Neuropathogical features of a rat model for perinatal hypoxic-ischemic encephalopathy with associated epilepsy

Hypoxic-ischemic (HI) encephalopathy is an important neurological problem of the perinatal period. Little is known of the long-term progression of HI insults or the maladaptive changes that lead to epilepsy. Using rats with unilateral carotid occlusion followed by hypoxia at postnatal day 7, this study provides an initial analysis of the epilepsy caused by a perinatal HI insult with chronic and continuous behavioral monitoring. The histopathology was investigated at postnatal day 30 and later at > or =6 months of age using cresyl violet, Timm, and rapid Golgi staining and immunocytochemistry. The resultant epilepsy showed an increase in seizure frequency over time, with a preponderance for seizure clusters and behavioral features of an ipsilateral cerebral syndrome. In addition to parasagittal infarcts and porencephalic cysts in severe lesions, columnar neuronal death was found with cytomegaly in isolated groups of dysmorphic cortical neurons. Cortical dysgenesis was seen in the form of deep laminar cell loss, microgyri, white matter hypercellularity, and blurring of the white and gray matter junction. Mossy fiber sprouting was not only detected in the atrophied ipsilateral dorsal hippocampus of HI rats with chronic epilepsy, but was also found in comparable grades in spared ipsi- and contralateral ventral hippocampi. The cortical lesions in this animal model show histological similarities with those found in humans after perinatal HI. The occurrence of cortical abnormalities that are associated with epilepsy in humans correlates with the consequent detection of spontaneous recurrent seizures.     

The Neuropathology of Hyperthermic Seizures in the Rat

Summary: Purpose: Single and repeated hyperthermic seizures were induced in rats beginning at age 22 days to determine the neuroanatomic consequences to the hippocampus and to compare these changes with those in the hippocampi of patients with temporal lobe epilepsy (TLE) experiencing febrile seizures.
Methods: Hyperthermic seizures were induced by placing rats in a bath of water at 45°C for 4 min. Seizures were visually observed, and some animals also were monitored electroen-cephalographically. Neurodegeneration was examined with a silver stain, whereas granule cell sprouting was detected with the Timm stain.
Results: In a majority of rats, hyperthermia-induced tonicclonic seizures ranged in duration from 30 s to 6 min; the seizure duration increased with the number of seizures. No neurodegeneration was detectable in these animals, although there was sprouting of granule cell collaterals into the inner molecular layer (IML) of the dentate. In a small number of animals, the short seizures evolved into status epilepticus, and neuronal degeneration was present in the hippocampus and other parts of the temporal lobe, and the mediodorsal thalamus.
Conclusions: This study confirms the relation between hyperthermia and seizure occurrence. It shows in particular that, as in the human, only prolonged seizures such as status epilepticus cause a pattern of neurodegeneration similar to that observed in human TLE.     

Kainic acid induced hippocampal seizures in rats: comparisons of acute and chronic seizures using intrahippocampal versus systemic injections

Hyppocampal epilepsy is a recently defined syndrome occurring in 65% of all temporal lobe epilepsies as defined by: 1) electrographic (EEG) onset in the hippocampus (HC) prior to EEG seizures elsewhere, 2) post-resection hippocampal sclerosis and mossy fiber synaptic reorganizations and 3) relief of typical complex partial seizures after surgical resection of the hyppocampus. We used intrahippocampal kainic acid injections V² in rats at different developmental ages (postnatal 7 through adult) to develop long term spontaneous HC EEG spikes, EEG seizures, and behavioral seizures. Split-screen video/EEG monitoring demonstrated that this intrahippocampal kainic acid model produced progressive development of: 1) ipsilateral interictal spikes, 2) later polyspike complexes, 3) bilaterally-asynchronous EEG spiking, 4) unilateral HC EEG seizure onsets with occasional secondarily generalized spread to apposite HC and motor cortex to elicit complex partial seizures, and 5) in all seizing rats there was mossy fiber synaptic reorganization, even when injected at age 7 days. These results indicate that the intrahippocampal kainic acid injection model is similar to human hippocampal epilepsy.Supported by NIH Grants NS 02808, NS 31655 (T.L.B.); K08 NS 1603 (G.W.M.); and Fogarty Fellowship TWO 4959 (J.P.L.).     

