Effects of A1 receptor agonist/antagonist on spontaneous seizures in pilocarpine-induced epileptic rats

Adenosine is an endogenous anticonvulsant that activates pre- and postsynaptic adenosine A₁ receptors. A₁ receptor agonists increase the latency for the development of seizures and status epilepticus following pilocarpine administration. Although hippocampal adenosine is increased in the chronic phase of the pilocarpine model, it is not known whether the modulation of A₁ receptors may influence the frequency of spontaneous recurrent seizures (SRS). Here, we tested the hypothesis that the A1 receptor agonist RPia ([R]-N-phenylisopropyladenosine) and the A1 antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine) administered to chronic pilocarpine epileptic rats would respectively decrease and increase the frequency of SRS and hippocampal excitability. Four months after Pilo-induced SE, chronic epileptic rats were video-monitored for the recording of SRS before (basal) and after a 2-week treatment with RPia (25 μg/kg) or DPCPX (50 μg/kg). Following sacrifice, brain slices were studied with electrophysiology. We found that rats given RPia had a 93% nonsignificant reduction in the frequency of seizures compared with their own pretreatment baseline. In contrast, the administration of DPCPX resulted in an 87% significant increase in seizure rate. Nontreated epileptic rats had a similar frequency of seizures along the study. Corroborating our behavioral data, in vitro recordings showed that slices from animals previously given DPCPX had a shorter latency to develop epileptiform activity, longer and higher DC shifts, and higher spike amplitude compared with slices from nontreated Pilo controls. In contrast, smaller spike amplitude was recorded in slices from animals given RPia. In summary, the administration of A1 agonists reduced hippocampal excitability but not the frequency of spontaneous recurrent seizures in chronic epileptic rats, whereas A1 receptor antagonists increased both.     

Alterations of hippocampal GAbaergic system contribute to development of spontaneous recurrent seizures in the rat lithium-pilocarpine model of temporal lobe epilepsy.

Reorganization of excitatory and inhibitory circuits in the hippocampal formation following seizure-induced neuronal loss has been proposed to underlie the development of chronic seizures in temporal lobe epilepsy (TLE). Here, we investigated whether specific morphological alterations of the GABAergic system can be related to the onset of spontaneous recurrent seizures (SRS) in the rat lithium-pilocarpine model of TLE. Immunohistochemical staining for markers of interneurons and their projections, including parvalbumin (PV), calretinin (CR), calbindin (CB), glutamic acid decarboxylase (GAD), and type 1 GABA transporter (GAT1), was performed in brain sections of rats treated with lithium-pilocarpine and sacrificed after 24 h, during the silent phase (6 and 12 days), or after the onset of SRS (10-18 days after treatment). Semiquantitative analysis revealed a selective loss of interneurons in the stratum oriens of CA1, associated with a reduction of GAT1 staining in the stratum radiatum and stratum oriens. In contrast, interneurons in CA3 were largely preserved, although GAT1 staining was also reduced. These changes occurred within 6 days after treatment and were therefore insufficient to cause SRS. In the dentate gyrus, extensive cell loss occurred in the hilus. The pericellular innervation of granule cells by PV-positive axons was markedly reduced, although the loss of PV-interneurons was only partial. Most strikingly, the density of GABAergic axons, positive for both GAD and GAT1, was dramatically increased in the inner molecular layer. This change emerged during the silent period, but was most marked in animals with SRS. Finally, supernumerary CB-positive neurons were detected in the hilus, selectively in rats with SRS. These findings suggest that alterations of GABAergic circuits occur early after lithium-pilocarpine-induced status epilepticus and contribute to epileptogenesis. In particular, the reorganization of GABAergic axons in the dentate gyrus might contribute to synchronize hyperexcitability induced by the interneuron loss during the silent period, leading to the onset of chronic seizures.     

