponsive period enhances the adrenocortical response to novelty in adult rats by affecting feedback regulation in the CA1 hippocampal field [Articolo su rivista]
Biagini, Giuseppe; MERLO PICH, Emilio; Carani, Cesare; Marrama, Paolo; Agnati, Luigi Francesco
abstract

The aim of the present experiment was to study the effects of early postnatal maternal separation on behavioural and adrenocortical responses to novelty in rats tested as adults. Sprague-Dawley rat pups were exposed to daily maternal separation (5 h/day) from postnatal day 2 to 6, during the stress hyporesponsive period. Since this procedure requires physical contact with the animals, a first control group of daily handled pups was introduced. A second control group, consisting of pups never handled or separated from the mother, was also considered. At postnatal day 45, the rats were tested in a two-compartment exploratory apparatus: the maternally separated and the non-handled rats, whose behavioural performance did not differ, showed higher emotional behaviour when compared with the handled rats (P < 0.05), suggesting that the handling procedure hut not maternal separation improved the capacity to cope with novelty. Corticosterone plasma levels were found to be higher in the maternally separated rats than in the other two groups (P < 0.05), either at resting conditions or at 30 min after novelty exposure (P < 0.05). Levels of nuclear glucocorticoid receptor immunoreactivity in the CA1 hippocampal field were shown to be regulated by novelty exposure, as expected, in both the handled and the non-handled rats but not in the maternally separated rats. In conclusion, repeated maternal separation periods of 5 h/day during the first week of life produced long-lasting effects on the hippocampal regulation of the hypothalamic-pituitary-adrenocortical axis, which appear to be associated with increased responsiveness to stress stimuli in adulthood.


1998 - Protective effects of delapril, indapamide and their combination on stroke occurrence and lifespan in salt-loaded stroke-prone spontaneously hypertensive rats [Articolo su rivista]
Razzetti, R; Bongrani, S; Oberto, G; Ferrari, R; Biagini, Giuseppe
abstract

The effects of long-term oral administration of delapril (CAS 83435-67-0), indapamide (CAS 26807-65-8) and their combination on the occurrence of stroke and on mortality were investigated in young salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp) for 31 weeks of treatment (8th-39th week of age) and up to 8 weeks thereafter. Body weight and saline consumption were investigated at regular intervals and cerebrovascular lesions, renal and heart weight were assessed after sacrifice. Untreated SHRsp served as controls. About 50 % of control animals died within 6 weeks of saline administration and in 56 % of surviving animals cerebral lesions were present at sacrifice, while no death and no cerebral lesions were observed in animals drinking saline, to which delapril, indapamide and their combination had been added, up to the end of treatment. This protective effect was maintained even in the withdrawal period. All treatments induced a highly significant (p < 0.001) reduction of heart weight/body weight and kidney weight/body weight ratios.


1997 - Neuron-glia cross talk in rat striatum after transient forebrain ischemia [Capitolo/Saggio]
Zoli, M.; Biagini, G.; Ferrari, R.; Pedrazzi, P.; Agnati, L. F.
abstract

Striatum is highly vulnerable to transient forebrain ischemia induced by the 4 vessel occlusion (4V0) method (Brierley 1976. Pulsinelli et al. 1982, Zini et al. 1990a). Massive degeneration and loss of Nissl-stained neurons occur within 24 hr from an ischemia of long duration (30 min) (Pulsinelli et al. 1982). Neuronal loss is mainly restricted to the lateral part of caudate-putamen (Pulsinelli et al. 1982, Zini et al. 1990a). Cellular alterations include loss of medium-size spiny projection neurons (Pulsinelli et al. 1982, Francis and Pulsinelli 1982), largely corresponding to dopaminoceptive neurons (Benfenati et al. 1989, Zoli et al. 1989), and increase in reactive astrocytes (Pulsinelli et al. 1982, Grimaldi et al. 1990) and microglia (Gehrmann et al. 1982). On the other hand, large cholinergie (Francis and Pulsinelli 1982) and medium-size aspiny somatostatin (SS)/neuropeptide Y (NPY)-containing interneurons are resistant to the ischemic insult (Pulsinelli et al. 1982, Grimaldi et al. 1990). In a few instances, such as in the case of SS and NPY immunoreactivity (IR), the initial loss is followed by full recovery within 7 (SS) or 40 (NPY) days post-ischemia (Grimaldi et al. 1990). However, it is not known whether some kind of recovery is present for the bulk of medium-size spiny projections neurons after the first days post-ischemia.


1997 - Protective effects of delapril, indapamide and their combination chronically administered to stroke-prone spontaneously hypertensive rats fed a high-sodium diet [Articolo su rivista]
Biagini, Giuseppe; Zoli, Michele; C., Torri; S., Boschi; G., Vantaggiato; M., Ballestri; A., Baraldi; Agnati, Luigi Francesco
abstract

