Sprouting of mossy fibers and the vacating of postsynaptic targets in the inner molecular layer of the dentate gyrus

Aberrant mossy fiber sprouting, which presumably results from hilar mossy cell death after status epilepticus (SE), is a frequently studied feature of temporal lobe epilepsy. Although mossy fiber sprouting can be suppressed by the protein synthesis inhibitor cycloheximide, spontaneous seizures remain unaltered. We have investigated the mechanisms underlying the ability of cycloheximide to block SE-induced mossy fiber sprouting in the inner molecular layer of dentate gyrus (IML). Pilocarpine-induced SE in the presence of cycloheximide resulted in a reduced number of injured hilar cells compared to rats not pretreated with cycloheximide. Presumed mossy cells, identified by calcitonin gene related peptide (CGRP) immunohistochemistry, were not significantly reduced in either group 60 days after SE. Whereas controls had a strong band of CGRP-positive fibers (putative mossy cell axons) and no neo-Timm stained fibers in the IML, pilocarpine-treated rats had no CGRP fibers and strong neo-Timm staining. Cycloheximide-pilocarpine-treated animals, in contrast, had CGRP and neo-Timm staining similar to controls. Cycloheximide might protect hilar CGRP-positive cells during SE and, by allowing those cells to retain their normal axonal projection, prevent mossy fiber sprouting. The recently suggested "irritable" mossy cell hypothesis relies on the survival of mossy cells for network hyperexcitability. We hypothesized that CGRP may be a marker for a subpopulation of relatively resistant mossy cells in rats, which, if they survive injury, may become irritable and contribute to hyperexcitability. We suggest that cycloheximide prevents SE-induced mossy fiber sprouting by preventing the loss of hilar CGRP-positive cells (putative mossy cells).     

Mossy fiber sprouting as a potential therapeutic target for epilepsy

Hippocampal mossy fibers, axons of dentate granule cells, converge in the dentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. In the hippocampal formation of temporal lobe epilepsy patients, however, this stereotyped pattern of projection is often collapsed; the mossy fibers branch out of the dentate hilus and abnormally innervate the dentate inner molecular layer, a phenomenon that is termed mossy fiber sprouting. Experimental studies have replicated this sprouting in animal models of temporal lobe epilepsy, including kindling and pharmacological treatment with convulsants. Because these axon collaterals form recurrent excitatory inputs into dendrites of granule cells, the circuit reorganization is assumed to cause epileptiform activity in the hippocampus, whereas some recent studies indicate that the sprouting is not necessarily associated with early-life seizures. Here we review the mechanisms of mossy fiber sprouting and consider its potential contribution to epileptogenesis. Based on recent findings, we propose that the sprouting can be regarded as a result of disruption of the molecular mechanisms underlying the axon guidance. We finally focus on the possibility that prevention of the abnormal sprouting might be a new strategy for medical treatment with temporal lobe epilepsy.     

Mossy fiber synaptic reorganization induced by kindling: time course of development, progression, and permanence

Recent studies have revealed that mossy fiber axons of granule cells in the dentate gyrus undergo reorganization of their terminal projections in both animal models of epilepsy and human epilepsy. This synaptic reorganization has been demonstrated by the Timm method, a histochemical technique that selectively labels synaptic terminals of mossy fibers because of their high zinc content. It has been generally presumed that the reorganization of the terminal projections of the mossy fiber pathway is a consequence of axonal sprouting and synaptogenesis by mossy fibers. To evaluate this possibility further, the time course for development of Timm granules, which correspond ultrastructurally to mossy fiber synaptic terminals, was examined in the supragranular layer of the dentate gyrus at the initiation of kindling stimulation with an improved scoring method for assessment of alterations in Timm histochemistry. The progression and permanence of this histological alteration were similarly evaluated during the behavioral and electrographic evolution of kindling evoked by perforant path, amygdala, or olfactory bulb stimulation. Mossy fiber synaptic terminals developed in the supragranular region of the dentate gyrus by 4 d after initiation of kindling stimulation in a time course compatible with axon sprouting. The induced alterations in the terminal projections of the mossy fiber pathway progressed with the evolution of behavioral kindled seizures, became permanent in parallel with the development of longlasting susceptibility to evoked seizures, and were observed as long as 8 months after the last evoked kindled seizure. The results demonstrated a strong correlation between mossy fiber synaptic reorganization and the development, progression, and permanence of the kindling phenomenon.     

Electrophysiology of dentate granule cells after kainate-induced synaptic reorganization of the mossy fibers.