Transplantation of bone marrow mononuclear cells decreases seizure incidence, mitigates neuronal loss and modulates pro-inflammatory cytokine production in epileptic rats

Approximately 30% of patients with mesial temporal lobe epilepsy do not respond to treatment with antiepileptic drugs. We have previously shown that transplantation of mononuclear bone marrow cells (BMC) has an anticonvulsant effect in acute epilepsy. Here, we used pilocarpine to induce epilepsy in rats and studied the effects of BMC injected intravenously either at the onset of seizures or after 10 months of recurrent seizures. BMC effectively decreased seizure frequency and duration. In addition, decreased levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and increased levels of anti-inflammatory cytokine (IL-10) were observed in the brain and serum of BMC-treated rats. Transplants performed at seizure-onset protected against pilocarpine-induced neuronal loss and gliosis and stimulated the proliferation of new neurons in epileptic rats. Our data demonstrate that BMC transplantation has potent therapeutic effects and could be a potential therapy for clinically intractable epilepsies.     

Kainic acid seizures in the developing brain: status epilepticus and spontaneous recurrent seizures.

Acute and chronic effects of seizures induced by intraperitoneal (i.p.) injection of kainic acid (KA) were studied in developing rats (postnatal days (P) 5, 10, 20, 30, and adult 60). For 3 months following KA-induced status epilepticus, spontaneous recurrent seizure (SRS) occurrence was quantified using intermittent video monitoring. Latency to generalized seizures was then tested using flurothyl, and brains were histologically analyzed for CA3 lesions. In P5-10 rats, KA caused generalized tonic-clonic ('swimming') seizures. SRS did not develop, and there was no significant difference between control and KA-treated rats in latency to flurothyl-induced seizures. In contrast, rats P20 and older exhibited limbic automatisms followed by limbic motor seizures which secondarily generalized. Incidence and frequency of SRS increased with age. P20-30 rats with SRS had shorter latencies to flurothyl seizures than did KA-treated P20-30 rats without SRS or controls. KA-treated P60 rats (with or without SRS) had shorter latencies than controls to flurothyl seizure onset. SRS in P60 rats occurred sooner after KA than in P20-30 rats. CA3 lesions were seen in P20-60 rats with and without SRS, but not in P5-10 rats. These data suggest that there are developmental differences in both acute and chronic responses to KA, with immature animals relatively protected from the long-term deleterious effects of this convulsant.     

Epilepsy induced by extended amygdala-kindling in rats: lack of clear association between development of spontaneous seizures and neuronal damage

Most patients with temporal lobe epilepsy (TLE), the most common type of epilepsy, show pronounced loss of neurons in limbic brain regions, including the hippocampus, amygdala, and parahippocampal regions. Hippocampal damage in patients with TLE is characterized by extensive neuronal loss in the CA3 and CA1 sectors and the hilus of the dentate gyrus. There is a long and ongoing debate on whether this type of hippocampal damage, referred to as hippocampal sclerosis, is the cause or consequence of TLE. Furthermore, hippocampal damage may contribute to the progressive features of TLE. The present study was designed to determine whether development of spontaneous recurrent seizures (SRS) after extended kindling of the amygdala in rats is associated with neuronal damage. The kindling model of TLE was chosen because previous studies have shown that only part of the rats develop SRS after extended kindling, thus allowing to compare the brain pathology of rats that received the same number of amygdala stimulation but did or did not develop SRS. For extended kindling, rats were stimulated twice daily 3-5 days a week for up to about 280 stimulations. During long-term EEG/video monitoring, SRS were observed in 50% of the rats over the period of extended kindling. SRS often started with myoclonic jerks or focal seizures and subsequently progressed into secondarily generalized seizures, so that the development of SRS recapitulated the earlier kindling of elicited seizures. No obvious neurodegeneration was observed in the CA1 and CA3 sectors of the hippocampus, the amygdala, parahippocampal regions or thalamus. A significant bilateral reduction in neuronal density was determined in the dentate hilus after extended kindling, but this reduction in hilar cell density did not significantly differ between rats with and without observed SRS. Determination of the total number of hilar neurons and of hilar volume indicated that the reduced neuronal density in the dentate hilus was due to expansion of hilar area but not to neuronal damage. The data demonstrate that extended kindling does not cause any hippocampal damage resembling hippocampal sclerosis, but that SRS develop in the absence of such damage.     