1. Stroke-prone spontaneously hypertensive rats (SHRsp) have been used widely to test agents putatively capable of vascular protection. These animals present an accelerated time course of hypertension and a reduced life-span. When fed a high-sodium diet from the eighth week of life, a further acceleration in blood pressure increase is obtained, and rats start to die after 5 weeks of diet as a consequence of cerebral haemorrhage. In this model, angiotensin-converting enzyme (ACE) inhibitors were repeatedly proved to prevent vascular lesions and death. Notably, this effect was independent of any hypotensive effect. On the contrary, diuretics were shown not to be equally effective. A combination of ACE inhibitors and diuretics, although known to have synergistic effects in the therapy of hypertension, has never previously been tested. 2. Our aim was to study the effects of long-term treatment with the ACE inhibitor delapril (12 mg day(-1) kg(-1)), the thiazide-like diuretic indapamide (1 mg day(-1) kg(-1)), and their combination (12 and 1 mg day(-1) kg(-1) respectively), on the survival of SHRsp rats fed a high-sodium diet from the eighth week of life onwards. The effects of the treatments on blood pressure, body weight, food and fluid intake, diuresis, proteinuria and the appearance of lesion signs and death were assessed weekly When control rats reached 50% mortality, they were killed, together with some drug-treated rats, to compare lesions in brain and kidney. The other drug-treated rats continued treatments until 50% mortality was reached in two treatment groups. 3. All drug treatments were able to delay death significantly when compared with control rats, which reached 50% mortality after 6 weeks of salt loading. This event was preceded by a highly significant increase in proteinuria, diuresis and fluid intake that took place 3 weeks after the increase in blood pressure over the initial range. In delapril-or indapamide-treated SHRsp these changes were never seen, even when animals started to die. In the combination-treated group, a significant increase (P<0.01) in fluid intake and diuresis, but not proteinuria, was observed from the third week of treatment onwards. 4. Treatment with delapril or indapamide did not block the progressive increase in blood pressure as observed in control animals. However, the increase in blood pressure was markedly retarded with respect to control rats. At variance with this, in combination-treated animals blood pressure levels were maintained until the end of the experiment within the 99% confidence interval initially observed in control animals. 5. Infarctual and haemorrhagic cerebral lesions were observed in 38% of control rats; no lesions were noted in brains of age-matched rats receiving a drug treatment. Kidneys from control animals presented major degenerative lesions of glomeruli and arteries, characterized by fibrinoid necrosis. This condition was absent in drug-treated animals, which presented minor signs of ischaemic lesion. Heart hypertrophy when heart weight was expressed as a percentage of body weight, was similar in saline-, delapril-or indapamide-treated rats. At variance with this, in combination-treated animals the heart weight to body weight ratio was significantly (P<0.01) lower than in the other groups. 6. In conclusion, the diuretic indapamide showed similar protective effects as the ACE inhibitor delapril on acute vascular lesions and survival of SHRsp. Moreover, their combination synergized in preventing heart hypertrophy consequent to long-term hypertension. This result is probably related to the enhanced diuresis and the better control of blood pressure levels selectively found in combination-treated animals.


1997 - Time-course of protective effects on the aorta wall by treatment with delapril, indapamide and their combination in stroke-prone spontaneously hypertensive rats (SHRsp) [Abstract in Rivista]
Contri, Miranda; Taparelli, Francesca; Boraldi, Federica; Biagini, Giuseppe
abstract

The investigation was aimed at characterizing long term changes in the aorta wall structure in hypertensive rats.


1996 - Computer-assisted mapping of basic fibroblast growth factor immunoreactive nerve cell populations in the rat brain [Articolo su rivista]
K., Fuxe; B., Tinner; Zoli, Michele; Rf, Pettersson; A., Baird; Biagini, Giuseppe; G., Chadi; Agnati, Luigi Francesco
abstract

We have performed a mapping of basic fibroblast growth factor (bFGF) immunoreactive (ir) glial and nerve cell populations in the male rat brain using a rabbit antibody raised against a synthetic peptide of bovine bFGF. Regional morphometric and microdensitometric analysis of the bFGF ir neuronal profiles in coronal brain sections was carried out by means of an automatic image analyser. The density and intensity of the bFGF ir glial profiles were subjectively evaluated. The bFGF immunoreactivity (IR) was detected within the cytoplasm of neurons, except within the pyramidal neurons of hippocampal CA2 region, the fasciola cinerea and the indusium griseum, where bFGF IR was present in the nucleus. In contrast, in glial cells bFGF IR was always found in the nucleus, Neuronal and glial IR was no longer observed after absorption of the bFGF antiserum with recombinant bFGF. Basic FGF IR was found in neuronal and glial cell populations throughout the brain as well as in the choroid plexus and in the ependymal cells lining the ventricles, Basic FGF ir nerve cells were found in all layers of both the neocortex and allocortex. Within the caudate putamen and the nucleus accumbens a low density of weak bFGF ir neuronal profiles was detected, The majority of the thalamic nuclei showed medium to high densities of moderate to strong bFGF ir neuronal profiles. All the hypothalamic nuclei, with the exception of the anterior and lateral hypothalamic area and of the ventral hypothalamic nucleus, contained a high density of bFGF ir profiles, The pens and the medulla oblongata were characterized by the presence of a large number of nuclei containing moderate to high densities of strong bFGF ir profiles. The Purkinje cell layer of the cerebellar cortex contained a high density of moderately bFGF ir profiles. A moderate density of strong bFGF ir nerve cell profiles was observed within all the laminae of the spinal cord, except within the II and III laminae where a high density of strongly ir profiles was found. Histogram analysis of total immunoreactivity showed that the distribution of bFGF ir profiles within the telencephalon and mesencephalon tend to be similar with regard to the central tendency and spread. Using Kendall's tau, a significant correlation between intensity and density values was obtained only in the diencephalon. The cytoplasmic bFGF IR found in distinct nerve cell populations all over the rat brain and spinal cord may represent forms of bFGF which can be released from the nerve cells via non-exocytotic mechanisms in view of the absence of an intracellular signal peptide in bFGF. The presence of nuclear bFGF IR within the glial cells all over the central nervous system (CNS) suggests an intracellular function of bFGF, such as the promotion of mitogenesis and/or participation in the transcriptional regulation of various genes.


