Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.     

Recurrent excitation in the dentate gyrus of a murine model of temporal lobe epilepsy

Similar to rats, systemic pilocarpine injection causes status epilepticus (SE) and the eventual development of spontaneous seizures and mossy fiber sprouting in C57BL/6 and CD1 mice, but the physiological correlates of these events have not been identified in mice. Population responses in granule cells of the dentate gyrus were examined in transverse slices of the ventral hippocampus from pilocarpine-treated and untreated mice. In Mg(2+)-free bathing medium containing bicuculline, conditions designed to increase excitability in the slices, electrical stimulation of the hilus resulted in a single population spike in granule cells from control mice and pilocarpine-treated mice that did not experience SE. In SE survivors, similar stimulation resulted in a population spike followed, at a variable latency, by negative DC shifts and repetitive afterdischarges of 3-60 s duration, which were blocked by ionotropic glutamate receptor antagonists. Focal glutamate photostimulation of the granule cell layer at sites distant from the recording pipette resulted in population responses of 1-30 s duration in slices from SE survivors but not other groups. These data support the hypothesis that SE-induced mossy fiber sprouting and synaptic reorganization are relevant characteristics of seizure development in these murine strains, resembling rat models of human temporal lobe epilepsy.     

One hour of pilocarpine-induced status epilepticus is sufficient to develop chronic epilepsy in mice, and is associated with mossy fiber sprouting but not neuronal death

Determining the minimal duration of status epilepticus (SE) that leads to the development of subsequent spontaneous seizures (i.e., epilepsy) is important, because it provides a critical time-window for seizure intervention and epilepsy prevention. In the present study, male ICR (Imprinting Control Region) mice were injected with pilocarpine to induce acute seizures. SE was terminated by diazepam at 10 min, 30 min, 1 h, 2 h and 4 h after seizure onset. Spontaneous seizures occurred in the 1, 2 and 4 h SE groups, and the seizure frequency increased with the prolongation of SE. Similarly, the Morris water maze revealed that the escape latency was significantly increased and the number of target quadrant crossings was markedly decreased in the 1, 2 and 4 h SE groups. Robust mossy fiber sprouting was observed in these groups, but not in the 10 or 30 min group. In contrast, Fluoro-Jade B staining revealed significant cell death only in the 4 h SE group. The incidence and frequency of spontaneous seizures were correlated with Timm score (P = 0.004) and escape latency (P = 0.004). These data suggest that SE longer than one hour results in spontaneous motor seizures and memory deficits, and spontaneous seizures are likely associated with robust mossy fiber sprouting but not neuronal death.     

Behavioral and cognitive alterations, spontaneous seizures, and neuropathology developing after a pilocarpine-induced status epilepticus in C57BL/6 mice

Many patients with epilepsy suffer from psychiatric comorbidities including depression, anxiety, psychotic disorders, cognitive, and personality changes, but the mechanisms underlying the association between epilepsy and psychopathology are only incompletely understood. Animal models of epilepsy, such as the pilocarpine model of acquired temporal lobe epilepsy (TLE), are useful to study the relationship between epilepsy and behavioral dysfunctions. In the present study, we examined behavioral and cognitive alterations, spontaneous seizures, and neuropathology developing after a pilocarpine-induced status epilepticus in the C57BL/6 (B6) inbred strain of mice, which is commonly used as background strain for genetically modified mice. For this study, we used the same pilocarpine ramping-up dosing protocol and behavioral test battery than in a previous study in NMRI mice, thus allowing direct comparison between these two mouse strains. All B6 mice that survived SE developed epilepsy with spontaneous recurrent seizures. Epileptic B6 mice exhibited significant increases of anxiety-related behavior in the open field and light–dark box, increased locomotor activity in the open field, elevated plus maze, hole board, and novel object exploration tests, and decreased immobility in the forced swimming and tail suspension tests. Furthermore, spatial learning and memory were severely impaired in the Morris water maze, although hippocampal damage was much less severe than previously determined in NMRI mice. B6 mice in which pilocarpine did not induce SE but only single seizures did not exhibit any detectable neurodegeneration, but differed behaviorally from sham controls in several tests of the test battery used. Our data indicate that the pilocarpine model of TLE in B6 mice is ideally suited to study the neurobiological mechanisms underlying the association between seizures, brain damage and psychopathology.     