Hippocampal Nogo-A and neo-Timm's staining in amygdala kindling rats

PURPOSE: Nogo-A, one of the axon regeneration inhibitors, has been shown to be up-regulated in both the experimental and human temporal lobe epilepsy. However, the role of Nogo-A in mossy fiber sprouting (MFS) relative to epileptogenesis is unknown. This work was designed to examine the relationship of the hippocampal Nogo-A protein expression with MFS during the development of amygdala kindling. METHODS: Using immunohistochemistry and neo-Timm's histological procedures, we evaluated the distribution and density of Nogo-A and Nogo-66 receptor (Ng-R) expression and MFS in the bilateral hippocampus of amygdala kindling rats. RESULTS: Nogo-A expression in the ipsilateral hippocampus gradually increased with the development of kindling in the sector CA2-3. In contrast, no increased Nogo-A expression was observed in the contralateral hippocampus as the rats advanced to stage 5 kindled seizures. Furthermore, poorer Nogo-A and Nogo-66 receptor (Ng-R) expression were observed in the dentate granule cells as aberrant MFS occurred. CONCLUSIONS: In amygdala kindling rats, generalized stage 5 seizures were not associated with increased Nogo-A expression in the contralateral hippocampus supporting the concept that seizures by themselves do not induce Nogo-A expression. Furthermore, in the ipsilateral hippocampus, the expression of Nogo-A relative to MSF suggests that this protein may partially control aberrant synaptic reorganization during epileptogenesis.     

Experimental Models of Temporal Lobe Epilepsy: New Insights from the Study of Kindling and Synaptic Reorganization

Summary: Temporal lobe epilepsy is a common localization-related epileptic syndrome characterized by complex partial seizures, ictal and interictal epileptic discharges arising from limbic structures of the temporal lobe, and association with hippocampal sclerosis. Temporal lobe epilepsy may follow perinatal injury and febrile convulsions, may be progressive, and frequently becomes refractory to standard antiepileptic therapy. The neurobiology that underlies these features of temporal lobe epilepsy is not known. Recent studies in experimental models have provided new insights that may help clarify the relationship of seizures, hippocampal sclerosis, and temporal lobe epilepsy. Observations from the study of the hippocampus with kainic acid-induced lesions, the kindling model, and other experimental models of epilepsy have demonstrated that seizures induce structural and electrophysiologic alterations in hippocampal pathways that may lead to increased excitability and could play a role in the development and progression of temporal lobe epilepsy. These alterations include mossy fiber synaptic reorganization, induction of NMDA-mediated synaptic transmission, and progressive hippocampal neuronal loss induced by brief kindled seizures. Some of the structural alterations induced by kindling have also been observed in the human epileptic temporal lobe, raising the possibility that mechanisms operative in kindling may play a role in the pathogenesis of hippocampal sclerosis and in the syndrome of human temporal lobe epilepsy.     

Mossy fiber synaptic reorganization in the epileptic human temporal lobe

The distribution of the mossy fiber synaptic terminals was examined using the Timm histochemical method in surgically excised hippocampus and dentate gyrus from patients who underwent lobectomy of the anterior part of the temporal lobe for refractory partial complex epilepsy. The dentate gyrus of epileptic patients demonstrated intense Timm granules and abundant mossy fiber synaptic terminals in the supragranular region and the inner molecular layer. In contrast, the dentate gyrus of presenescent nonepileptic primates demonstrated no Timm granules in the supragranular region. In nonepileptic senescent primates, occasional very sparse supragranular Timm granules were results are morphological evidence of mossy fiber synaptic reorganization in the temporal lobe of epileptic humans, and suggest the intriguing possibility that mossy fiber sprouting and synaptic reorganization induced by repeated partial complex seizures may play a role in human epilepsy.     