Electrophysiological observations in hippocampal slices from rats treated with the ketogenic diet

The electrophysiological effects of the high-fat, low-carbohydrate ketogenic diet (KD) were assessed in normal and epileptic [kainic-acid(KA)-treated] adult rats using hippocampal slices. In the first set of experiments, normal rats were fed the KD or a standard control diet for 6-8 weeks (beginning on postnatal day 56, P56), after which they were sacrificed for hippocampal slices. All rats on the KD became ketotic. The baseline effects of the KD were determined by comparing extracellular measures of synaptic transmission and responses to evoked stimulation, and hippocampal excitability was tested in Mg(2+)-free medium. There were no differences in EPSP slope, input/output relationship, responses to evoked stimulation or Mg(2+)-free burst frequency between slices from control and KD-fed rats. In another set of experiments, rats were made epileptic by intraperitoneal injection of kainic acid (KA) on P54, which caused status epilepticus followed by the development of spontaneous recurrent seizures (SRS) over the next few weeks. Two days after KA-induced status, rats were divided into a control-fed group and a KD-fed group. Animals on the KD had significantly fewer SRS over the ensuing 8 weeks. In hippocampal slices from KA-treated, KD-fed rats, there were fewer evoked CA1 population spikes than from slices of control-fed rats. These results suggest that the KD does not alter baseline electrophysiological parameters in normal rats. In rats made chronically epileptic by administration of KA, KD treatment was associated with fewer spontaneous seizures and reduced CA1 excitability in vitro. Therefore, at least part of the KD mechanism of action may involve long-term changes in network excitability. Copyright Copyright 1999 S. Karger AG, Basel     

Relationship between neuronal loss and interictal glucose metabolism during the chronic phase of the lithium-pilocarpine model of epilepsy in the immature and adult rat

The lithium-pilocarpine (Li-Pilo) model of epilepsy reproduces most of the features of human temporal lobe epilepsy. After having studied the metabolic changes occurring during the silent phase, in the present study, we explored the relationship between interictal metabolic changes and neuronal loss during the chronic phase following status epilepticus (SE) induced by Li-Pilo in 10-day-old (P10), 21-day-old (P21), and adult rats. Rats were observed and their EEG was recorded to detect the occurrence of spontaneous recurrent seizures (SRS). Local cerebral glucose utilization was measured during the interictal period of the chronic phase, between 2 and 7 months after SE, by the [(14)C]2-deoxyglucose method in rats subjected to SE at P10, P21, or as adults. Neuronal damage was assessed by cell counting on adjacent cresyl violet stained sections. When SE was induced at P10, rats did not become epileptic, did not develop lesions and cerebral glucose utilization was in the normal range 7 months later. When SE was induced in adult rats, they all became epileptic after a mean duration of 25 days and developed lesions in the forebrain limbic areas, which were hypometabolic during the interictal period of the chronic phase, 2 months after SE. When SE was induced in P21 rats, 24% developed SRS, and in 43% seizures could be triggered (TS) by handling, after a mean delay of 74 days in both cases. The remaining 33% did not become epileptic (NS). The three groups of P21 rats developed quite comparable lesions mainly in the hilus of the dentate gyrus, lateral thalamus, and entorhinal cortex; at 6 months after SE, the forebrain was hypometabolic in NS and TS rats while it was normo- to slightly hypermetabolic in SRS rats. These data show that interictal metabolic changes are age-dependent. Moreover, there is no obvious correlation, in this model, between interictal hypometabolism and neuronal loss, as reported previously in human temporal lobe epilepsy.     