1995 - Age-related alterations in tanycytes of the mediobasal hypothalamus of the male rat [Articolo su rivista]
Zoli, Michele; F., Ferraguti; A., Frasoldati; Biagini, Giuseppe; Agnati, Luigi Francesco
abstract

By means of semiquantitative immunocytochemistry, possible age-related changes in dopamine and cyclic AMP-regulated phosphoprotein mr 32 (DARPP-32) and glial fibrillary acidic protein (GFAP) immunoreactivities (IR) were investigated in tanycytes of the arcuate nucleus. These two markers showed opposite changes during aging. DARPP-32 IR decreased by around 70%, whereas GFAP IR increased by around 300% in 24-month-old vs. 3-month-old rats. These changes were accompanied by a progressive loss in the number of tanycytes, measured by counting of their long processes in the arcuate nucleus. No significant age-related change was observed either in GFAP IR in astrocytic populations of the mediobasal hypothalamus or in tyrosine hydroxylase IR in dopaminergic neurons of the dorsal arcuate nucleus. These observations indicate that the tanycytic population of the arcuate nucleus undergoes important modifications during aging, which include cell loss, impairment in the intracellular signalling cascade linked to DARPP-32, and hypertrophy. These changes may be related to the alterations in the neuroendocrine systems known to occur during aging.


1995 - Changes in glucocorticoid receptor immunoreactivity after adrenalectomy and corticosterone treatment in the rat testis [Articolo su rivista]
Biagini, Giuseppe; MERLO PICH, Emilio; A., Frasoldati; Agnati, Luigi Francesco; Marrama, Paolo
abstract

The distribution of glucocorticoid receptor (GR) in the rat testis was investigated by means of immunocytochemistry (IR) and computer-assisted image analysis. A monoclonal antibody against rat liver GR showed the presence of GR IR selectively in the nuclei of interstitial and tubular cells. The semiquantitative microdensitometry of GR IR revealed that 77% of the specific staining was localized in the tubular compartment of rat testis. After adrenalectomy, GR IR was greatly reduced both in interstitial and tubular cells, roughly at the same degree. Corticosterone treatment (50 mg/kg, for 5 d) of adrenalectomized animals yielded a recovery of nuclear immunopositivity without changing the cellular distribution of GR, as observed in control rats. Nevertheless, the high dose of corticosterone administered produced a significant (p<0.01) decrease of GR IR with respect to control rats. These results provide evidence for a prevalent nuclear binding of GR in the tubular compartment in basal conditions. On the other hand, adrenalectomy or repeated corticosterone treatment seem to affect GR similarly in all positive cells without changing significantly the proportion of GR IR in the different testicular compartments. This uneven distribution of GR IR suggests that tubular cells can be a major target of corticosterone when affecting directly testicular functions in the rat.


1995 - Diethyldithiocarbamate, a superoxide dismutase inhibitor, counteracts the maturation of ischemic-like lesions caused by endothelin-1 intrastriatal injection. [Articolo su rivista]
Biagini, Giuseppe; D., Sala; Zini, Isabella
abstract

The effects of a focal lesion induced by endothelin-1 (ETI1, 0.8 mu g/0.8 mu l) on superoxide dismutase (SOD) were studied in the neostriatum of male rats. SOD activity was analyzed at several time intervals (5, 20, 60 min, 4, 24 h and 7 days) after the lesion. No significant changes were observed early after the injection, but SOD activity started to rise significantly at the 60-min time interval reaching a peak 24 h after the injection. In a second experiment the volume of ET-1-induced lesion was evaluated following treatments which induce variations of SOD activity. ET-1 caused a large lesion (9.20 +/- 1.32 mm(3)) in the neostriatum 24 h after the injection that was 3-fold greater than that observed 1 h after. Rats treated with the SOD inhibitor diethyldithiocarbamate showed a lesion equivalent to that observed 1 h after ET-1 injection, suggesting that SOD may be involved in the maturation of ET-1-induced neuronal damage.


1995 - EFFECT OF ENDOTHELIN-1 INDUCED ISCHEMIA ON PEROXIDATIVE DAMAGE AND MEMBRANE-PROPERTIES IN RAT STRIATUM SYNAPTOSOMES [Articolo su rivista]
P., Viani; Zini, Isabella; G., Cervato; Biagini, Giuseppe; Agnati, Luigi Francesco; B., Cestaro
abstract

Synaptosomes obtained from rat striata lesioned by central injection of endothelin-1 (ET-1) were analyzed for the levels of lipid peroxidation products, the susceptibility to lipid peroxidation, the phospholipid and free fatty acid composition and the activity of Na+,K+-ATPase one hour after ET-1 treatment. The intrastriatal injection of ET-1 promoted an increase of endogenous thiobarbituric reactive substances (TEARS), as index of free radical mediated lipid damage, and a greater susceptibility to iron/ascorbate-induced lipid peroxidation. The pattern of free fatty acids showed a significant decrease of arachidonic and docosahexaenoic acid consequent to ET-1 treatment. The analysis of lipid composition showed a significant loss of phospholipids: among phospholipid species, sphingomyelin and phosphatidylethanolamine plasmalogen were particularly reduced by ET-1 treatment. The activity of membrane-bound Na+,K+-ATPase was also significantly reduced in synaptosomes obtained from ET-1 lesioned striata. Taken together these results indicate a significant modification of synaptosomal membrane of ET-1 treated rat striata, possibly due to a free radical mediated damage.