Association Between the Density of Mossy Fiber Sprouting and Seizure Frequency in Experimental and Human Temporal Lobe Epilepsy

Summary: Purpose : If the sprouting of granule cell axons or mossy fibers in the dentate gyrus is critical for the generation of spontaneous seizures in temporal lobe epilepsy (TLE), one could hypothesize that epileptic animals or humans with increased sprouting would have more frequent seizures. This hypothesis was tested by analyzing the data gathered from experimental and human epilepsy.
Methods : In experiment I (rats with "newly diagnosed" TLE), self-sustained status epilepticus was induced in rats by electrically stimulating the amygdala. Thereafter, the appearance of spontaneous seizures was monitored by continuous video-electroencephalography (EEG) until the animal developed two spontaneous seizures and for 11 d thereafter. Rats were perfused for histology, and mossy fibers were stained using the Timm method. In experiment II (rats with "recently diagnosed" TLE), status epilepticus was induced in rats and the development of seizures was monitored by video-EEG for 24 h/d every other day for 60 days. All animals were then perfused for histology. In experiment III (rats with "chronic" TLE), animals were monitored by video-EEG for 24 h/d every other day for 6 months before histologic analysis. To assess mossy fiber sprouting in human TLE, hippocampal sections from 31 patients who had undergone surgery for drug-refractory TLE were stained with an antibody raised against dynorphin.
Results and Conclusions : Our data indicate that the density of mossy fiber sprouting is not associated with the total number of lifetime seizures or the seizure frequency in experimental or human TLE.     

Genetic dissection of the signals that induce synaptic reorganization

Synaptic reorganization of mossy fibers following kainic acid (KA) administration has been reported to contribute to the formation of recurrent excitatory circuits, resulting in an epileptogenic state. It is unclear, however, whether KA-induced mossy fiber sprouting results from neuronal cell loss or the seizure activity that KA induces. We have recently demonstrated that certain strains of mice are resistant to excitotoxic cell death, yet exhibit seizure activity similar to what has been observed in rodents susceptible to KA. The present study takes advantage of these strain differences to explore the roles of seizure activity vs cell loss in triggering mossy fiber sprouting. In order to understand the relationships between gene induction, cell death, and the sprouting response, we assessed the regulation of two molecules associated with the sprouting response, c-fos and GAP-43, in mice resistant (C57BL/6) and susceptible (FVB/N) to KA-induced cell death. Following administration of KA, increases in c-fos immunoreactivity were observed in both strains, although prolonged induction of c-fos was present only in the hippocampal neurons of FVB/N mice. Mossy fiber sprouting following KA administration was also only observed in FVB/N mice, while induction of GAP-43, a marker associated with mossy fiber sprouting, was not observed in either strain. These results indicate that: (i) KA-induced seizure activity alone is insufficient to induce mossy fiber sprouting; (ii) mossy fiber sprouting may be due to the loss of hilar neurons following kainate administration; and (iii) induction of GAP-43 is not a necessary component of the sprouting response that occurs following KA in mice.     

Circuit Mechanisms of Seizures in the Pilocarpine Model of Chronic Epilepsy: Cell Loss and Mossy Fiber Sprouting