The duration of sustained convulsive seizures determines the pattern of hippocampal neurogenesis and the development of spontaneous epilepsy in rats

The duration of sustained seizures (SS) plays a crucial role in the occurrence of spontaneous recurrent seizures (SRS) in experimental animals. We tested whether rats with varying durations of initial convulsive SS exhibited differential neurogenesis patterns in the hippocampal dentate gyrus that may be related to subsequent epileptogenesis. Sprague-Dawley rats with pilocarpine-induced convulsive SS were divided into short SS (30 min) and long SS (2 h) groups. Their behavior was monitored to identify convulsive SRS. From 1 to 28 days post-SS, cell proliferation was evaluated by 5'-bromo-2'-deoxyuridine (BrdU) labeling and immature neuroblasts in the dentate gyrus were identified by doublecortin immunohistochemistry. Convulsive SRS was detected in 8 out of the 9 long SS rats, but not in the 9 short SS rats. During day 1-3, proliferative cells were diffusely localized throughout the hippocampus in the long SS rats but were primarily confined within the subgranular zone in the short SS rats. Within the subgranular zone, a significant increase in the number of BrdU-positive cells was found at days 3 and 7 after the long SS and on day 1 after the short SS. Notably, abnormal dendritic outgrowth and hilar-ectopic localization of doublecortin-positive cells were present in the long SS rats. In conclusion, aberrant hippocampal neurogenesis following long SS may contribute to the development of SRS.     

Epileptogenesis and neuropathology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats

It has previously been shown that prolonged (60-min) low-intensity electrical stimulation of a kindled focus in the basolateral nucleus of the amygdala (BLA) of Wistar rats resulted in the development of self-sustained status epilepticus (SSSE) with predominantly partial seizures and subsequent brain damage in the ipsilateral hemisphere. In the present study, using high-intensity (700 microA) pulsed-train electrical stimulation of the BLA for 25 min, SSSE was induced in both kindled and non-kindled Wistar rats, demonstrating that under these experimental conditions prior kindling is not necessary to induce SSSE. Thus, all subsequent experiments were done in non-kindled rats of different strains (Wistar, Sprague-Dawley) and genders. Three distinct behavioral types of SSSE were observed: (1) continuous partial seizures; (2) continuous partial seizures, repeatedly interrupted by generalized convulsive seizures; and (3) continuous generalized convulsive seizures. These three forms of SSSE were seen in both strains and genders, although the percentage of rats in each strain and gender developing a specific type of SSSE differed. Rats spontaneously recovered from SSSE after between 3 and 8h on average, the SSSE duration depending on SSSE type, rat strain and gender. Following SSSE, rats were monitored with a video- and EEG-recording system for occurrence of spontaneous recurrent seizures (SRS). Overall, about 80% of the rats developed epilepsy with SRS after SSSE, but the proportion of rats developing SRS depended on the type of SSSE. Only 33% of the rats developed SRS after a partial SSSE, compared to >90% in case of either type 2 or type 3 SSSE with generalized convulsive seizures. Interruption of different forms of SSSE with diazepam after 90 min prevented development of epilepsy, while a generalized SSSE duration of 4h consistently produced epilepsy in >90% of rats. Histologic analysis of rat brains after the different SSSE types indicated that neuronal loss after partial SSSE was much more regionally restricted and less severe compared to neuronal damage after SSSE with generalized convulsive seizures, which was similar to the brain damage seen in the kainate and pilocarpine models of temporal lobe epilepsy. These experiments establish that prolonged electrical stimulation of the BLA induces different forms of SSSE that resemble nonconvulsive and convulsive types of SE in humans. These different forms of SSSE induce epilepsy with SRS and brain pathology reminiscent of temporal lobe epilepsy with hippocampal sclerosis. The rat model provides a new tool to mimic different types of SE and investigate the pathogenesis underlying their long-term complications.     