1995 - Ex-vivo demonstration of nitric oxide in the rat brain: Effects of intrastriatal endothelin-1 injection [Articolo su rivista]
A. V., Kozlov; Biagini, Giuseppe; Tomasi, Aldo; Zini, Isabella
abstract

Nitric oxide (NO.) is a novel transmitter with multiple functions in endothelium and neuronal tissue. In particular, it has been implicated in the pathogenesis of neurodegenerative diseases. The aim of the present study was to demonstrate the ex vivo detection Df NO. in basal conditions and after ET-1 intrastriatal injection by means of electron paramagnetic resonance (EPR) spectroscopy using locally injected hemoglobin (Hb) as a NO. trapping agent. The extent of neostriatal damage after Hb and ET-1 injections was assessed by means of immunocytochemistry with a monoclonal antibody against dopamine and cAMP-phosphoprotein M(r) 32 (DARPP-32), which is considered a marker of striatal intrinsic neurons. In the absence of local Hb injection, no signal related to endogenous NO. was detected in the neostriatum, suggesting that endogenous NO. trapping agents are not sufficiently concentrated to allow NO. detection with the present technique. Instead, 1 h after Hb injection, a clear nitrosyl-Hb signal can be detected in neostriatal homogenates. ET-1, a powerful vasoconstrictor agent, was used to cause neuronal loss in the neostriatum. No change in nitrosyl-Hb signal was observed in neostriata 1 h after ET-1 injection, whereas an almost 3-fold increase in the signal intensity was present 24 h after ET-1 injection. The analysis of neostriatal damage showed that Hb injection did not cause either significant damage of striatal tissue or potentiation of ET-1-induced lesions. In conclusion, the present technique allows ex vivo detection of NO. in the brain. The delayed increase in NO. observed after ET-1 injection indicates that this molecule may participate in the development of slowly progressive neuronal damage occurring at late post-ischemic times.


1995 - TEMPORAL CHANGES IN SULFATED GLYCOPROTEIN-2 (CLUSTERIN) AND ORNITHINE DECARBOXYLASE MESSENGER-RNA LEVELS IN THE RAT TESTIS AFTER ETHANE-DIMETHANE SULFONATE-INDUCED DEGENERATION OF LEYDIG-CELLS [Articolo su rivista]
A., Frasoldati; Zoli, Michele; F. F. G., Rommerts; Biagini, Giuseppe; M. F., Fustini; Carani, Cesare; Agnati, Luigi Francesco; Marrama, Paolo
abstract

Short- (3-24 h) and long-term (4-50 days) changes in sulphated glycoprotein-2 (SGP3) and ornithine decarboxylase (ODC) mRNA levels in the adult rat testis were studied following a single dose of ethane-dimethane sulphonate (EDS), to destroy the Leydig cells. Distribution patterns of SGP-2 and ODC labelling were consistent with prevailing expression of the two transcripts in Sertoli cells and germ cells, respectively This pattern did not show appreciable changes following EDS administration. No labelling of SGP-2 mRNA was noted in the interstitium of control and EDS-treated rats. This finding indicates that Leydig cell death induced by EDS is not associated with increased SGP-2 mRNA levels, a phenomenon related to apoptotic cell death in many tissues. Semi-quantitative densitometric analysis of the preparations demonstrated differential changes in SGP-2 and ODC mRNA levels in:the tubular compartment following EDS treatment. At 6, but not at 3 and 12, h following EDS administration, SGP-2 mRNA levels showed a significant increase, possibly secondary to a direct effect of the alkylating agent on Sertoli cells. A significant decrease in ODC mRNA levels was observed from day 7 to day 28, matching degenerative changes in the seminiferous epithelium. In contrast, a decrease in SGP-2 transcript levels was observed from days 21-35 after treatment. In conclusion, our findings demonstrate that SGP3 mRNA, a putative marker of apoptosis, is not altered in the testicular interstitium during EDS-induced degeneration of Leydig cells. In the tubular compartment, the content of both ODC and SGP-2 mRNAs following EDS administration appears to be dependent mostly on the integrity of the germ cell complement and not to be influenced directly by changes in testosterone levels.


1994 - DIFFERENT CLASSES OF VOLUME TRANSMISSION SIGNALS EXIST IN THE CENTRAL NERVOUS SYSTEM AND ARE AFFECTED BY METABOLIC SIGNALS, TEMPERATURE GRADIENTS AND PRESSURE WAVES [Articolo su rivista]
Agnati, Luigi Francesco; Cortelli, Pietro; Biagini, Giuseppe; Bjelke, B; Fuxe, K.
abstract

VOLUME transmission (VT) is the mode of intercellular communication involving the diffusion of transmitters, via extracellular fluid (ECF) pathways, from nerve cells selectively capable of producing the signal (signal source) to nerve and glial cells selectively capable of recognizing it (signal target). The proposal is now put forward that at least two classes of VT signals can be distinguished: (a) the private-code signals, exemplified by neurotransmitters, which are released by a limited group of nerve cells and recognized, via high affinity G-protein coupled receptors or by cytosolic enzymes such as guanylate cyclase in the case of nitric oxide, by a specific group of cells; and (b) the accessible-code signals, such as the electrical signals that are released by all neuronal cells and decoded by almost every CNS cell. In the present paper, it will be underlined that carbon dioxide, hydrogen ions, temperature gradients and pressure waves may be regulators of wiring transmission and VT.


1994 - Le basi razionali della terapia delle malattie neurodegenerative e il concetto di farmaco astrocita-cinetico [The treatment of neurodegenerative diseases and the astrocytokinetic drug] [Articolo su rivista]
Agnati, L. F.; Frasoldati, A.; Biagini, G.; Zini, I.
abstract


1994 - Possible mechanisms for the powerful actions of neuropeptides [Articolo su rivista]
K., Fuxe; X. M., Li; B., Bjelke; P. B., Hedlund; Biagini, Giuseppe; Agnati, Luigi Francesco
abstract

In order to understand the mechanisms underlying the powerful actions of neuropeptides, the present article has emphasized the unique ability of neuropeptides to act as VT signals, which via high-affinity G-protein coupled receptors can exert long-lasting actions and control synaptic transmission via receptor-receptor interactions. Also of substantial importance is the ability of neuropeptides to act as a set of signals via the formation of different types of active fragments, which can act as negative-feedback or positive-feedback signals to modulate the response elicited by the parent peptide and to give origin to syndromic responses. Also in the actions of the fragments on the neuronal network, receptor-receptor interactions may play an important role both by modulating the parent peptide receptors and by modulating other types of VT and/or WT receptors. Future work will have to evaluate the role of neuropeptides as transcellular signals and as regulators of neuronal excitabilities after the formation of carbamates, but certainly new important developments are within the horizon of today's research.


