Carveol Attenuates Seizure Severity and Neuroinflammation in Pentylenetetrazole-Kindled Epileptic Rats by Regulating the Nrf2 Signaling Pathway.

Epilepsy is a neurodegenerative brain disorder characterized by recurrent seizure attacks. Numerous studies have suggested a strong correlation between oxidative stress and neuroinflammation in several neurodegenerative disorders including epilepsy. This study is aimed at investigating the neuroprotective effects of the natural compound carveol against pentylenetetrazole- (PTZ-) induced kindling and seizure model. Two different doses of carveol (10 mg/kg and 20 mg/kg) were administered to male rats to determine the effects and the effective dose of carveol and to further demonstrate the mechanism of action of nuclear factor E2-related factor (Nrf2) in PTZ-induced kindling model. Our results demonstrated reduced levels of innate antioxidants such as superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), and glutathione (GSH), associated with elevated lipid peroxidation (LPO) and inflammatory cytokines level such as tumor necrosis factor-alpha (TNF-α), and mediators like cyclooxygenase (COX-2) and nuclear factor kappa B (NFκB). These detrimental effects exacerbated oxidative stress and provoked a marked neuronal alteration in the cortex and hippocampus of PTZ-intoxicated animals that were associated with upregulated Nrf2 gene expression. Furthermore, carveol treatment positively modulated the antioxidant gene Nrf2 and its downstream target HO-1. To further investigate the role of Nrf2, an inhibitor of Nrf2 called all-trans retinoic acid (ATRA) was used, which further exacerbated PTZ toxicity. Moreover, carveol treatment induced cholinergic system activation by mitigating acetylcholinesterase level which is further linked to attenuated neuroinflammatory cascade. The extent of blood-brain barrier disruption was evaluated based on vascular endothelial growth factor (VEGF) expression. Taken together, our findings suggest that carveol acts as an Nrf2 activator and therefore induces downstream antioxidants and mitigates inflammatory insults through multiple pathways. This eventually alleviates PTZ-induced neuroinflammation and neurodegeneration.

Neuroendocrine changes in the hypothalamic-neurohypophysial system in the Wistar audiogenic rat (WAR) strain submitted to audiogenic kindling.

The Wistar audiogenic rat (WAR) strain is used as an animal model of epilepsy, which when submitted to acute acoustic stimulus presents tonic-clonic seizures, mainly dependent on brainstem (mesencephalic) structures. However, when WARs are exposed to chronic acoustic stimuli (audiogenic kindling-AK), they usually present tonic-clonic seizures, followed by limbic seizures, after recruitment of forebrain structures such as the cortex, hippocampus and amygdala. Although some studies have reported that hypothalamic-hypophysis function is also altered in WAR through modulating vasopressin (AVP) and oxytocin (OXT) secretion, the role of these neuropeptides in epilepsy still is controversial. We analyzed the impact of AK and consequent activation of mesencephalic neurocircuits and the recruitment of forebrain limbic (LiR) sites on the hypothalamic-neurohypophysial system and expression of Avpr1a and Oxtr in these structures. At the end of the AK protocol, nine out of 18 WARs presented LiR. Increases in both plasma vasopressin and oxytocin levels were observed in WAR when compared to Wistar rats. These results were correlated with an increase in the expressions of heteronuclear (hn) and messenger (m) RNA for Oxt in the paraventricular nucleus (PVN) in WARs submitted to AK that presented LiR. In the paraventricular nucleus, the hnAvp and mAvp expressions increased in WARs with and without LiR, respectively. There were no significant differences in Avp and Oxt expression in supraoptic nuclei (SON). Also, there was a reduction in the Avpr1a expression in the central nucleus of the amygdala and frontal lobe in the WAR strain. In the inferior colliculus, Avpr1a expression was lower in WARs after AK, especially those without LiR. Our results indicate that both AK and LiR in WARs lead to changes in the hypothalamic-neurohypophysial system and its receptors, providing a new molecular basis to better understaind epilepsy.

Ultrastructural GABA immunogold labeling in the substantia nigra pars reticulata of kindled genetic absence epilepsy rats.

Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is a well-known animal model of absence epilepsy and they are resistant to electrical kindling stimulations. The present study aimed to examine possible differences in gamma-aminobutyric acid (GABA) levels and synapse counts in the substantia nigra pars reticulata anterior (SNRa) and posterior (SNRp) regions between GAERS and Wistar rats receiving kindling stimulations. Animals in the kindling group either received six stimulations in the amygdala and had grade 2 seizures or they were kindled, having grade five seizures. Rats were decapitated one hour after the last stimulation. SNR regions were obtained after vibratome sectioning of the brain tissue. GABA immunoreactivity was detected by immunogold method and synapses were counted. Sections were observed by transmission electron microscope and analyzed by Image J program. GABA density in the SNRa region of fully kindled GAERS and Wistar groups increased significantly compared to that of their corresponding grade 2 groups. The number of synapses increased significantly in kindled and grade 2 GAERS groups, compared to kindled and grade 2 Wistar groups, respectively, in the SNRa region. GABA density in the SNRp region of kindled GAERS group increased significantly compared to that of GAERS grade 2 group. In the SNRp region, both kindled and grade 2 GAERS groups were found to have increased number of synapses compared to that of GAERS control group. We concluded that both SNRa and SNRp regions may be important in modulating resistance of GAERS to kindling stimulations.

Characterization of kindled VGAT-Cre mice as a new animal model of temporal lobe epilepsy.

OBJECTIVE: Development of novel therapies for temporal lobe epilepsy is hindered by a lack of models suitable for drug screening. While testing the hypothesis that "inhibiting inhibitory neurons" was sufficient to induce seizures, it was discovered that a mild electrical kindling protocol of VGAT-Cre mice led to spontaneous motor and electrographic seizures. This study characterizes these seizures and investigates the mechanism. METHODS: Mice were implanted with electroencephalographic (EEG) headsets that included a stimulating electrode in the hippocampus before being electrically kindled. Seizures were evaluated by review of EEG recordings and behavior. γ-Aminobutyric acidergic (GABAergic) neurotransmission was evaluated by quantitative polymerase chain reaction, immunocytochemistry, Western blot, and electrophysiology. RESULTS: Electrical kindling of VGAT-Cre mice induces spontaneous recurring seizures after a short latency (6 days). Seizures occur 1-2 times per day in both male and female mice, with only minimal neuronal death. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. The insertion of Cre disrupts the expression of VGAT mRNA and protein, and impairs GABAergic synaptic transmission in the hippocampus. SIGNIFICANCE: Kindled VGAT-Cre mice can be used to study the mechanisms involved in epileptogenesis and may be useful for screening novel therapeutics.

Pentylenetetrazol kindling induces cortical astrocytosis and increased expression of extracellular matrix molecules in mice.

Although epilepsy is one of the most common chronic neurological disorders with a prevalence of approximately 1.0 %, the underlying pathophysiology remains to be elucidated. Understanding the molecular and cellular mechanisms involved in the development of epilepsy is important for the development of appropriate therapeutic strategy. In this study, we investigated the effects of status epilepticus on astrocytes, microglia, and extracellular matrix (ECM) molecules in the somatosensory cortex and piriform cortex of mice. Activation of astrocytes was observed in many cortices except the retrosplenial granular cortex after pentylenetetrazol (PTZ)-induced kindling in mice. Activated astrocytes in the cortex were found in layers 1-3 but not in layers 4-6. In the somatosensory and piriform cortices, no change was observed in the number of parvalbumin (PV)-positive neurons and PV-positive neurons covered with perineuronal nets. However, the amount of ECM in the extracellular space increased. The expression of VGLUT1- and GAD67-positive synapses also increased. Thus, in the PTZ-kindling epilepsy mice model, an increase in the number of ECM molecules and activation of astrocytes were observed in the somatosensory cortex and piriform cortex. These results indicate that PTZ-induced seizures affect not only the hippocampus but also other cortical areas. Our study findings may help to develop new therapeutic approaches to prevent seizures or their sequelae.

The role of dopamine D2-like receptors in a "depotentiation-like effect" of deep brain stimulation in kindled rats.