海马干细胞移植对癫癎鼠脑电影响的初步研究

目的研究神经干细胞移植对癫癎鼠脑电的影响,为神经干细胞移植治疗癫癎提供理论依据.方法分离、培养新生鼠海马干细胞,将其移植至海人酸(KA)所致癫癎模型鼠的右侧海马,比较移植组及未移植组大鼠在移植前和移植后1周、4周、8周及24周海马及杏仁核的脑电变化.结果海马干细胞移植可减少癫癎动物脑电的痫性发放,并降低癫癎波幅约50%.结论神经干细胞移植对于KA诱发癫癎鼠的癫癎具有一定的抑制作用,但其具体作用机制还有待于进一步的研究.     

GFP转基因骨髓基质干细胞移植对癫痫鼠脑电的影响

目的　观察绿色荧光蛋白（GFP）转基因骨髓基质干细胞（BMSCs）移植至致痫鼠后的存活、迁移及其对癫痫鼠脑电的影响。方法　分离、培养GFP转基因小鼠BMSCs，移植至青霉素致痫鼠的右侧海马内，比较移植后1w、2w、4wBMSCs在脑内的存活和迁移情况及大鼠脑电改变。结果　BMSCs可以在致痫鼠脑内存活和迁移，随移植时间延长，细胞存活数逐渐减少（P〈0．01）；BMSCs移植可减少癫痫大鼠脑电的痫性放电，降低癫痫波波幅。结论　BMSCs移植于青霉素诱发的癫痫鼠脑内后能够存活、迁移，并能够改善癫痫鼠的脑电生理功能，提示干细胞移植可能成为一种有效的癫痫     

Effect of long-term spontaneous recurrent seizures or reinduction of status epilepticus on the development of supragranular mossy fiber sprouting.

In a recent report we have shown that a protein synthesis inhibitor, cycloheximide (CHX), is able to block the mossy fiber sprouting (MFS) that would otherwise be triggered by pilocarpine (Pilo)-induced status epilepticus (SE), and also gives relative protection against hippocampal neuronal death. Under this condition animals still showed spontaneous recurrent seizures (SRS) which led us to question the role played by sprouted mossy fibers in generating those seizures. In both patients and animal models of epilepsy the relative contribution of SE (when present) and/or SRS for the development of MFS is not known. In the present study we investigated the relationship between MFS, SE and SRS, and evaluated whether the CHX-induced blockade of MFS was transient or permanent in nature. We performed a chronic study which included animals subject to Pilo-induced SE in the presence of CHX and sacrificed between 8 and 10 months later, and animals that were subject to Pilo-induced SE in the presence of CHX and underwent a reinduction of SE with Pilo, 45 days after the first induction, but this time in the absence of CHX. Re-induction of SE or a long period of chronic seizures, were able to trigger supragranular MFS even in animals where the first (or only) SE event was triggered in the presence of CHX. MFS did not show any association with the frequency of SRS, and thus seemed to depend more critically on time. Our current findings allow us to suggest that MFS are neither the cause nor the consequence of SRS in the pilocarpine model.     

Neuronal cell death in a rat model for mesial temporal lobe epilepsy is induced by the initial status epilepticus and not by later repeated spontaneous seizures

PURPOSE: To determine whether repeated seizures contribute to hippocampal sclerosis, we investigated whether cell loss in the (para) hippocampal region was related to the severity of chronic seizure activity in a rat model for temporal lobe epilepsy (TLE). METHODS: Chronic epilepsy developed after status epilepticus (SE) that was electrically induced 3-5 months before. The presence of neuronal damage was assessed by using Fluoro-Jade and dUTP nick end-labeling (TUNEL) of brain sections counterstained with Nissl. RESULTS: We found a negative correlation between the numbers of surviving hilar cells and the duration of the SE (r = -0.66; p < 0.01). In the chronic phase, we could discriminate between rats with occasional seizures (0.15 +/- 0.05 seizures per day) without progression and rats with progressive seizure activity (8.9 +/- 2.8 seizures/day). In both groups, the number of TUNEL-positive cells in parahippocampal regions was similar and higher than in controls. In the hippocampal formation, this was not significantly different from controls. Fluoro-Jade staining showed essentially the same pattern at 1 week and no positive neurons in chronic epileptic rats. CONCLUSIONS: Cell death in this rat model is related to the initial SE rather than to the frequency of spontaneous seizures. These results emphasize that it is of crucial importance to stop the SE as soon as possible to prevent extended cell loss and further progression of the disease. They also suggest that neuroprotectants can be useful during the first week after SE, but will not be very useful in the chronic epileptic phase.     