Morphological data from humans with temporal lobe epilepsy and from animal models of epilepsy suggest that seizure-induced damage to dentate hilar neurons causes granule cells to sprout new axon collaterals that innervate other granule cells. This aberrant projection has been suggested to be an anatomical substrate for epileptogenesis. This hypothesis was tested in the present study with intra- and extracellular recordings from granule cells in hippocampal slices removed from rats 1-4 months after kainate treatment. In this animal model, hippocampal cell loss leads to sprouting of mossy fiber axons from the granule cells into the inner molecular layer of the dentate gyrus. Unexpectedly, when slices with mossy fiber sprouting were examined in normal medium, extracellular stimulation of the hilus or perforant path evoked relatively normal responses. However, in the presence of the GABAA-receptor antagonist, bicuculline, low-intensity hilar stimulation evoked delayed bursts of action potentials in about one-quarter of the slices. In one-third of the bicuculline-treated slices with mossy fiber sprouting, spontaneous bursts of synchronous spikes were superimposed on slow negative field potentials. Slices from normal rats or kainate-treated rats without mossy fiber sprouting never showed delayed bursts to weak hilar stimulation or spontaneous bursts in bicuculline. These data suggest that new local excitatory circuits may be suppressed normally, and then emerge functionally when synaptic inhibition is blocked. Therefore, after repeated seizures and excitotoxic damage in the hippocampus, synaptic reorganization of the mossy fibers is consistently associated with normal responses; however, in some preparations, the mossy fibers may form functional recurrent excitatory connections, but synaptic inhibition appears to mask these potentially epileptogenic alterations.     

Stereologic estimation of hippocampal GluR2/3- and calretinin-immunoreactive hilar neurons (presumptive mossy cells) in two mouse models of temporal lobe epilepsy

Purpose: Hippocampal mossy cells receive dense innervation from dentate granule cells and, in turn, mossy cells innervate both granule cells and interneurons. Mossy cell loss is thought to trigger granule cell mossy fiber sprouting, which may affect granule cell excitability. The aim of this study was to quantify mossy cell loss in two animal models of temporal lobe epilepsy, and determine whether there exists a relationship between mossy cell loss, mossy fiber sprouting, and granule cell dispersion. Methods: Representative hippocampal sections from p35 knockout mice and mice with unilateral intrahippocampal kainate injection were immunolabeled for GluR2/3, two subunits of the amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor and calretinin to identify mossy cells. Mossy fibers were immunostained against synaptoporin. Key Findings: p35 Knockout mice showed no hilar cell death, but moderate mossy fiber sprouting and granule cell dispersion. In the kainate‐injected hippocampus, there was an 80% and 85% reduction of GluR2/3‐ and GluR2/3/calretinin‐positive hilar neurons, respectively, and dense mossy fiber sprouting and significant granule cell dispersion. In the contralateral hippocampus there was a 52% loss of GluR2/3‐, but only a 20% loss of GluR2/3‐calretinin‐immunoreactive presumptive mossy cells, and granule cell dispersion; no mossy fiber sprouting was observed. Significance: These results indicate a probable lack of causality between mossy cell death and mossy fiber sprouting.     

Strong paired pulse depression of dentate granule cells in slices from patients with temporal lobe epilepsy

Summary. Recurrent, feedback excitation by sprouted mossy fibers may contribute to the hyperexcitability observed in human temporal lobe epilepsy. Observations in rodent models of epilepsy mimic the findings in human tissue and reveal that dentate granule cells sprout axons which innervate fibers in their own dendritic layer. However, recent evidence in rodents suggest that these sprouted fibers may form connections which cause inhibition of dentate granule cells, not excitation. Thus, the net effect of sprouting in human epileptic tissue may not only be recurrent excitation. We analyzed paired pulse depression in dentate slices from 9 patients with temporal lobe epilepsy and found evidence for strong feedback inhibition. We also noted failure of high frequency stimulation induced inhibition in our human specimens. These data challenge the concept that human epileptic dentate granule cells are excited by recurrent mossy fiber sprouting. Accepted February 20, 1998; received December 1997     

Manganese-enhanced magnetic resonance imaging detects mossy fiber sprouting in the pilocarpine model of epilepsy

Purpose: Mossy fiber sprouting (MFS) is a frequent finding following status epilepticus (SE). The present study aimed to test the feasibility of using manganese‐enhanced magnetic resonance imaging (MEMRI) to detect MFS in the chronic phase of the well‐established pilocarpine (Pilo) rat model of temporal lobe epilepsy (TLE). Methods: To modulate MFS, cycloheximide (CHX), a protein synthesis inhibitor, was coadministered with Pilo in a subgroup of animals. In vivo MEMRI was performed 3 months after induction of SE and compared to the neo‐Timm histologic labeling of zinc mossy fiber terminals in the dentate gyrus (DG). Key Findings: Chronically epileptic rats displaying MFS as detected by neo‐Timm histology had a hyperintense MEMRI signal in the DG, whereas chronically epileptic animals that did not display MFS had minimal MEMRI signal enhancement compared to nonepileptic control animals. A strong correlation (r = 0.81, p < 0.001) was found between MEMRI signal enhancement and MFS. Significance: This study shows that MEMRI is an attractive noninvasive method for detection of mossy fiber sprouting in vivo and can be used as an evaluation tool in testing therapeutic approaches to manage chronic epilepsy.     

Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2‐specific markers Purkinje cell protein 4 (PCP4) and regulator of G‐Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin‐expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE‐associated pathological restructuring. © 2015 Wiley Periodicals, Inc.     