The mechanisms involved in the anti-seizure effects of low-frequency stimulation (LFS) have not been completely determined. However, Gi-protein-coupled receptors, including D2-like receptors, may have a role in mediating these effects. In the present study, the role of D2-like receptors in LFS' anti-seizure action was investigated. Rats were kindled with semi-rapid (6 stimulations per day), electrical stimulation of the hippocampal CA1 area. In LFS-treated groups, subjects received four trials of LFS at 5 min, 6 h, 24 h, and 30 h following the last kindling stimulation. Each LFS set occurred at 5 min intervals, and consisted of 4 trains. Each train contained 200, 0/1 ms long, monophasic square wave pulses at 1 Hz. Haloperidol (D2-like receptors antagonist, 2 µm) and/or bromocriptine (D2-like receptors agonist 2 µg/µlit) were microinjected into the lateral ventricle immediately after the last kindling, before applying LFS. Obtained results showed that applying LFS in fully-kindled subjects led to a depotentiation-like decrease in kindling-induced potentiation and reduced the amplitude and rise slope of excitatory and inhibitory post-synaptic currents in whole-cell recordings from CA1 pyramidal neurons. In addition, LFS restored the kindling-induced, spatial learning and memory impairments in the Barnes maze test. A D2-like receptor antagonist inhibited these effects of LFS, while a D2-like receptor agonist mimicked these effects. In conclusion, a depotentiation-like mechanism may be involved in restoring LFS' effects on learning and memory, and synaptic plasticity. These effects depend on D2-like receptors activity.

Alteration of Extracellular Matrix Molecules and Perineuronal Nets in the Hippocampus of Pentylenetetrazol-Kindled Mice.

The pathophysiological processes leading to epilepsy are poorly understood. Understanding the molecular and cellular mechanisms involved in the onset of epilepsy is crucial for drug development. Epileptogenicity is thought to be associated with changes in synaptic plasticity; however, whether extracellular matrix molecules-known regulators of synaptic plasticity-are altered during epileptogenesis is unknown. To test this, we used a pentylenetetrazole- (PTZ-) kindling model mouse to investigate changes to hippocampal parvalbumin- (PV-) positive neurons, extracellular matrix molecules, and perineuronal nets (PNNs) after the last kindled seizure. We found an increase in Wisteria floribunda agglutinin- (WFA-) and Cat-315-positive PNNs and a decrease in PV-positive neurons not surrounded by PNNs, in the hippocampus of PTZ-kindled mice compared to control mice. Furthermore, the expression of WFA- and Cat-315-positive molecules increased in the extracellular space of PTZ-kindled mice. In addition, consistent with previous studies, astrocytes were activated in PTZ-kindled mice. We propose that the increase in PNNs after kindling decreases neuroplasticity in the hippocampus and helps maintain the neural circuit for recurrent seizures. This study shows that possibility of changes in extracellular matrix molecules due to astrocyte activation is associated with epilepticus in PTZ-kindled mice.

Increased expression of DOC2A in human and rat temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of intractable epilepsy. Currently, the molecular mechanisms underlying epileptogenesis in TLE remain elusive; however, synaptic transmission may play an important role in the pathogenesis of epilepsy. Synaptic transmission is regulated by diverse mechanisms, including presynaptic modulators of synaptic vesicle formation and release, modulators of neurotransmission and distinct Ca2+ sensors. DOC2A, a novel Ca2+ sensor, can regulate spontaneous synaptic transmission and has been implicated in Ca2+-dependent neurotransmitter release. In this study, we demonstrate for the first time that DOC2A expression is significantly increased in human TLE and in two different rat models of TLE (pilocarpine- and kindling-induced) compared to the control groups. Localization of DOC2A in the human TLE patients and pilocarpine post-SE rat model was observed in neurons but not in astrocytes; DOC2A was also concentrated at the presynaptic terminals and colocalized with VMAT2. Our results suggest that the abnormal protein expression of DOC2A in epileptic brain tissue may play an important role in epilepsy.

Sustained somatostatin gene expression reverses kindling-induced increases in the number of dividing Type-1 neural stem cells in the hippocampi of behaviorally responsive rats.