Sprouting of GABAergic and mossy fiber axons in dentate gyrus following intrahippocampal kainate in the rat.

The present study examined the bilateral synaptic rearrangements of presumed gamma-aminobutyric acid (GABAergic) inhibitory axons and mossy fiber (presumed excitatory) recurrent collaterals following intrahippocampal kainic acid (KA) injection. Glutamate decarboxylase immunoreactivity (GAD-IR) was used to study inhibitory axon terminal sprouting, following 0.5 microgram KA/0.2 microliter injected unilaterally into the posterior hippocampus of rats (n = 16), with survival periods of 14, 28, and 120 days. The age-matched control animals (n = 9) received intrahippocampal 0.2 microliter saline (sham, n = 4) or no injection (normal, n = 5). To study mossy fiber synaptic rearrangements, 0.5 microgram KA/0.2 microliter volumes were injected unilaterally into the posterior hippocampus of rats (n = 10), with survival periods from 14, 28, and 120 days, and Timm sulfide-stained tissue sections were compared to age-matched sham (n = 4) or normal controls (n = 4). At 14 through 120 days after posterior KA injection, GAD-IR puncta were significantly increased in the ipsi- and contralateral inner molecular layers (IML) of the fascia dentata (FD) when compared to sham or normal controls. KA lesion also induced mossy fiber recurrent collateral sprouting into the ipsi- and contralateral FD IMLs. The loss of both the commissural and ipsilateral associational afferents to the FD apparently induced sprouting into their ipsi- and contralateral termination zones by granule cell mossy fibers and GAD-IR axons, thus establishing an abnormal circuitry near the observed pathology in the kainate model of epilepsy. Although reactive synaptogenesis of mossy fibers producing monosynaptic excitation may be one mechanism for KA epileptogenicity, the concurrent sprouting of GABAergic terminals in the same IML zone of the FD suggests that anomalous inhibitory synapses may contribute to chronic KA hippocampal hyperexcitability.     

Focal neocortical epilepsy affects hippocampal volume, shape, and structural integrity: a longitudinal MRI and immunohistochemistry study in a rat model

Purpose: Extratemporal epilepsy often coincides with cognitive decline, which may be associated with hippocampal dysfunction. Severe hippocampal sclerosis can be detected with conventional neuroimaging in some patients with chronic extrahippocampal epilepsy (so‐called dual pathology). However, subtle structural hippocampal changes may already develop at a much earlier phase, and in a larger number of patients. Our goal was to longitudinally characterize the development of bilateral hippocampal pathology in an experimental neocortical focal epilepsy model. Methods: Focal unilateral neocortical epilepsy was induced by microinjection of tetanus toxin in the primary motor cortex in adult male Sprague‐Dawley rats. Another group of age‐matched rats served as controls. In both groups, structural magnetic resonance imaging (MRI) was performed at 1, 3, 7, and 10 weeks of follow‐up. Bilateral hippocampi were outlined and macroscopically analyzed using a state‐of‐the‐art point‐based morphometry model. Hippocampal microstructural changes at the end of follow‐up, 10 weeks after epilepsy induction, were assessed with postmortem standard cresyl‐violet, Fluoro‐Jade, proteolipid protein 1, vimentin, glial fibrillary acidic protein, and ionized calcium binding adaptor molecule 1 stainings. Key Findings: All rats in the injected group developed seizures. The ipsilateral hippocampal volume was on average 8.76 (mean) ± 3.32% (standard deviation) smaller in the epileptic animals as compared to controls (p = 0.01) during the 10 weeks of follow‐up. The contralateral hippocampus showed a similar reduction of 8.49 (mean) ± 3.27% (standard deviation) in total volume (p = 0.02). Clear hippocampal shape differences were found between the two groups. The most affected areas after epilepsy induction were the bilateral dorso‐mediorostral, dorsolateral, and ventrolateral areas of the hippocampi. Normal developmental shape changes of the hippocampus, as detected in control rats, were largely absent in the ipsilateral hippocampus of epileptic rats. Quantitative histologic analysis revealed significant neuronal loss in the hippocampus, most pronounced in the hilar subregion, globally impaired myelination, reactive astrocytosis, and activated microglia. We found a weak but significant correlation between the number of neurons and hippocampal volume (r = 0.25, p = 0.0025). Significance: We found evidence of hippocampal pathology in both hemispheres following experimental focal neocortical epilepsy. The observed development of bilateral hippocampal pathology, with onset in the early stages of focal neocortical epilepsy, may be a significant factor in comorbidities, such as cognitive dysfunction, found in patients with extratemporal localization‐related epilepsy.     