Differential migration of astrocytes grafted into the developing rat brain

Fetal and neonatal astrocytes migrate in specific patterns when transplanted into the adult rat host brain. However, it is unclear whether these astrocytes demonstrate the same degree of mobility during early brain development. In the present study, neonatal cortical, hippocampal, and hypothalamic astrocytes were collected from the brains of 1- to 3-day-old rats and placed in tissue culture. After 14 to 21 days, cultures enriched in astrocytes were harvested and labelled with either the fluorescent dye Fast Blue or fluorescein-labelled latex beads. They were then transplanted into the right frontal cerebrum of neonatal rats at 2,5,8, and 11 days postpartum. Seven days after transplantation, animals were sacrificed and their brains were fixed by immersion in aldehydes, sectioned on a cryostat, and examined with fluorescence microscopy. Transplanted astrocytes migrated along the corpus callosum, internal capsule, glial limitans, ventricular linings, and hippocampal structure. Labelled cells were also found in the contralateral hemisphere in day 2 brains. Migration in a radial fashion from the injection site toward the periphery was a particularly obvious pattern, and was most pronounced in these younger hosts. In days 5 and 8 rat brains, astrocyte migration became more restricted to the hemisphere of implantation. In 11-day-old host brains hemispheric restriction and other region-specific influences became manifest and specifically modulated migration. Radial migration was absent in the 11-day-old host group except for cells of cortical origin. The observed results demonstrate that neonatal cortical, hippocampal, and hypothalamic astrocytes transplanted into the neonatal cerebrum migrate in patterns that are more extensive than in the adult brain. This suggests that cellular migration in the neonatal brain is governed by factors that are less restrictive than those regulating migration in the adult brain. In particulur, our observations imply that radial glia may provide migratory substrates for transplanted astrocytes, and region-specific regulation of migration may begin around 11 days after birth. © 1993 Wiley-Liss, Inc.     

Fluorescent tracer in pilocarpine-treated rats shows widespread aberrant hippocampal neuronal connectivity

Neuronal fibres of the hippocampal formation of normal and chronic epileptic rats were investigated by fluorescent tracing methods using the pilocarpine model of limbic epilepsy. Two months after onset of spontaneous limbic seizures, hippocampal slices were prepared and maintained in vitro for 10 h. Small crystals of fluorescent dye [fluorescein (fluoro-emerald) and tetramethylrhodamine (fluoro-ruby)] were applied to different hippocampal regions. The main findings were: (i) in control rats there was no supragranular labelling when the mossy fibre tract was stained in stratum radiatum of area CA3. However, in epileptic rats a fibre network in the inner molecular layer of the dentate gyrus was retrogradely labelled; (ii) a retrograde innervation of area CA3 by CA1 pyramidal cells was disclosed by labelling remote CA1 neurons after dye injection into the stratum radiatum of area CA3 in chronic epileptic rats; (iii) labelling of CA1 neurons apart from the injection site within area CA1 was observed in epileptic rats but not in control animals; and (iv), a subicular-hippocampal projection was present in pilocarpine-treated rats when the tracer was injected just below the stratum pyramidale of area CA1. The findings show that fibre rearrangement in distinct regions of the epileptic hippocampal formation can occur as an aftermath of pilocarpine-induced status epilepticus.     