Neurogenesis persists throughout life in the hippocampi of all mammals, including humans. In the healthy hippocampus, relatively quiescent Type-1 neural stem cells (NSCs) can give rise to more proliferative Type-2a neural progenitor cells (NPCs), which generate neuronal-committed Type-2b NPCs that mature into Type-3 neuroblasts. Many Type-3 neuroblasts survive and mature into functionally integrated granule neurons over several weeks. In kindling models of epilepsy, neurogenesis is drastically upregulated and many new neurons form aberrant connections that could support epileptogenesis and/or seizures. We have shown that sustained vector-mediated hippocampal somatostatin (SST) expression can both block epileptogenesis and reverse seizure susceptibility in fully kindled rats. Here we test whether adeno-associated virus (AAV) vector-mediated sustained SST expression modulates hippocampal neurogenesis and microglial activation in fully kindled rats. We found significantly more dividing Type-1 NSCs and a corresponding increased number of surviving new neurons in the hippocampi of kindled versus sham-kindled rats. Increased numbers of activated microglia were found in the granule cell layer and hilus of kindled rats at both time points. After intrahippocampal injection with either eGFP or SST-eGFP vector, we found similar numbers of dividing Type-1 NSCs and -2 NPCs and surviving BrdU+ neurons and glia in the hippocampi of kindled rats. Upon observed variability in responses to SST-eGFP (2/4 rats exhibited Grade 0 seizures in the test session), we conducted an additional experiment. We found significantly fewer dividing Type-1 NSCs in the hippocampi of SST-eGFP vector-treated responder rats (5/13 rats) relative to SST-eGFP vector-treated non-responders and eGFP vector-treated controls that exhibited high-grade seizures on the test session. The number of activated microglia was upregulated in the GCL and hilus of kindled rats, regardless of vector treatment. These data support the hypothesis that sustained SST expression exerts antiepileptic effects potentially through normalization of neurogenesis and suggests that abnormally high proliferating Type-1 NSC numbers may be a cellular mechanism of epilepsy.

Identification of 2-(2'-fluoro-[1,1'-biphenyl]-2-yl)acetamide as a Sodium Valproate-like broad spectrum anti-epileptic drug candidate.

By further optimizing compound A [2'-fluoro-N-methyl-[1,1'-biphenyl]-2-sulfonamide], we identified DSP-0565 [2-(2'-fluoro-[1,1'-biphenyl]-2-yl)acetamide, 17a] as a strong, broad-spectrum anti-epileptic drug (AED) candidate. Our efforts mainly focused on finding an alternative polar group for the sulfonamide in order to improve ADME profile of compound A including good metabolic stability and no reactive metabolic production. This led to the identification of biphenyl acetamide as a new scaffold for development of broad-spectrum AED candidates. DSP-0565 showed anti-convulsant activity in various models (scPTZ, MES, 6 Hz and amygdala kindling) with good safety margin, and was therefore selected as a clinical candidate.

Astrocytic mobilization of glutathione peroxidase-1 contributes to the protective potential against cocaine kindling behaviors in mice via activation of JAK2/STAT3 signaling.

Compelling evidence indicates that oxidative stress contributes to cocaine neurotoxicity. The present study was performed to elucidate the role of the glutathione peroxidase-1 (GPx-1) in cocaine-induced kindling (convulsive) behaviors in mice. Cocaine-induced convulsive behaviors significantly increased GPx-1, p-IkB, and p-JAK2/STAT3 expression, and oxidative burdens in the hippocampus of mice. There was no significant difference in cocaine-induced p-IkB expression between non-transgenic (non-TG) and GPx-1 overexpressing transgenic (GPx-1 TG) mice, but significant differences were observed in cocaine-induced p-JAK2/STAT3 expression and oxidative stress between non-TG and GPx-1 TG mice. Cocaine-induced glial fibrillary acidic protein (GFAP)-labeled astrocytic level was significantly higher in the hippocampus of GPx-1 TG mice. Triple-labeling immunocytochemistry indicated that GPx-1-, p-STAT3-, and GFAP-immunoreactivities were co-localized in the same cells. AG490, a JAK2/STAT3 inhibitor, but not pyrrolidone dithiocarbamate, an NFκB inhibitor, significantly counteracted GPx-1-mediated protective potentials (i.e., anticonvulsant-, antioxidant-, antiapoptotic-effects). Genetic overexpression of GPx-1 significantly attenuated proliferation of Iba-1-labeled microglia induced by cocaine in mice. However, AG490 or astrocytic inhibition (by GFAP antisense oligonucleotide and α-aminoadipate) significantly increased Iba-1-labeled microglial activity and M1 phenotype microglial mRNA levels, reflecting that proinflammatory potentials were mediated by AG490 or astrocytic inhibition. This microglial activation was less pronounced in GPx-1 TG than in non-TG mice. Furthermore, either AG490 or astrocytic inhibition significantly counteracted GPx-1-mediated protective potentials. Therefore, our results suggest that astrocytic modulation between GPx-1 and JAK2/STAT3 might be one of the underlying mechanisms for protecting against convulsive neurotoxicity induced by cocaine.