Development of later life spontaneous seizures in a rodent model of hypoxia-induced neonatal seizures

Purpose: To study the development of epilepsy following hypoxia‐induced neonatal seizures in Long‐Evans rats and to establish the presence of spontaneous seizures in this model of early life seizures. Methods: Long‐Evans rat pups were subjected to hypoxia‐induced neonatal seizures at postnatal day 10 (P10). Epidural cortical electroencephalography (EEG) and hippocampal depth electrodes were used to detect the presence of seizures in later adulthood (>P60). In addition, subdermal wire electrode recordings were used to monitor age at onset and progression of seizures in the juvenile period, at intervals between P10 and P60. Timm staining was performed to evaluate mossy fiber sprouting in the hippocampi of P100 adult rats that had experienced neonatal seizures. Key Findings: In recordings made from adult rats (P60–180), the prevalence of epilepsy in cortical and hippocampal EEG recordings was 94.4% following early life hypoxic seizures. These spontaneous seizures were identified by characteristic spike and wave activity on EEG accompanied by behavioral arrest and facial automatisms (electroclinical seizures). Phenobarbital injection transiently abolished spontaneous seizures. EEG in the juvenile period (P10–60) showed that spontaneous seizures first occurred approximately 2 weeks after the initial episode of hypoxic seizures. Following this period, spontaneous seizure frequency and duration increased progressively with time. Furthermore, significantly increased sprouting of mossy fibers was observed in the CA3 pyramidal cell layer of the hippocampus in adult animals following hypoxia‐induced neonatal seizures. Notably, Fluoro‐Jade B staining confirmed that hypoxic seizures at P10 did not induce acute neuronal death. Significance: The rodent model of hypoxia‐induced neonatal seizures leads to the development of epilepsy in later life, accompanied by increased mossy fiber sprouting. In addition, this model appears to exhibit a seizure‐free latent period, following which there is a progressive increase in the frequency of electroclinical seizures.     

Neuron loss,granule cell axon reorganization,and functional changes in the dentate gyrus of epileptic kainate-treated rats

We sought to describe quantitatively the morphological and functional changes that occur in the dentate gyrus of kainate-treated rats, an experimental model of temporal lobe epilepsy. Adult rats were treated systemically with kainic acid, and, months later, after displaying spontaneous recurrent motor seizures, their dentate gyri were examined. Histological, immunocytochemical, and quantitative stereological techniques were used to estimate numbers of neurons per dentate gyrus of various classes and to estimate the extent of granule cell axon reorganization along the septotemporal axis of the hippocampus in control rats and epileptic kainate-treated rats. Compared with control rats, epileptic kainate-treated rats had fewer Nissl-stained hilar neurons and fewer somatostatin-immunoreactive neurons. There was a correlation between the extent of hilar neuron loss and the extent of somatostatin-immunoreactive neuron loss. However, functional inhibition in the dentate gyrus, assessed with paired-pulse responses to perforant-pathway stimulation, revealed enhanced, and not the expected reduced, inhibition in epileptic kainate-treated rats. Numbers of parvalbumin- and cholecystokinin-immunoreactive neurons were similar in control rats and in most kainate-treated rats. A minority (36%) of the epileptic kainate-treated rats had fewer parvalbumin- and cholecystokinin-immunoreactive neurons than control rats, and those few (8%) with extreme loss in these interneuron classes showed markedly hyperexcitable dentate gyrus field-potential responses to orthodromic stimulation. Compared with control rats, epileptic kainate-treated rats had larger proportions of their granule cell and molecular layers infiltrated with Timm stain. There was a correlation between the extent of abnormal Timm staining and the extent of hilar neuron loss. Granule cell axon reorganization and dentate gyrus neuron loss were more severe in temporal vs. septal hippocampus. These findings from the dentate gyrus of epileptic kainate-treated rats are strikingly similar to those reported for human temporal lobe epilepsy, and they suggest that neuron loss and axon reorganization in the temporal hippocampus may be important in epileptogenesis. J. Comp. Neurol. 385:385–404, 1997. © 1997 Wiley-Liss, Inc.     

Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of ‘excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.     

Reduced function of L-AP4-sensitive metabotropic glutamate receptors in human epileptic sclerotic hippocampus

Human temporal lobe epilepsy is characterized by strong synaptic reorganization that leads to abnormal recurrent excitatory synaptic connections among hippocampal neurons. In addition, electrophysiological studies show that synaptic activity of the main afferent input to the hippocampus, the perforant path, is prolonged and amplified by changes in postsynaptic glutamate receptors. The current view is that these morphological and physiological abnormalities contribute significantly to the hyperexcitability seen in the hippocampus of temporal lobe epilepsy. Recently, it was found that presynaptic inhibitory metabotropic glutamate receptors are an important negative feedback mechanism that controls synaptic release of glutamate in the hippocampus. In this study, we assessed the functionality of this feedback system by investigating the metabotropic glutamate receptor mediated depression of excitatory synaptic transmission in surgically removed hippocampi from patients with marked synaptic reorganization (Ammon's horn sclerosis group) and from patients without detectable reorganization (lesion group). We report here that this control of synaptic transmission is lost in hippocampi from the Ammon's horn sclerosis group whereas this control is preserved in hippocampi from the lesion group. The data presented here suggest that the loss of feedback inhibition mediated by metabotropic glutamate receptors could be a further, previously not recognized, mechanism in the pathophysiology of temporal lobe epilepsy.     

The effect of hypoxic-ischemic brain injury in perinatal rats on the abundance and proteolysis of brevican and NG2

Oligodendrocyte (OL) progenitor cells are particularly susceptible to perinatal hypoxia/ischemia (H-I) resulting in decreased myelination and attenuated development of white matter fiber tracts. Brevican is an aggregating chondroitin sulfate proteoglycan (CSPG) secreted by OLs and their progenitors prior to and during active developmental myelination whereas neuron-glia antigen 2 (NG2) is a transmembrane CSPG produced by early OL progenitors. Although both proteoglycans are associated with maturation of OLs, it is not known if they are altered by H-I brain injury in the neonate. We have therefore examined the time course of changes in brevican and NG2 abundance and proteolysis in the neonatal rat hippocampus after H-I. In a standard H-I model of unilateral carotid artery ligation and exposure to hypoxia, a cavitary infarct involving the ipsilateral parietal and temporal regions of cerebral cortex, hippocampus, and striatum of most rat pups was clearly evident 4 days after H-I. The abundance of total extractable brevican was markedly reduced in the ipsilateral hippocampus at 1 and 14 days after H-I (relative to the contralateral side). At these times, the total G1 proteolytic fragment of brevican was lower in the ipsilateral hippocampus and the level of a protease-generated brevican fragment was significantly diminished in the OL-rich hippocampal fimbria. Hippocampal NG2 levels were also lower at 1 and 4 days after H-I, but were not different from the contralateral side at 14 days. Since brevican, brevican G1 fragment, and NG2 loss occur around the time of progressive cell death and the appearance of the infarct, it may be that H-I rapidly induces a cellular response that actively depletes these proteoglycans from the hippocampal matrix. While the mechanism of this loss is unclear, it would appear to be an early event in the process that could be involved in apoptotic cell death and/or tissue injury.     