1994 - The concept of astrocyte-kinetic drug in the treatment of neurodegenerative diseases: evidence for L-deprenyl-induced activation of reactive astrocytes [Articolo su rivista]
Biagini, Giuseppe; A., Frasoldati; K., Fuxe; Agnati, Luigi Francesco
abstract

Basic fibroblast growth factor (bFGF) increases neuronal survival and growth in cell cultures and stimulates functional recovery From brain lesion. In addition, bFGF is able to induce glial cell proliferation and differentiation. Recently, L-deprenyl has been shown to potentiate astrocyte reaction to a mechanical lesion and to possess a trophic-like activity in several experimental models. In the present paper, we have therefore investigated if the enhancing effect of L-deprenyl on astrocyte reactivity is accompanied by increased levels of bFGF. The effect of L-deprenyl (0.25 mg/kg/day) on bFGF immunoreactivity (IR) after the insertion of an injection cannula in rat neostriatum have been investigated. It has been found that subchronic L-deprenyl treatment potentiates both the lesion-induced increase of glial fibrillary acidic protein (GFAP) and bFGF IRs (P < 0.01). These data suggest that a possible mechanism for L-deprenyl-induced neuroprotection may be the activation of astrocytes associated with increased secretion of trophic factors that promote neuronal survival and growth. This ''astrocyte-kinetic'' action of L-deprenyl could represent a new therapeutical approach to increase trophic support of lesioned neurons.


1994 - The treatment of neurodegenerative diseases and the astrocytokinetic drug [Articolo su rivista]
Agnati, L. F.; Frasoldati, A.; Biagini, G.; Zini, I.
abstract


1993 - Effects of polyamine synthesis blockade on neuronal loss and astroglial reaction after transient forebrain ischemia [Articolo su rivista]
Zoli, Michele; Zini, Isabella; R., Grimaldi; Biagini, Giuseppe; Agnati, Luigi Francesco
abstract

Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated to increase in the early phase of several types of brain lesion. However, their role in the pathogenesis of tissue damage is still debated. In the present paper the effects of treatments with alpha-difluoromethylomithine, a suicide inhibitor of omithine decarboxylase, have been investigated in a model of transient forebrain ischemia. Three treatment schedules were used: alpha-difluoromethylomithine treatment was either started 3 hr before and repeated 1 hr after the insult, or started at the time of the insult and continued for 3 or 7 days after post-ischemic reperfusion. The rats were sacrificed 4 hT, 7 or 40 days after reperfusion, respectively. The acute experiment demonstrated that alpha-difluoromethylomithine can reduce the increase of glial fibrillary acidic protein immunoreactivity, an early marker of astroglial reaction, in ischemic striatum. Subchronic and chronic alpha-difluoromethylomithine treatments induced a worsening of the morphological outcome of the ischemic lesion. In caudate-putamen a trend for an increase of the area of neuronal loss was present after both treatments. In the hippocampal formation, a significant increase in the severity of neuronal lesion was observed in the mildly lesioned CA3 field. In addition, other alterations of lesioned tissue were observed in alpha-difluoromethylomithine-treated animals, including increases of non-neuronal cells at 7 and especially 40 days post-lesion in striatum and CA3 hippocampal field. In conclusion, present data indicate that omithine decarboxylase activation after ischemic lesion is a crucial factor for survival of mildly lesioned neurons and proper tissue reaction to the ischemic lesion. The experiment on acute alpha-difluoromethylomithine treatment suggests that these effects may be, at least in part, related to putrescine-induced activation of astroglial cells in the early post-lesion period.


1993 - Indole-pyruvic acid treatment reduces damage in striatum but not in hippocampus after transient forebrain ischemia in the rat [Articolo su rivista]
Zoli, Michele; MERLO PICH, Emilio; F., Ferraguti; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

The effects of treatment with indole-pyruvic acid, an endogenous metabolite of tryptophan converted into kynurenic acid in the brain, were studied in rats after transient forebrain ischemia induced by the 4-vessel occlusion procedure. The histological analysis showed a significant protective effect of indole pyruvic acid treatment on striatal ischemic lesions assessed by the extent of regional atrophy and the area of neuronal disappearance 14 days after ischemia. Striatal neurons were labelled by dopamine and adenosine 3':5'monophosphate regulated phosphoprotein-32 immunoreactivity. Conversely, increased neuronal loss, regional atrophy and glial fibrillary acidic protein immunoreactivity, an index of post-injury astroglial activation, were observed in the hippocampal formation, especially the CA3 field, of indole-pyruvic acid-treated rats when compared with vehicle-treated ischemic rats. The treatment with indole-pyruvic acid did not produce any improving effects in a test assessing short-term impairments after transient ischemia (motor test score at 24 h and 48 h post-ischemia). Furthermore, no significant effects of indole-pyruvic acid treatment were found on performance in water T-maze studied at 7 and 14 days post-ischemia. The opposite effects of indole pyruvic acid on ischemic lesion in different brain regions may be related to its multiple neurochemical actions in the brain. The protective effect of indole pyruvic acid on ischemic damage in striatum may be due to its conversion into kynurenic acid, a broad spectrum glutamate receptor antagonist. At hippocampal level, where glutamate receptor antagonists have been proved ineffective in the present lesion model, indole-pyruvic acid-induced changes in monoamine availability may lead to a worsening of neuronal damage.