Pentylenetetrazole-Induced Kindling Mouse Model.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

Genetic Modulation of HSPA1A Accelerates Kindling Progression and Exerts Pro-convulsant Effects.

Strong evidence exists that Toll-like receptor (TLR)-mediated effects on microglia functional states can promote ictogenesis and epileptogenesis. So far, research has focused on the role of high-mobility group box protein 1 as an activator of TLRs. However, the development of targeting strategies might need to consider a role of additional receptor ligands. Considering the fact that heat shock protein A1 (hsp70) has been confirmed as a TLR 2 and 4 ligand, we have explored the consequences of its overexpression in a mouse kindling paradigm. The genetic modulation enhanced seizure susceptibility with lowered seizure thresholds prior to kindling. In contrast to wildtype (WT) mice, HSPA1A transgenic (TG) mice exhibited generalized seizures very early during the kindling paradigm. Along with an increased seizure severity, seizure duration proved to be prolonged in TG mice during this phase. Toward the end of the stimulation phase seizure parameters of WT mice reached comparable levels. However, a difference between genotypes was still evident when comparing seizure parameters during the post-kindling threshold determination. Surprisingly, HSPA1A overexpression did not affect microglia activation in the hippocampus. In conclusion, the findings demonstrate that hsp70 can exert pro-convulsant effects promoting ictogenesis in naïve animals. The pronounced impact on the response to subsequent stimulations gives first evidence that genetic HSPA1A upregulation may also contribute to epileptogenesis. Thus, strategies inhibiting hsp70 or its expression might be of interest for prevention of seizures and epilepsy. However, conclusions about a putative pro-epileptogenic effect of hsp70 require further investigations in models with development of spontaneous recurrent seizures.

Neuropathological profile of the pentylenetetrazol (PTZ) kindling model.

Introduction: There are three phases of seizure developing in pentylenetetrazol (PTZ)-induced kindling animal model: (i) pre-kindling phase; (ii) kindling phase or after animals are fully kindled; (iii) post-kindling phase with non-provoked spontaneous recurrent seizures. The aims of this review were to summarize the progress over time of the electroencephalographic features and neuropathological alterations in kindled PTZ treated animals. Materials and methods: Keywords relevant to PTZ kindling were used to a guide a literature search on Pubmed, Medline and Cochrane Library. Results: Clonic seizures induced PTZ at kindling phase led to a strong c-Fos expression in the hippocampus. Although, decline hippocampal neuron and metabolism disturbances were detected at pre-kindlig phase. Repeated PTZ induced seizures alter the GABA-mediated inhibition and glutamate-mediated excitation, which may contribute to increased seizure susceptibility. Similar to chemical animal models such as the pilocarpine and the kainic acid models, mossy fiber sprouting, hippocampal damage, and glucose hypometabolism had been seen after PTZ induced seizures. Conclusion: PTZ kindling model may improve understanding of the seizures development provided that the differences existing between the phases of kindling model are taken into account.

Piperine-loaded chitosan-STPP nanoparticles reduce neuronal loss and astrocytes activation in chemical kindling model of epilepsy.

Recent evidence suggests that encapsulation of hydrophobic drugs in biodegradable polymers opens a new horizon in nanomedicine filed. Piperine, a main alkaloid form of black pepper possesses potent anticonvulsant activity. However, the low water solubility of piperine has limited its clinical application. In this study, piperine was loaded on chitosan-sodium tripolyphosphate nanoparticles (CS-STPP NPs) and the effect of piperine NPs on seizures behavior and astrocytes activation was assessed in pentylentetrazol (PTZ)-induced kindling model. Animals have received the daily injection of free piperine or piperine NPs at doses of 5 or 10mg/kg, 10days before PTZ injections and their intraperitoneally (i.p) administration continued until the last PTZ injection. The neuroprotective effects of piperine NPs were evaluated using nissl staining and immunostaining against NeuN. Astrocytes activation was examined by GFAP immunostaining. Behavioral data showed that piperine NPs have inhibited the development of seizure parameters compared to the free piperine groups. In addition, the levels of cell loss and astrocytes activation were reduced in piperine NPs groups. In conclusion, these data suggest that piperine NPs enhance the neuroprotection and ameliorate the astrocytes activation in chemical kindling model of epilepsy. This may provide an effective therapeutic strategy for the treatment of epilepsy disorder.