Pentylenetetrazol-induced recurrent seizures in rat pups: time course on spatial learning and long-term effects

PURPOSE: Recurrent seizures in infants are associated with a high incidence of neurocognitive deficits. Animal models have suggested that the immature brain is less vulnerable to seizure-induced injury than is that in adult animals. We studied the effects of recurrent neonatal seizures on cognitive tasks performed when the animals were in adolescence and adulthood. METHODS: Seizures were induced by intraperitoneal injection of pentylenetetrazol (PTZ) for 5 consecutive days, starting from postnatal day 10 (P10). At P35 and P60, rats were tested for spatial memory by using the Morris water maze task. In adulthood, motor performance was examined by the Rotarod test, and activity level was assessed by the open field test. Seizure threshold was examined by inhalant flurothyl. To assess presence or absence of spontaneous seizures, rats were video recorded for 4 h/day for 10 consecutive days for the detection of spontaneous seizures. Finally, brains were examined for histologic evidence of injury with cresyl violet stain and Timm staining in the supragranular zone and CA3 pyramidal cell layers of the hippocampus. RESULTS: PTZ-treated rats showed significant spatial deficits in the Morris water maze at both P35 and P60. There were no differences in seizure threshold, motor balance, or activity level during the open field test. Spontaneous seizures were not recorded in any rat. The cresyl violet stain showed no cell loss in either the control or experimental rats. PTZ-treated rats exhibited more Timm staining in the CA3 subfield. However, the control and experimental rats showed similar Timm staining within the supragranular zone. CONCLUSIONS: Our findings indicate that recurrent PTZ-induced seizures result in long-term cognitive deficits and morphologic changes in the developing brain. Furthermore, these cognitive deficits could be detected during pubescence.     

Structural consequences of Kcna1 gene deletion and transfer in the mouse hippocampus

PURPOSE: Mice lacking the Kv1.1 potassium channel alpha subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1(-/-) (knock-out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. METHODS: Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1(-/-) animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1(-/-) brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E. coli lacZ reporter gene. Vector-injected mice were examined to determine the extent of neuronal infection. RESULTS: Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1(-/-) mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed "ectopic" Kv1.1 alpha channel subunit expression. CONCLUSIONS: Kcna1 deletion in mice results in a seizure disorder that resembles--electrographically and neuropathologically--the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection.     

电刺激构建大鼠皮层网络癫痫的海马细胞电生理特征

本文旨在探讨急性强直电刺激右后背海马（hippocampus，HPC）诱导出现双侧皮层网络癫痫的前背HPC神经元电生理机制。实验共用雄性SD大鼠35只，急性强直电刺激（60Hz，2s，0．4-0．6mA）大鼠右侧后背HPC（a—cute tetanization of the right posterior dorsal hippocampus，ATPDH）诱发癫痫模型。四通道同步记录双侧皮层电图（electrocorticogram，ECoG）或前背HPC深部电图和双侧前背HPC单位放电。结果显示，ATPDH可以引起以下效应：（1）双侧皮层癫痫样电活动起源于同侧HPC单位后放电，继而引起对侧HPC单位后放电，最终形成对侧和同侧皮层电图癫痫发作样电振荡；（2）增强双侧皮层4—10Hz节律性电振荡或诱发双侧HPCI00—250Hz电振荡，与此同时双侧HPC神经元均表现为非对称性电活动；（3）双侧HPC神经元锋电位间期（interspike interval，ISI）点呈现非规则性环状分布。同侧HPC爆发式单位放电IsI点分布的环状分层较规则、发生率较高，并与同侧HPC电振荡最大振幅的正弦式波动形成明显的时间对应关系。结果表明：ATPDH可诱导双侧皮层和HPC电图形成癫痫电网络。当皮层或HPC癫痫网络重建过程中，HPC神经元放电具有特征性ISI点环状分布等编码形式。     