We used the pilocarpine model of chronic spontaneous recurrent seizures to evaluate the time course of supragranular dentate sprouting and to assess the relation between several changes that occur in epilep tic tissue with different behavioral manifestations of this experimental model of temporal lobe epilepsy. Pilo carpine-induced status epilepticus (SE) invariably led to cell loss in the hilus of the dentate gyrus (DG) and to spontaneous recurrent seizures. Cell loss was often also noted in the DG and in hippocampal subfields CA1 and CA3. The seizures began to appear at a mean of 15 days after SE induction (silent period), recurred at variable frequencies for each animal, and lasted for as long as the animals were allowed to survive (325 days). The granule cell layer of the DG was dispersed in epileptic animals, and neo-Timm stains showed supra-and intragranular mossy fiber sprouting. Supragranular mossy fiber sprout ing and dentate granule cell dispersion began to appear early after SE (as early as 4 and 9 days, respectively) and reached a plateau by 100 days. Animals with a greater degree of cell loss in hippocampal field CAS showed later onset of chronic epilepsy (r= 0.83, p < 0.0005), suggest ing that CA3 represents one of the routes for seizure spread. These results demonstrate that the pilocarpine model of chronic seizures replicates several of the fea tures of human temporal lobe epilepsy (hippocampal cell loss, suprar and intragranular mossy fiber sprouting, den tate granule cell dispersion, spontaneous recurrent sei zures) and that it may be a useful model for studying this human condition. The results also suggest that even though a certain amount of cell loss in specific areas may be essential for chronic seizures to occur, excessive cell loss may hinder epileptogenesis.     

Locus Coeruleus and Neuronal Plasticity in a Model of Focal Limbic Epilepsy

Summary:  Purpose: A lesion of the noradrenergic nucleus Locus Coeruleus (LC) converts sporadic seizures evoked by microinfusion of bicuculline into the anterior piriform cortex (APC) of rats into limbic status epilepticus (SE). The purpose of this study was to evaluate the chronic effects of this new model of SE on the onset of secondary epileptogenesis. We further related the loss of noradrenaline (NE) with hippocampal mossy fiber sprouting.
Methods: Male Sprague Dawley rats were treated with systemic saline or DSP-4 (a neurotoxin selective for noradrenergic terminals originating from the LC), microinfused with bicuculline into the APC three days later, and sacrificed after 45 days. Naïve and DSP-4 pretreated sham-operated rats served as respective controls. The following evaluations were performed: (a) monitoring of acute seizures and delayed occurrence of spontaneous recurrent seizures (SRS); (b) NE levels in the hippocampus, frontal and olfactory cortex; (c) occurrence of mossy fiber sprouting into the inner molecular layer of the dentate gyrus of the dorsal hippocampus.
Results: In 30% of rats lacking noradrenergic terminals, SE evoked from the APC was followed by SRS. Conversely, seizures evoked in intact rats did not result in chronic epileptogenesis. Seizures/SE did not modify NE levels as compared with baseline levels both in naïve and DSP-4-pretreated rats. Rats undergoing SE following DSP-4 + bicuculline developed SRS which were accompanied by hippocampal mossy fiber sprouting.
Conclusions: Noradrenergic loss converts focally induced sporadic seizures into an epileptogenic SE, which is accompanied by mossy fiber sprouting within the dentate gyrus.     

Posttraumatic mossy fiber sprouting is related to the degree of cortical damage in three mouse strains

Controlled cortical impact injury was used to examine relationships between focal posttraumatic cortical damage and mossy fiber sprouting (MFS) in the dentate gyrus in three mouse strains. Posttraumatic MFS was more robust when cortical injury impinged upon the hippocampus, versus contusions restricted to neocortex, and was qualitatively similar among CD-1, C57BL/6, and FVB/N background strains. Impact parameters influencing injury severity may be critical in reproducing epilepsy-related changes in neurotrauma models.     

Effects of early-life LiCl-pilocarpine-induced status epilepticus on memory and anxiety in adult rats are associated with mossy fiber sprouting and elevated CSF S100B protein