Regulation of kindling epileptogenesis by hippocampal Toll-like receptors 2.

This study examined whether Toll-like receptors 2 (TLR2) contribute to rapid kindling epileptogenesis. A TLR2 agonist, lipoteichoic acid (LTA), LTA antibody (LTA-A), or normal saline (control) was administered daily over 3 consecutive days, unilaterally into ventral hippocampus of adult male Wistar rats. Thirty minutes after the last injection, the animals were subjected to a rapid kindling procedure. The ictogenesis was gauged by comparing afterdischarge threshold (ADT) and afterdischarge duration (ADD) before the treatments, after the treatments prior to kindling, and 24 h after kindling. Kindling progression and retention were analyzed using video recording. The results showed that before kindling, LTA produced an ADT reduction. Neither LTA nor LTA-A affected baseline ADD. On kindling progression, LTA accelerated occurrence of generalized seizures, whereas LTA-A delayed this effect. Treatment with LTA-A reduced the number of secondary generalized complex partial seizures. Twenty-four hours after kindling, the rats of both the saline and LTA groups showed increased hippocampal excitability as compared with prekindling parameters. Administration of LTA-A prevented kindling-induced increase of hippocampal excitability. Immunostaining revealed that LTA-A attenuated the inflammatory response produced by seizures. These findings suggest that the activation of TLR2 in the hippocampus may facilitate limbic epileptogenesis.

Neuroanatomical differences in FAST and SLOW rat strains with differential vulnerability to kindling and behavioral comorbidities.

OBJECTIVE: The neurobiological factors underlying a predisposition towards developing epilepsy and its common behavioral comorbidities are poorly understood. FAST rats are a strain that has been selectively bred for enhanced vulnerability to kindling, while the SLOW strain has been bred to be resistant to kindling. FAST rats also exhibit behavioral traits reminiscent of those observed in neurodevelopmental disorders (autism spectrum disorder (ASD)/attention-deficit/hyperactivity disorder (ADHD)) commonly comorbid with epilepsy. In this study, we aimed to investigate neuroanatomical differences between these strains that may be associated with a differential vulnerability towards these interrelated disorders. METHODS: Ex vivo high-resolution magnetic resonance imaging on adult male FAST and SLOW rat brains was performed to identify morphological differences in regions of interest between the two strains. Behavioral examination using open-field, water consumption, and restraint tests was also conducted on a subgroup of these rats to document their differential ASD/ADHD-like behavior phenotype. Using optical stereological methods, the volume of cerebellar granule, white matter, and molecular layer and number of Purkinje cells were compared in a separate cohort of adult FAST and SLOW rats. RESULTS: Behavioral testing demonstrated hyperactivity, impulsivity, and polydipsia in FAST versus SLOW rats, consistent with an ASD/ADHD-like phenotype. Magnetic resonance imaging analysis identified brain structural differences in FAST compared with SLOW rats, including increased volume of the cerebrum, corpus callosum, third ventricle, and posterior inferior cerebellum, while decreased volume of the anterior cerebellar vermis. Stereological measurements on histological slices indicated significantly larger white matter layer volume, reduced number of Purkinje cells, and smaller molecular layer volume in the cerebellum in FAST versus SLOW rats. SIGNIFICANCE: These findings provide evidence of structural differences between the brains of FAST and SLOW rats that may be mechanistically related to their differential vulnerability to kindling and associated comorbid ASD/ADHD-like behaviors.

Critical Evaluation of P2X7 Receptor Antagonists in Selected Seizure Models.