Electrophysiologic Analysis of a Chronic Seizure Model After Unilateral Hippocampal KA Injection

PURPOSE: Unilateral intrahippocampal injections of kainic acid (KA) in rats produce spontaneous recurrent limbic seizures and morphologic changes in hippocampus that resemble hippocampal sclerosis in patients with medically refractory mesial temporal lobe epilepsy (MTLE), that form of temporal lobe epilepsy (TLE) associated with hippocampal sclerosis. Interictal in vivo electrophysiologic studies have revealed high-frequency (250-500 Hz) oscillations, termed fast ripples (FRs). These oscillations may uniquely occur in or adjacent to the site of hippocampal KA injection, in areas that generate spontaneous seizures. Similar field potentials also have been demonstrated in the epileptogenic region of patients with TLE. We have now characterized ictal electrographic patterns in this rat model for comparison with those in human TLE and begun to evaluate the role of FRs in the transition to ictus in the KA-treated rat. METHODS: Rats received unilateral intrahippocampal injections of KA and, after the development of spontaneous seizures, were implanted with multiple fixed and moveable microelectrodes for single unit, field potential, and EEG recording. They were then monitored by using video-EEG telemetry for several weeks to capture and evaluate electrographic and behavioral seizure types. Results were correlated with Timm's stain demonstration of mossy fiber sprouting. RESULTS: Low-voltage fast (LVF) and hypersynchronous electrographic ictal-onset patterns were seen in the KA-treated rat that resembled similar ictal-onset patterns in patients with TLE. Hypersynchronous, but not LVF, ictal discharges were associated with recurrent FRs. As in the human, hypersynchronous ictal onsets originated predominantly in hippocampus, whereas LVF ictal onsets more often involved extrahippocampal structures. LVF ictal onsets occurred during wakefulness or paradoxical sleep and were usually associated with motor behavior, whereas hypersynchronous ictal onsets occurred during slow-wave sleep or periods of immobility and were not associated with motor behavior unless there was transition to another ictal electrographic pattern. Mossy fiber sprouting did not correlate with the frequency of ictal EEG discharges exhibited by each rat but was greater in those rats that demonstrated frequent behavioral seizures. CONCLUSIONS: The electrographic features of spontaneous seizures in the KA-treated rat resemble those of patients with medically refractory TLE with respect to EEG pattern and localization. Our data suggest that hypersynchronous ictal onsets represent epileptogenic disturbances in hippocampal circuits, whereas LVF ictal onsets may involve extrahippocampal areas having more direct connections to the motor system. Hypersynchronous seizures may involve the same neuronal mechanisms that generate interictal FRs.     

Sequential changes in glutamate transporter protein levels during Fe(3+)-induced epileptogenesis

Severe head injury in humans causes recurrent seizures; this form of epilepsy appears to correlate with occurrence of parenchymal hemorrhage. Injection of ferric cations, one component of hemoglobin, into rat amygdala, causes lipid peroxidation, and recurrent spontaneous seizures. We wondered whether regulation of extracellular glutamate might be perturbed as a mechanism of chronic epileptogenesis, therefore levels of glutamate transporter proteins GLT-1, GLAST and EAAC-1 were measured in ipsilateral and contralateral hippocampi removed from rats having spontaneous iron-induced limbic seizures. The neuronal transporter EAAC-1 was elevated bilaterally up to 30 days following the microinjection that initiated seizures. The neuronal transporter EAAC-1 was elevated bilaterally up to 30 days following the microinjection that initiated seizures. The glial transporter GLT-1 increased 5 and 15 days after iron injection on the side contralateral to the injection then returned to basal levels 30 days after the lesion. GLAST also showed an initial increase but at 15 and 30 days after injection, when experimental animals were experiencing spontaneous limbic behavioral seizures, this protein was down-regulated. The results suggest that iron-induced epileptogenesis involves alteration in glial glutamate transport that may lead to enhanced excitation within the hippocampus.     