Purpose: This study investigated putative correlations among behavioral changes and: (1) neuronal loss, (2) hippocampal mossy fiber sprouting, and (3) reactive astrogliosis in adult rats submitted to early‐life LiCl‐pilocarpine‐induced status epilepticus (SE). Methods: Rats (P15) received LiCl (3 mEq/kg, i.p.) 12–18 h prior pilocarpine (60 mg/kg; s.c.). At adulthood, animals were submitted to behavioral tasks and after the completion of tasks biochemical and histological analysis were performed. Results: In SE group, it was observed an increased number of degenerating neurons in the CA1 subfield and in the hilus of animals 24 h after SE. At adulthood, SE group presented an aversive memory deficit in an inhibitory avoidance task and the animals that presented lower latency to the step down showed a higher score for mossy fiber sprouting. In the light‐dark exploration task, SE rats returned less and spent less time in the light compartment and present an increased number of risk assessment behavior (RA). There was a negative correlation between the time spent in the light compartment and the score for mossy fiber sprouting and a positive correlation between score for mossy fiber sprouting and number of RA. LiCl‐pilocarpine‐treated animals showed higher levels of S100B immunocontent in the CSF as well as a positive correlation between the score for sprouting and the GFAP immunocontent in the CA1 subfield, suggesting an astrocytic response to neuronal injury. Conclusions: We showed that LiCl‐pilocarpine‐induced SE during development produced long‐lasting behavioral abnormalities, which might be associated with mossy fiber sprouting and elevated CSF S100B levels at adulthood.     

Early-life status epilepticus induces ectopic granule cells in adult mice dentate gyrus

A large number of aberrant hilar granule cells (GCs) are found in the patients and animal models of adult temporal lobe epilepsy (TLE), and these “ectopic” GCs have synchronous epileptiform bursting with other hippocampal neurons. In this study, we investigated whether early-life status epilepticus (SE) induces hilar ectopic GCs that remain in the adulthood because TLE patients frequently experience seizures in the early childhood when a large number of postnatally born GCs migrate in the hilus. To label newborn GCs, bromodeoxyuridine (BrdU) was injected daily for three consecutive days to C57BL/6J mice at different postnatal days starting at postnatal-0-day-old (P0) (Group1), P7 (Group2), or P35 (Group3). Mice in each group underwent pilocarpine-induced SE at P14. Six months later, to determine whether SE induces ectopic GCs, we plotted the distribution of postnatally born GCs which were immunohistochemically defined as BrdU- and the GC marker Prox1-colabeled cells. We also examined whether SE causes the granule cell layer (GCL) dispersion and/or the mossy fiber (MF) sprouting, other representative pathologies of TLE hippocampus. Only SE-experiencing mice in Group1 had significantly more neonatally born ectopic GCs compared with control mice. Neither control nor SE mice had dispersed GCL. All mice that underwent SE had sprouted MFs in CA3. We conclude that early-life SE disrupts a normal incorporation of GCs born pre-SE but not post-SE, inducing ectopic GCs in the adult hilus. Interestingly, the results also indicate that developmentally earlier born GCs are more responsive to early-life SE in terms of the emergence of ectopic GCs.     

Strain differences affect the induction of status epilepticus and seizure-induced morphological changes

Genetic deficits have been discovered in human epilepsy, which lead to alteration of the balance between excitation and inhibition, and ultimately result in seizures. Rodents show similar genetic determinants of seizure induction. To test whether seizure‐prone phenotypes exhibit increased seizure‐related morphological changes, we compared two standard rat strains (Long–Evans hooded and Wistar) and two specially bred strains following status epilepticus. The special strains, namely the kindling‐prone (FAST) and kindling‐resistant (SLOW) strains, were selectively bred based on their amygdala kindling rate. Although the Wistar and Long–Evans hooded strains experienced similar amounts of seizure activity, Wistar rats showed greater mossy fiber sprouting and hilar neuronal loss than Long–Evans hooded rats. The mossy fiber system was affected differently in FAST and SLOW rats. FAST animals showed more mossy fiber granules in the naïve state, but were more resistant to seizure‐induced mossy fiber sprouting than SLOW rats. These properties of the FAST strain are consistent with those observed in juvenile animals, further supporting the hypothesis that the FAST strain shares circuit properties similar to those seen in immature animals. Furthermore, the extent of mossy fiber sprouting was not well correlated with sensitivity to status epilepticus, but was positively correlated with the frequency of spontaneous recurrent seizures in the FAST rats only, suggesting a possible role for axonal sprouting in the development of spontaneous seizures in these animals. We conclude that genetic factors clearly affect seizure development and related morphological changes in both standard laboratory strains and the selectively bred seizure‐prone and seizure‐resistant strains.     