Is mossy fiber sprouting present at the time of the first spontaneous seizures in rat experimental temporal lobe epilepsy?

The contribution of mossy fiber sprouting to the generation of spontaneous seizures in the epileptic brain is under dispute. The present study addressed this question by examining whether sprouting of mossy fibers is present at the time of appearance of the first spontaneous seizures in rats, and whether all animals with increased sprouting have spontaneous seizures. Epileptogenesis was induced in 16 rats by electrically stimulating the lateral nucleus of the amygdala for 20-30 min until the rats developed self-sustained status epilepticus (SSSE). During and after SSSE, rats were monitored in long-term by continuous video-electroencephalography until they developed a second spontaneous seizure (8-54 days). Thereafter, monitoring was continued for 11 days to follow seizure frequency. The density of mossy fiber sprouting was analyzed from Timm-stained preparations. The density of hilar neurons was assessed from thionin-stained sections. Of 16 rats, 14 developed epilepsy. In epileptic rats, the density of mossy fiber sprouting did not correlate with the severity or duration (115-620 min) of SSSE, delay from SSSE to occurrence of first (8-51 days) or second (8-54 days) spontaneous seizure, or time from SSSE to perfusion (20-63 days). In the temporal end of the hippocampus, the sprouting correlated with the severity of neuronal damage (ipsilateral: r = -0.852, P < 0.01 contralateral: r = -0.748, P < 0.01). The two animals without spontaneous seizures also had sprouting. Increased density of sprouting in animals without seizures, and its association with the severity of neuronal loss was confirmed in another series of 30 stimulated rats that were followed-up with video-EEG monitoring for 60 d. Our data indicate that although mossy fiber sprouting is present in all animals with spontaneous seizures, its presence is not necessarily associated with the occurrence of spontaneous seizures.     

Persistence and atrophy of septal/diagonal band neurons expressing the p75 neurotrophin receptor in pilocarpine-induced chronic epilepsy in the rat

Systemic administration of pilocarpine, which results in status epilepticus followed by recurrent seizures in rats, is a widely used experimental model of chronic limbic epilepsy. Marked structural alterations have been documented in pilocarpine-induced epilepsy, and these include cell loss in the hippocampus and other brain areas, and sprouting of mossy and cholinergic fibers in the hippocampus. Evidence is accumulating that neurotrophins and neurotrophin receptors are involved in the cascade of these events. Two and 4 months after pilocarpine-induced epilepsy, neurons containing the 75-kDa low affinity neurotrophin receptor (p75^NTR) were investigated with immunohistochemistry in the medial septal and diagonal band nuclei. No significant differences in the distribution and number of immunoreactive neurons were found in the epileptic rats compared to control saline-treated animals. However, in the epileptic animals, a significant decrease in the perikaryal size of p75^NTR-immunoreactive neurons of the septal/diagonal band region was found by 60 days, and such atrophic changes were more marked in the diagonal band nuclei by 120 days. These findings indicate that the p75^NTR-containing cell bodies, which include the neurons projecting to the hippocampal formation and are cholinergic in the normal brain, survive after months of spontaneous recurrent seizures, during which, therefore, a supply of p75^NTR to target regions is maintained in the chronic epileptic brain. However, the present data point out that these p75^NTR-containing neurons undergo a significant shrinkage in pilocarpine-induced chronic epilepsy, thus indicating that they are involved in the brain pathology of temporal lobe epilepsy.     