The ATP-gated P2X7 receptor (P2X7R) is a non-selective cation channel which senses high extracellular ATP concentrations and has been suggested as a target for the treatment of neuroinflammation and neurodegenerative diseases. The use of P2X7R antagonists may therefore be a viable approach for treating CNS pathologies, including epileptic disorders. Recent studies showed anticonvulsant potential of P2X7R antagonists in certain animal models. To extend this work, we tested three CNS-permeable P2X7R blocker (Brilliant Blue G, AFC-5128, JNJ-47965567) and a natural compound derivative (tanshinone IIA sulfonate) in four well-characterized animal seizure models. In the maximal electroshock seizure threshold test and the pentylenetetrazol (PTZ) seizure threshold test in mice, none of the four compounds demonstrated anticonvulsant effects when given alone. Notably, in combination with carbamazepine, both AFC-5128 and JNJ-47965567 increased the threshold in the maximal electroshock seizure test. In the PTZ-kindling model in rats, useful for testing antiepileptogenic activities, Brilliant Blue G and tanshinone exhibited a moderate retarding effect, whereas the potent P2X7R blocker AFC-5128 and JNJ-47965567 showed a significant and long-lasting delay in kindling development. In fully kindled rats, the investigated compounds revealed modest effects to reduce the mean seizure stage. Furthermore, AFC-5128- and JNJ-47965567-treated animals displayed strongly reduced Iba 1 and GFAP immunoreactivity in the hippocampal CA3 region. In summary, our results show that P2X7R antagonists possess no remarkable anticonvulsant effects in the used acute screening tests, but can attenuate chemically-induced kindling. Further studies would be of interest to support the concept that P2X7R signalling plays a crucial role in the pathogenesis of epileptic disorders.

[(18)F]FDG PET Neuroimaging Predicts Pentylenetetrazole (PTZ) Kindling Outcome in Rats.

PURPOSE: Epileptogenesis, i.e., development of epilepsy, involves a number of processes that alter the brain function in the way that triggers spontaneous seizures. Kindling is one of the most used animal models of temporal lobe epilepsy (TLE) and epileptogenesis, although chemical kindling suffers from high inter-assay success unpredictability. This study was aimed to analyze the eventual regional brain metabolic changes during epileptogenesis in the pentylenetetrazole (PTZ) kindling model in order to obtain a predictive kindling outcome parameter. PROCEDURES: In vivo longitudinal positron emission tomography (PET) scans with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) along the PTZ kindling protocol (35 mg/kg intraperitoneally (i.p.), 18 sessions) in adult male rats were performed in order to evaluate the regional brain metabolism. RESULTS: The half of the PTZ-injected rats reached the kindled state. In addition, a significant decrease of [(18)F]FDG uptake at the end of the protocol in most of the brain structures of kindled animals was found, reflecting the characteristic epilepsy-associated hypometabolism. However, PTZ-injected animals but not reaching the kindled state did not show this widespread brain hypometabolism. Retrospective analysis of the data revealed that hippocampal [(18)F]FDG uptake normalized to pons turned out to be a predictive index of the kindling outcome. Thus, a 19.06 % reduction (p = 0.008) of the above parameter was found in positively kindled rats compared to non-kindled ones just after the fifth PTZ session. CONCLUSION: Non-invasive PET neuroimaging was a useful tool for discerning epileptogenesis progression in this animal model. Particularly, the [(18)F]FDG uptake of the hippocampus proved to be an early predictive parameter to differentiate resistant and non-resistant animals to the PTZ kindling.

Decreased Expression of Sox-1 in Cerebellum of Rat with Generalized Seizures Induced by Kindling Model.

The single feature of all malformations in cortical development is the clinical association with epilepsy. It has been proven that Sox-1 expression is essential during neurodevelopment and it is reported that Sox-1 knockout mice present spontaneous generalized seizures. Particularly in cerebellum, Sox-1 plays a key role in the Bergmann´s glia (BG) function, which allows the correct function of the Purkinje cells (PC). The targets of PC are the dentate and interpositus nuclei, which form the main cerebellar efferents involved in the physiopathology of epilepsy. Here we present the Sox-1 expression in cerebellum of rats during electric amygdala-kindling. We obtained seizures and once they had 3, 15 and 45 electric stimuli, the animals were sacrificed; the cerebellum was processed for inmunohistochemistry and Western blot analysis was performed to determine Sox-1 expression. Liquid chromatography was performed to examine gammaaminobutyric acid (GABA) and glutamate concentration. According to the literature, a progressive increase was observed in the electrographic and behavioral parameters. We found that Sox-1 expression in 15 and 45-stimuli groups had a statistically significant decrease as compared with controls, while the 3-stimuli group was similar to the control group. The concentration of glutamate was increased in rats with 45 stimuli. We can conclude that Sox-1 expression decreases as the number of seizures increases, and this is probably due to an altered glutamate regulation by a dysfunctional BG. In this way, we can suggest this mechanism as a one possible explanation of how the cerebellum participates in the pathophysiology of epilepsy.