Localized overexpression of FGF-2 and BDNF in hippocampus reduces mossy fiber sprouting and spontaneous seizures up to 4 weeks after pilocarpine-induced status epilepticus

Purpose: We have recently reported that viral vector–mediated supplementation of fibroblast growth factor‐2 (FGF‐2) and brain‐derived neurotrophic factor (BDNF) in a lesioned, epileptogenic rat hippocampus limits neuronal damage, favors neurogenesis, and reduces spontaneous recurrent seizures. To test if this treatment can also prevent hippocampal circuit reorganization, we examined here its effect on mossy fiber sprouting, the best studied form of axonal plasticity in epilepsy. Methods: A herpes‐based vector expressing FGF‐2 and BDNF was injected into the rat hippocampus 3 days after an epileptogenic insult (pilocarpine‐induced status epilepticus). Continuous video–electroencephalography (EEG) monitoring was initiated 7 days after status epilepticus, and animals were sacrificed at 28 days for analysis of cell loss (measured using NeuN immunofluorescence) and mossy fiber sprouting (measured using dynorphin A immunohistochemistry). Key Findings: The vector expressing FGF‐2 and BDNF decreased both mossy fiber sprouting and the frequency and severity of spontaneous seizures. The effect on sprouting correlated strictly with the cell loss in the terminal fields of physiologic mossy fiber innervation (mossy cells in the dentate gyrus hilus and CA3 pyramidal neurons). Significance: These data suggest that the supplementation of FGF‐2 and BDNF in an epileptogenic hippocampus may prevent epileptogenesis by decreasing neuronal loss and mossy fiber sprouting, that is, reducing some forms of circuit reorganization.     

Plastic Changes and Disease-modifying Effects of Scopolamine in the Pilocarpine Model of Epilepsy in Rats

Summary:  Purpose: We describe the use of a clinically relevant pharmacological intervention that alters the clinical history of status epilepticus (SE)-induced spontaneous recurrent seizures (SRS) in the pilocarpine model and the possible plastic changes underlying such an effect.
Methods: Two hours after pilocarpine-induced SE (320–350 mg/kg, i.p.), rats received scopolamine 1–2 mg/kg i.p. or saline, every 6 h for 3 days. After that, osmotic minipumps were implanted for continuous delivery of scopolamine or saline for an additional 14 days. Animals were video-monitored for 12 h/week during the following 3-month period for the occurrence of SRS and, thereafter, were perfused, processed, and coronal brain sections were stained for acetylcholinesterase (AChE) and for the presence of supragranular mossy fibers (Timm).
Results: Treatment with scopolamine led to significantly fewer SRS. Staining for AChE in the dentate gyrus was significantly more intense in naïve animals. The scopolamine group had the least intense AChE staining of all groups. However, regression analysis of the AChE staining for this group did not correlate with the presence or absence of SRS, or the latency or frequency of SRS. Supragranular mossy fiber sprouting developed in all animals experiencing pilocarpine-induced SE, irrespective of whether or not they were treated with scopolamine.
Conclusions: Pilocarpine-induced SE in the presence of scopolamine might produce animals that, despite mossy fiber sprouting, were not seen to exhibit spontaneous seizures. In addition, our data suggest that the encountered changes in the AChE staining in the dentate gyrus that followed treatment with scopolamine do not help to explain its disease-modifying effects.     

Effect of theophylline and trimethobenzamide when given during kainate-induced status epilepticus: an improved histopathologic rat model of human hippocampal sclerosis