1993 - Indole-pyruvic acid, a tryptophan ketoanalogue, antagonizes the endocrine but not the behavioral effects of repeated stress in a model of depression [Articolo su rivista]
Biagini, Giuseppe; MERLO PICH, Emilio; Carani, Cesare; Marrama, Paolo; J. A., Gustafsson; K., Fuxe; Agnati, Luigi Francesco
abstract

Increased glucocorticoid secretion is frequent in mood disorders and is normalized by long-term antidepressant therapy. Many antidepressants act by increasing central serotonin transmission. We investigated the effects of a serotonin precursor, indole-pyruvic acid (IPA), in an animal model of depression based on repeated exposure to unpredictable stress. Rats were divided in groups, and IPA (20 mg/kg), the tricyclic antidepressant imipramine (IMI) (5 mg/kg), or vehicle was administered daily during 3 weeks of repeated exposure to various stressors according to the procedure described by Katz et al [Katz RJ, Roth KA, Carroll BJ (1981): Neurosci Biobehav Rev 5:247-251]. After treatment, rats were evaluated for stress-induced exploratory behavior and killed 24 hr later. Serum corticosterone levels and glucocorticoid receptor (GR) immunoreactivity (IR) in the nuclei of neurons located in the hippocampal subregion CA1 were also measured. Rats exposed to repeated stress showed a lower exploratory behavior score (p < 0.01), higher basal corticosterone levels (p < 0.01), and stronger GR IR in the hippocampus (p < 0.05) than control rats. All of these effects were antagonized by IMI treatment. IPA administration did not affect the behavioral response induced by repeated stress (p < 0.01) but normalized serum corticosterone levels. In addition, IPA treatment produced a decrease in GR IR (p < 0.05 versus control group) that was not modified by exposure to repeated stress. Differences in the drug effects suggest that (1) the dose of IPA used for treatment was not sufficient to produce behavior effects; (2) a side product of IPA in vivo transformation, kynurenic acid, may have affected glutamate transmission, interacting with the behavioral outcomes; (3) the serotonin precursor IPA and the uptake blocker IMI may have produced different adaptive changes in hippocampal serotonin transmission, as indicated by nuclear GR IR measurements.


1993 - L-deprenyl increases GFAP immunoreactivity selectively in activated astrocytes in rat brain [Articolo su rivista]
Biagini, Giuseppe; Zoli, Michele; K., Fuxe; Agnati, Luigi Francesco
abstract

L-DEPRENYL is a selective inhibitor of monoamine oxidase (MAO) B, an enzyme predominantly localized in astrocytes. We have investigated the effect of treatment with L-deprenyl (0.25 mg kg-1 day-1) on glial fibrillary acidic protein (GFAP) immunoreactivity (IR) after lesioning the rat striatum with an injection cannula. No effect of drug treatment on GFAP IR was found in unlesioned striata. A significant increase (p < 0.01 vs saline treated rats) in GFAP IR was found in the tissue surrounding the lesion in striata of rats treated with L-deprenyl for 4 days after the lesion. When post-treated for 42 days, however, L-deprenyl no longer increased GFAP IR although reactive astrocytes were still present in the lesioned area. These results suggest that L-deprenyl can enhance the activation of astrocytes during a critical time-period following a striatal injury.


1992 - Brain aging and neuronal plasticity [Articolo su rivista]
Agnati, Luigi Francesco; Benfenati, Fabio; V., Solfrini; Biagini, Giuseppe; K., Fuxe; D., Guidolin; Carani, Cesare; Zini, Isabella
abstract

In the present paper three aspects of the aging processes and their physiopathological implications will be presented: neuronal death, the information handling capabilityof neurons, and the processes of interneuronal communication.


1992 - Central mechanisms subserving the impaired growth hormone secretion induced by persistent blockade of NMDA receptors in immature male rats. [Articolo su rivista]
L., Cocilovo; V., DE GENNARO COLONNA; Zoli, Michele; Biagini, Giuseppe; B. P., Settembrini; E. E., Muller; D., Cocchi
abstract

Recently, we have reported in immature female rats that short-term blockade of glutamate receptors of the N-methyl-D-aspartic acid (NMDA) subtype by the noncompetitive antagonist MK-801 induced a reduction of growth rate, basal and stimulated growth hormone (GH) release and plasma somatomedin C levels. In the present study, we investigated in immature male rats the mechanism(s) through which agonists and antagonists of glutamate receptors affect GH secretion. In 21-day-old male rats, administration of MK-801 (0.2 mg/kg i.p. b.i.d.) for 10 days induced a significant impairment of growth rate, which was unrelated to a significant reduction of food intake. GH secretion from anterior pituitary fragments of MK-801-treated rats was not significantly reduced under basal conditions but was significantly less under stimulation by 40 mM K+. Incubation of dispersed pituitary cells of 31-day-old rats with N-methyl-aspartic acid (1 and 100-mu-M), alone or associated with MK-801 (1-mu-M) did not change GH secretion. Semi quantitative densitometric analysis of hypothalami of MK-801-treated rats evidenced a clearcut decrease in the intensity of GHRH-like immuno-reactivity (LI) staining in the median eminence (ME), whereas no differnece was observed in the ME-somatostatin (SS)-LI. Finally, GHRH mRNA but not SS-mRNA, evaluated by slot-blot hybridization, was reduced in the hypothalamus of MK-801-treated rats. These and our previous data would demonstrate that NMDA glutamate receptors play an important role in the neuroendocrine control of GH secretion in the rat, and suggest an action mediated by GHRH-secreting neurons.