Prolonged low-dose caffeine exposure protects against hippocampal damage but not against the occurrence of epilepsy in the lithium-pilocarpine model in the rat

PURPOSE: Acute caffeine exposure has proconvulsant effects and worsens epileptic and ischemic neuronal damage. Surprisingly, prolonged caffeine exposure decreases the susceptibility to seizures and the extent of ischemic damage. We explored whether the exposure to a low long-term dose of caffeine could protect the brain from neuronal damage and epileptogenesis in the lithium-pilocarpine model of temporal lobe epilepsy. METHODS: Rats received either plain tap water or water containing caffeine (0.3 g/L) for 15 days before the induction of status epilepticus (SE) by lithium-pilocarpine and for 7 days after SE. The extent of neuronal damage was assessed in the hippocampus and piriform and entorhinal cortices in brain sections stained with thionine and obtained from animals killed 7 days after SE. The latency to spontaneous recurrent seizures was controlled by video monitoring. RESULTS: Caffeine treatment induced a marked, almost total neuroprotection in CA1 and a very limited protection in the hilus of the dentate gyrus, whereas damage in layers III-IV of the piriform cortex was slightly worsened by the treatment. All rats, whether they received caffeine or plain tap water, became epileptic after the same latency (17-19 days). CONCLUSIONS: Thus these data extend the neuroprotective effects of low long-term caffeine exposure to epileptic damage and confirm that the sole protection of the Ammon's horn has no influence on the genesis of spontaneous recurrent seizures in this model.     

Enhanced epileptogenic susceptibility in a genetic model of reactive synaptogenesis: the spastic Han-Wistar rat

Our laboratory has been studying the spastic Han-Wistar (sHW) rat as a model of neuronal degeneration. Mutant sHW rats display a number of developmental abnormalities that eventually lead to hippocampal pyramidal cell death and synaptic reorganization starting around 30 days of age. The present study examined the contribution of hippocampal reorganization to the expression of seizures induced by systemic injections of kainic acid. Behavioral observations, EEG recordings and hippocampal Fos protein expression in these animals indicated that mutants develop paroxysmal discharges and seizures earlier than controls and the intensity of epileptic manifestations is greater. Kainate injections were lethal in 50% of mutants compared to only 5% of controls. Fos expression was increased approximately twofold in the mutant hippocampus, implicating abnormal excitation in this region. Additional studies in untreated animals indicated that GluR2 mRNA expression was significantly increased throughout the hippocampus in mutant animals, possibly contributing to the enhanced susceptibility to kainate treatment. These results confirm the role of synaptic reorganization in the increased propensity to develop epileptic discharges. Our data also underscore the usefulness of this natural model of cell degeneration and reactive synaptogenesis for understanding the mechanisms of neuronal hyperexcitability.     

Single injection of a low dose of pentylenetetrazole leads to epileptogenesis in an animal model of cortical dysplasia

Purpose:   Cortical dysplasia (CD) is one of the most frequent causes of pharmacoresistent focal epilepsy. Despite significant advances in various diagnostic and therapeutic methods, the basic mechanisms of higher susceptibility for seizures in patients with CD are unknown. Animal models of CD present with a lower threshold for seizure induction. The purpose of this study is to further characterize the animal model of in utero radiation-induced CD and to illustrate the effect of a late postnatal second hit (low dose of pentylenetetrazole, PTZ) on the development of spontaneous seizures.
Methods:   Pregnant Sprague–Dawley rats were irradiated on E17 (145 cGy; control group was left untreated). Litters were implanted with bifrontal epidural and hippocampal depth electrodes. After baseline electroencephalography (EEG) recording, animals received 30 mg/kg PTZ and were chronically monitored. Histopathology of the brains was verified.
Results:   No seizures were detected in animals that did not receive PTZ. PTZ-injected irradiated (XRT) rats showed severe prolonged, repetitive seizures during the acute period. During the chronic period, XRT rats had recurrent seizures and epileptiform spikes. PTZ-injected control animals exhibited milder and fewer acute seizures and did not show seizures during the chronic period. Histology was consistent with cortical and hippocampal dysplasia.
Conclusions:   This study shows that a single treatment with a low dose of PTZ renders XRT rats (but not age-matched controls) epileptic, exhibiting spontaneous epileptiform spikes and seizures on EEG. These results might mirror the natural history of patients with CD thought to be caused by prenatal/congenital or perinatal insults.