PURPOSE: The most common pathology in temporal lobe epilepsy (TLE) is hippocampal sclerosis. It is controversial whether status epilepticus (SE) or prolonged seizures plus secondary cerebral injuries are pathogenic mechanisms of hippocampal sclerosis. This study addressed this question in rat models of TLE. METHODS: Hippocampal neuron densities and supragranular mossy fiber sprouting were determined in adult rats subjected to systemic kainate-induced SE (KA-only) and KA-induced SE followed 75 minutes later by theophylline (KA/Theo) or trimethobenzamide (KA/Tri). These drugs probably decrease seizure-induced cerebral hyperemia or hypertension. RESULTS: Compared with controls and KA-only rats, KA/Tri and KA/Theo rats showed decreased CA3b and CA1 neuron densities (i.e., greater Sommer's sector injury). In addition, KA/Tri rats showed that increased trimethobenzamide dosages were associated with decreased hilar, CA3c, CA3b, CA1, and subiculum neuron densities. There were no significant differences in supragranular mossy fiber sprouting between KA-only, KA/Tri, and KA/Theo rats. CONCLUSIONS: Pharmacologic manipulations during KA-induced SE are associated with differences in hippocampal pathology, especially in Sommer's sector, and the final pattern of damage and axon sprouting shows histopathologic similarities to that in patients with hippocampal sclerosis. Our findings support the hypothesis that secondary physiologic insults during SE that are likely to decrease seizure-induced cerebral hyperemia and hypertension may generate greater hippocampal neuronal injury compared with SE alone, and this may be a pathogenic mechanism of human hippocampal sclerosis in patients with TLE.     

Kainic acid induction of mossy fiber sprouting: dependence on mouse strain

After seizures caused by kindling or kainic acid (KA), hippocampal granule-cell axons, the mossy fibers, sprout into the supragranular layer of the rat. The mechanisms underlying this phenomenon remain elusive, but excitotoxic loss of hilar cells, which project to this supragranular layer, is suspected to be a critical determinant. Consistent with this hypothesis, we previously reported that while rats show mossy fiber sprouting after kainate, ICR mice do not. This may be associated with the observation that ICR mice, unlike rats, do not appear to show hilar cell death after KA (McNamara et al., Mol Brain Res 1996;40:177-187). Other strains of mice, however, such as 129/SvEMS, do show hilar cell death after KA (Schauwecker and Steward, Proc Natl Acad Sci USA 1997;94:4103-4108). We examined the possibility that the 129/SvEMS mouse strain would show granule-cell sprouting, in contrast to ICR mice. After administration of KA, mossy fiber sprouting was indeed observed in strain 129/SvEMS, but only in animals displaying evident hilar cell death. In contrast, neither hilar cell death nor mossy fiber sprouting was observed in ICR mice, confirming previous results. Both mouse strains demonstrated comparable behavioral seizures. These results strengthen the view that hilar cell death, together with epileptogenesis, triggers reactive synaptogenesis and mossy fiber sprouting.     

Effects of herbimycin A in the pilocarpine model of temporal lobe epilepsy

Pilocarpine-induced status epilepticus (SE) causes widespread tyrosine phosphorylation in the brain. It has been postulated that this intracellular signal may mediate potentially epileptogenic changes in the morphology and physiology of particular brain regions, including the hippocampus. The present study evaluated the effects of herbimycin A, a protein tyrosine kinase (PTK) inhibitor, over the acute (during which intense biochemical and electrophysiological activation occurs) and the chronic phase (characterized by spontaneous and recurrent epileptic seizures and the presence of synaptic reorganization, e.g., mossy fiber sprouting) of the pilocarpine model of epilepsy. The administration of a single dose of 1.74 nmol of herbimycin A (i.c.v., 5 microL) 5 min after the onset of SE did not change the acute behavioral manifestation of seizures despite significantly decreasing c-Fos immunoreactivity in different areas of the hippocampus and of the limbic cortex. Herbimycin-treated animals developed spontaneous recurrent seizures, as did control animals, with a similar latency for the appearance of the first seizure and similar seizure frequency. Neo-Timm staining revealed that all animals experiencing SE, regardless of whether or not injected with herbimycin, showed aberrant mossy fiber sprouting in the supragranular region of the dentate gyrus. Herbimycin did not obviously affect neuronal cell death as evaluated in Nissl-stained sections. These results indicate that the PTK blockade achieved with the current dose of herbimycin reduced the acute c-Fos expression but failed to alter the spontaneous seizure frequency or to attenuate the morphological modifications triggered by the SE.     