1992 - Feeding and drinking responses to neuropeptide Y injections in the paraventricular hypothalamic nucleus of aged rats [Articolo su rivista]
MERLO PICH, Emilio; B., Messori; Zoli, Michele; F., Ferraguti; Marrama, Paolo; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

Neuropeptide Y (NPY), a peptide of the pancreatic polypeptide family, exerts a potent stimulatory action on eating when injected into the paraventricular hypothalamic nucleus (PVN) in rats. Several NPY-containing systems are altered with advancing age, and aged rodents develop anorexia and a modified daily cycle pattern of feeding. These findings suggest that a relationship may exist between the aging-related anorexia and the reduced function of NPY-containing systems projecting to the PVN. In the present study eating and drinking behavior in satiated or fasted young (3 months) and aged (24 months) rats have been investigated over 22 h after NPY injection into the PVN. The levels of NPY immunoreactivity (IR) in PVN were also evaluated by means of semiquantitative immunocytochemistry. NPY injections into PVN increased food and water consumption in both young and aged satiated rats 30, 90 and 240 min after injection. However, the feeding and drinking responses elicited by 0.05, 0.10 and 1.0 nmol of NPY were significantly attenuated in the aged rats when compared to young rats. In aged rats, 24 h of food and water deprivation produced significant increase of food consumption measured at 30, 90 min and 22 h, which was equivalent to that induced by 1:0 nmol NPY injection. Administration of 1.0 nmol NPY in PVN did not further increase the 24 h deprivation effect on feeding in both groups of rats, but enhanced drinking in deprived young rats. This effect was not present in aged rats. In addition, aged rats showed a stronger response to 24 h deprivation than to 1.0 nmol NPY administration. These results and the severe reduction of NPY IR levels in PVN nerve terminals of aged rats suggest that NPY deficiency may be a factor responsible for anorexia in the aged.


1992 - NEURONAL PLASTICITY AND AGING PROCESSES IN THE FRAME OF THE RED QUEEN THEORY [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; Biagini, Giuseppe; Fuxe, K.
abstract

On the basis of the morphofunctional evidence obtained in old brains of humans and mammals the present hypothesis has been introduced. This hypothesis states that neuronal plasticity can be used either to compensate for neuronal degeneration or to store new information. Thus, in pathological ageing the marked rate of degeneration has fully exhausted the already reduced plasticity capability of neural networks. In this way marked impairments of memory trace formation take place in pathological ageing conditions such as Alzheimer's disease. The essence of this hypothesis is that a competition for the available plasticity exists between the compensatory responses to ageing-induced degeneration and the processes necessary for memory trace formation. We have called this hypothesis the 'Red Queen Theory', an analogy borrowed from Lewis Carroll's book Through the Looking Glass. Thus, in ageing, processes responsible for plasticity must be forced to run at the highest possible rate to maintain the morphofunctional substrate of the existing networks as well as to allow the formation of new memory traces.


1992 - Siagoside selectively attenuates morphological and functional striatal impairments induced by transient forebrain ischemia in rats [Articolo su rivista]
MERLO PICH, Emilio; R., Grimaldi; Zoli, Michele; Biagini, Giuseppe; V., Solfrini; G., Toffano; K., Fuxe; Agnati, Luigi Francesco
abstract

Background and Purpose: Transient forebrain ischemia induced in rats by the four-vessel occlusion method is known to produce severe neural damage in the hippocampus and striatum and a behavioral syndrome the major symptom of which is a working memory deficit. Recent evidence suggests that monosialogangliosides can ameliorate postischemic symptoms. Our purpose was to study the effect of siagoside, the inner ester of GM1 ganglioside, on some behavioral and morphological impairments induced by four-vessel occlusion in rats. Methods: Rats were injected daily with 5 mg/kg i.p. siagoside starting 4 hours after the cerebral ischemia. After 14 days the rats were tested for working memory in a water T maze or scored for apomorphine-induced stereotypy. The rats were killed 21 days after the cerebral ischemia. Histological and computer-assisted morphometric analyses were performed on cresyl violet-stained brain sections, which were graded according to a neuropathologic score, and on sections stained with a monoclonal antiserum against dopamine and cyclic adenosine-3',5'-monophosphate-regulated phosphoprotein, a marker for striatal dopaminoceptive neurons. Results: Siagoside treatment reduced the stereotypy score induced by low doses of apomorphine and the extent of striatal lesions but did not affect the working memory deficit or the extent of hippocampal lesions. Conclusion: Daily siagoside treatment after acute cerebral ischemia attenuates some morphological and functional deficits related to striatal damage. These effects can be interpreted as a selective protective action on striatal neural populations or as a modulatory action on neural systems involved in striatal control. These data are consistent with preliminary clinical reports showing that monosialogangliosides enhance motor recovery after acute ischemic stroke.


1991 - A NEW MODEL OF FOCAL BRAIN ISCHEMIA BASED ON THE INTRACEREBRAL INJECTION OF ENDOTHELIN-1 [Articolo su rivista]
Agnati, Luigi Francesco; Zoli, Michele; Kurosawa, M; Benfenati, Fabio; Biagini, Giuseppe; Zini, Isabella; Hallstrom, A; Ungerstedt, U; Toffano, G; Fuxe, K.
abstract

Endothelin-1 and its receptors are widely distributed in the brain of rodents and humans. In view of its potent and long-lasting vasoconstrictor activity, a role of endothelin-1 has been proposed in brain ischemia. In the present paper, the local ijnection of endothelin-1 was utilized to induce ischemia in rat striatum. An evaluation of the rostrocaudal extension of the lesion is reported. By using intracerebral microdialysis, a marked increase of lactate and dopamine, but not glutamate, was observed in this region upon endothelin-1 administration. Moreover, preliminary data reported show a protective effect of ganglioside treatment on endothelin-1 lesion of rat striatum. The characteristics of the present model of brain ischemia are discussed in comparison with well characterized models, such as the Pulsinelli's four vessel occlusion and the middle cerebral artery occlusion.