Drebrin A expression is altered after pilocarpine-induced seizures: time course of changes is consistent for a role in the integrity and stability of dendritic spines of hippocampal granule cells

We used a pathophysiological model of temporal lobe epilepsy induced by pilocarpine in adult rats in order to assess the in vivo role of drebrin A (DA), one of the major regulators of F-actin. This model displays a dynamic reorganization of the glutamatergic network including neo-spinogenesis, morphogenesis, and neo-synaptogenesis associated with an aberrant sprouting of granule cell axons in the dentate gyrus (DG). This reactive plasticity contributes in dentate granule-cell hyperexcitability that could lead to the emergence of recurrent spontaneous seizures. We investigated the hippocampal DA expression changes in pilocarpine animals using immunohistochemical, Western blot, and in situ hybridization analyses. We showed that DA immunoreactivity was decreased in the inner molecular layer (IML) and in the hilus (H) of the DG, at latent stage, when spinogenesis and morphogenesis occur. Western blot analysis confirmed these overall hippocampal decreases of DA protein expression. At chronic stage, when newly formed glutamatergic synapses are being established, the levels of immunolabeling for DA in the H and the IML were similar to control rats. This recovery is likely due to the increase of DA mRNA in perikarya of hilar and granule cells. Interestingly, our data showed that the changes pattern of labeling for Bassoon, a specific marker for presynaptic active zone, in the IML of pilocarpine-treated animals paralleled those found for DA at all time points examined. Furthermore, our double and triple immunofluorescence studies showed that the recovery in DA levels in the IML occurred within the dendritic spines involved in glutamatergic active synapses of presumed granule cells. Altogether, our results indicate that in vivo DA is not critical for spinogenesis and morphogenesis but instead is consistent with an involvement in synaptic structural integrity, stabilization, and function. Thus, DA appears as a novel modulator of reactive synaptic plasticity associated with epilepsy.     

Hippocampal and thalamic diffusion abnormalities in children with temporal lobe epilepsy

PURPOSE: Previous studies using diffusion MRI in patients with temporal lobe epilepsy have shown abnormal water diffusion in the hippocampus. Because thalamus and lentiform nuclei are considered important for the regulation of cortical excitability and seizure propagation, we analyzed diffusion tensor imaging (DTI) abnormalities in these subcortical structures and in hippocampus of children with partial epilepsy with and without secondary generalization. METHODS: Fourteen children with partial epilepsy involving the temporal lobe underwent MRI including a DTI sequence. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values were obtained in the hippocampus, thalamus, and lentiform nucleus, and compared with DTI data of 14 control children with no epilepsy, as well as glucose positron emission tomography (PET) findings. RESULTS: Decreased FA (p < 0.001) and increased ADC (p = 0.003) values were found in the hippocampi ipsilateral to the seizure focus. Significant FA decreases (p = 0.002) also were seen in the contralateral hippocampi, despite unilateral seizure onset and excellent surgical outcome in patients who underwent surgery. ADC values showed a trend for increase in the thalami ipsilateral to the epileptic focus in the seven children with secondarily generalized seizures (p = 0.09). No group differences of ADC or FA were found in the lentiform nuclei. The DTI variables did not correlate with regional glucose metabolism in any of the structures analyzed. CONCLUSIONS: Increased ADC values in hippocampus can assist in lateralizing the seizure focus, but decreased FA in the contralateral hippocampus suggests that it too may be dysfunctional despite unilateral seizure onset. Less-robust thalamic abnormalities of water diffusion in patients with secondarily generalized seizures suggest secondary involvement of the thalamus, perhaps due to recruitment of this structure into the epileptic network; however, this must be confirmed in a larger population. DTI appears to be a sensitive method for detection abnormalities in children with partial epilepsy, even in structures without apparent changes on conventional MRI.