N-cadherin在颞叶癫痫海马苔藓纤维出芽和突触重组中的作用

目的 探讨神经性钙粘附分子(N-cadherin)在癫痫状态后海马苔藓纤维出芽和突触重组中的作用。方法取锂一匹罗卡品诱导大鼠癫痫持续状态及慢性自发性颞叶癫痫发作期的大鼠脑片，用Timm染色和免疫组化的方法分别检测苔藓纤维出芽和N-cadherin在大鼠海马组织中的表达。结果癫痫状态后第2周和第4周的实验组大鼠可见到苔藓纤维出芽，穿越齿状回颗粒细胞层到达内分子层，并在此形成一条致密的层状带(Timm染色)。免疫组化染色发现实验组大鼠在第2周和第4周，海马齿状回内分子层可以看到强染色，并形成一条致密带，与Timm染色时观察到的条带一致。结论癫痫状态后在海马齿状回内分子层N-cadherin的表达上调．N-cadherin可能参与了癫痫后苔藓纤维出芽和突触重组过程。     

Hippocampal injury,atrophy, synaptic reorganization,and epileptogenesis after perforant pathway stimulation‐induced status epilepticus in the mouse

Prolonged dentate granule cell discharges produce hippocampal injury and chronic epilepsy in rats. In preparing to study this epileptogenic process in genetically altered mice, we determined whether the background strain used to generate most genetically altered mice, the C57BL/6 mouse, is vulnerable to stimulation‐induced seizure‐induced injury. This was necessary because C57BL/6 mice are reportedly resistant to the neurotoxic effects of kainate‐induced seizures, which we hypothesized to be related to strain differences in kainate's effects, rather than genetic differences in intrinsic neuronal vulnerability. Bilateral perforant pathway stimulation‐induced granule cell discharge for 4 hours under urethane anesthesia produced degeneration of glutamate receptor subunit 2 (GluR2)‐positive hilar mossy cells and peptide‐containing interneurons in both FVB/N (kainate‐vulnerable) and C57BL/6 (kainate‐resistant) mice, indicating no strain differences in neuronal vulnerability to seizure activity. Granule cell discharge for 2 hours in C57BL/6 mice destroyed most GluR2‐positive dentate hilar mossy cells, but not peptide‐containing hilar interneurons, indicating that mossy cells are the neurons most vulnerable to this insult. Stimulation for 24 hours caused extensive hippocampal neuron loss and injury to the septum and entorhinal cortex, but no other detectable damage. Mice stimulated for 24 hours developed hippocampal sclerosis, granule cell mossy fiber sprouting, and chronic epilepsy, but not the granule cell layer hypertrophy (granule cell dispersion) produced by intrahippocampal kainate. These results demonstrate that perforant pathway stimulation in mice reliably reproduces the defining features of human mesial temporal lobe epilepsy with hippocampal sclerosis. Experimental studies in transgenic or knockout mice are feasible if electrical stimulation is used to produce controlled epileptogenic insults. J. Comp. Neurol. 515:181–196, 2009. © 2009 Wiley‐Liss, Inc.     

红藻氨酸致颞叶癫(癎)大鼠脑内Semaphorin3A、Semaphorin4C基因表达的研究

目的研究轴索导向分子Semaphorin3A(Sema3A)、4C(Sema4C)对癫大鼠海马苔藓纤维重建的调控作用及对皮层神经元的保护作用。方法大鼠侧脑室内注射红藻氨酸制备颞叶癫模型,原位杂交法检测致痫间后1d,1、2、3、4周大鼠脑内Sema3A/Sema4C mRNA表达。结果致痫间后1周Sema3A、Sema4CmRNA分别在齿状回(DG),CA3区表达明显下降(P<0.01),持续至3、4周时恢复至正常(P>0.05);致痫间后1d Sema3A mRNA在皮层表达明显下降(P<0.01),持续至1、2周后恢复至正常(P>0.05)。结论红藻氨酸致痫间后DG及CA3区神经元分别下调Sema3A/Sema4C mRNA的表达,促进癫大鼠苔藓纤维重建;皮层神经元通过下调Sema3A mRNA的表达来维持自身存活。