1991 - Corticosterone treatment counteracts lesions induced by neonatal treatment with monosodium glutamate in the mediobasal hypothalamus of the male rat [Articolo su rivista]
Zoli, Michele; F., Ferraguti; Biagini, Giuseppe; A., Cintra; K., Fuxe; Agnati, Luigi Francesco
abstract

The effects of glucocorticoids on monosodium glutamate-induced neurotoxicity in the neonatal basal hypothalamus were studied by means of semiquantitative immunocytochemistry for tyrosine hydroxylase, growth hormone releasing factor and luteinizing hormone releasing hormone. Neonatal monosodium glutamate treatment induced a marked decrease in tyrosine hydroxylase immunoreactive neurons in the arcuate nucleus and growth hormone releasing factor immunoreactive nerve terminals in the median eminence. These effects were significantly antagonized by the coadministration of corticosterone. Corticosterone alone had no effect on the parameters studied. No significant change in luteinizing hormone releasing hormone immunoreactivity in the median eminence was detected after any treatment. These results demonstrate that corticosterone, possibly acting via type II corticosterone receptors which are highly enriched in the arcuate neurons, can exert a protective action on glutamate-induced neurotoxicity.


1991 - PAIN, ANALGESIA, AND STRESS - AN INTEGRATED VIEW [Articolo su rivista]
Agnati, Luigi Francesco; Tiengo, M; Ferraguti, F; Biagini, Giuseppe; Benfenati, Fabio; Benedetti, C; Rigoli, M; Fuxe, K.
abstract

The different theories on the neuroanatomical substrate of pain have been revised in the frame of new concepts on the intercellular communication in the central nervous system. In fact, it has recently been proposed that two kinds of electrochemical transmission exist in the brain: the first one, called wiring transmission (WT), uses neuronal chains (neuronal plasma membranes and synaptic contacts), whereas the second one, called volume transmission (VT), uses the extracellular fluid as physical substrate. The old concept of a separate system of afferents and central cells that constitute the pain mechanism is no more longer tenable. To reach a better understanding of the psychophysiological basis of pain, we should consider a view where WT and VT cooperate within neuronal systems functionally affected by the pervading modulatory action of endocrine signals.


1990 - EFFECTS OF CENTRALLY ADMINISTERED CLONIDINE AND NEUROPEPTIDE-Y ON ARTERIAL BLOOD-PRESSURE IN THE RAT AFTER TRANSIENT FOREBRAIN ISCHEMIA [Articolo su rivista]
Grimaldi, R; Zini, Isabella; Biagini, Giuseppe; Toffano, G; Agnati, Luigi Francesco
abstract

In the present study the response of central cardiovascular system to the hypotensive drugs clonidine neuropeptide Y (NPY) was evaluated in ischemic rats at various time intervals after reperfusion.


1990 - Effects of indole-pyruvic acid on sleep and food intake in the rat [Articolo su rivista]
MERLO PICH, Emilio; V., Solfrini; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

Indole-pyruvic acid was studied for its short- and long-term effects on electroencephalographic sleep and on food intake in rats implanted with cortical and muscular electrodes. Following a single injection, indole-pyruvic acid (10-50 mg kg-1 i.p.) reduced by 16-23 min (range) the latency of the first slow-wave episode in a dose-related fashion and produced a significant increase in slow-wave sleep time (12-40%) in doses of 10-30 mg kg-1. Rapid eye movement sleep latency and rapid eye movement sleep time were increased (by 23-37 min) and reduced (57-71%) respectively. The effects of indole-pyruvic acid on slow-wave sleep time were still present after 3, 7 and 14 days of chronic administration (10 mg kg-1 day-1), whereas tolerance to the effect of indole-pyruvic acid on rapid eye movement sleep was observed. Daily food consumption was reduced (20-28%) by acute administration of indole-pyruvic acid (15-30 mg kg-1 i.p.), but tolerance developed after 5 days of repeated injections. These findings are in accordance with previous evidence suggesting that indole-pyruvic acid effects may be related to the activation of central serotonin neurons, which are involved in the inhibitory control of sleep and food intake.


1990 - Evidence for a role of neosynthetized putrescine in the increase of glial fibrillary acid protein immunoreactivity induced by a mechanical lesion in the rat brain [Articolo su rivista]
Zini, Isabella; Zoli, Michele; R., Grimaldi; MERLO PICH, Emilio; Biagini, Giuseppe; K., Fuxe; Agnati, Luigi Francesco
abstract

The effect of alpha-difluoromethylornithine (alpha-DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) the rate limiting enzyme of polyamine biosynthesis, was studied on the astroglial reaction in a model of mechanical brain injury. alpha-DFMO markedly decreased the astroglial activation induced by the microdialysis probe implantation in the striatum of the male rat, as studied by glial fibrillary acidic protein (GFAP) immunocytochemistry. This response was restored by putrescine (20 nmol/ml) administered via the microdialysis probe. These results suggest that the astroglial reaction and the polyamine biosynthesis activation induced by a localized mechanical lesion are causally linked phenomena.


1990 - Long-lasting reduction of glucocorticoid receptor immunoreactivity in the hippocampal field CA1 but not in the dentate gyrus after neonatal treatment with corticosterone in the rat [Articolo su rivista]
Zoli, Michele; Agnati, Luigi Francesco; K., Fuxe; F., Ferraguti; Biagini, Giuseppe; A., Cintra; Ja, Gustafsson
abstract

The aim of the present immunohistochemical study was to investigate the effects of neonatal treatment with corticosterone on the long-term regulation of glucocorticoid receptor in the hippocampus.