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.     

Nogo-A及其受体与癫痫大鼠海马苔藓纤维发芽的关系

目的研究神经轴突再生抑制因子Nogo-A及其受体NgR蛋白在癫痫大鼠海马细胞内的表达水平,探讨Nogo-A及NgR与癫痫大鼠海马苔藓纤维发芽(mossy fiber sprouting,MFS)发生、发展的关系,为进一步明确癫痫发病机制提供研究依据。方法采用红藻氨酸(KA)颈部皮下注射法建立大鼠癫痫模型。(1)采用链霉素抗生物素蛋白-过氧化物酶(SP)法对大鼠海马中Nogo-A及NgR蛋白进行检测,并用图像分析仪对染色结果进行分析;(2)应用Timm's硫化银法对大鼠海马脑片进行染色,并对结果进行半定量分析。结果(1)实验组Nogo-A蛋白表达与对照组差别无显著性意义(P>0.05);(2)注射KA后4h NgR蛋白表达降低,至造模后7d,NgR蛋白表达升高至正常水平,实验组与对照组之间比较,组内、组间表达均有显著性差异(P<0.01);(3)Timm's硫化银染色结果显示:最早在造模2w后可见海马苔藓状纤维发芽,染色结果为淡染,至造模后2个月发芽脑片染色为深染。结论红藻氨酸造模后癫痫大鼠海马Nogo-A蛋白表达无改变;NgR蛋白表达在造模初期呈现降量调节;癫痫大鼠海马苔藓状纤维发芽的发生可能与NgR蛋白水平...     

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.     

Physiologic and Morphologic Characteristics of Granule Cell Circuitry in Human Epileptic Hippocampus

Summary: Morphological and electrophysiological techiques were used to examine granule cells and their mossy fiber axons in nine surgically resected hippocampal specimens from temporal lobe epilepsy (TLE) patients. Timm histochemistry showed mossy fiber sprouting into the inner molecular layer (IML) of the dentate in a subset of tissue samples. In slices from five tissue samples, stimulus-induced bursting activity could be induced with a low concentration (2.5 μM) of bicuculline; bursts were sensitive to the N -methyl- d -aspartate (NMDA) blocker, APV. There was a general correlation between such sprouting and experimentally induced yperexcit ability. Fourteen granule cells from five tissue samples were intracellularly stained [with lucifer yellow (LY) or neurobiotin]. Axons from a subset of these neurons showed axon collaterals reaching into the IML, but this axon projection pattern for single cells was not directly correlated with degree of mossy fiber sprouting shown grossly by Timm staining. Electron microscopic examination of intracellularly stained elements showed mossy fiber axon terminals making asymmetric synaptic contacts (including autapses on the granule cell dendrite) with dendritic shafts and spines in both apical and basal domains. These data are consistent with the hypothesis that mossy fiber sprouting provides a structural basis for recurrent excitation of granule cells, but does not provide direct support of the hypothesis that mossy fiber sprouting causes hyperexcitability. The data suggest that granule cell bursting activity is at least in part a function of compromised synaptic inhibition, since levels of γ-aminobutyric acid (GABA) blockade that are generally subthreshold for burst induction were epileptogenic in some tissue samples from human epileptic hippocampus.     

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.     

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.     

Characterization of Target Cells for Aberrant Mossy Fiber Collaterals in the Dentate Gyrus of Epileptic Rat

Previous studies have demonstrated formation of recurrent excitatory circuits between sprouted mossy fibers and granule cell dendrites in the inner molecular layer of the dentate gyrus (9, 28, 30). In addition, there is evidence that inhibitory nonprincipal cells also receive an input from sprouted mossy fibers (39). This study was undertaken to further characterize possible target cells for sprouted mossy fibers, using immunofluorescent staining for different calcium-binding proteins in combination with Timm histochemical staining for mossy fibers. Rats were injected intraperitoneally with kainic acid in order to induce epileptic convulsions and mossy fiber sprouting. After 2 months survival, hippocampal sections were immunostained for parvalbumin, calbindin D28k, or calretinin followed by Timm-staining. Under a fluorescent microscope, zinc-positive mossy fibers in epileptic rats were found to surround parvalbumin-containing neurons in the granule cell layer and to follow their dendrites, which extended toward the molecular layer. In addition, dendrites of calbindin D28k-containing cells were covered by multiple mossy fiber terminals in the inner molecular layer. However, the calretinin-containing cell bodies in the granule cell layer did not receive any contacts from the sprouted fibers. Electron microscopic analysis revealed that typical Timm-positive mossy fiber terminals established several asymmetrical synapses with the soma and dendrites of nonpyramidal cells within the granule cell layer. These results provide direct evidence that, in addition to recurrent excitatory connections, inhibitory circuitries, especially those responsible for the perisomatic feedback inhibition, are formed as a result of mossy fiber sprouting in experimental epilepsy.     

Fetal Hippocampal Cells Grafted to Kainate-Lesioned CA3 Region of Adult Hippocampus Suppress Aberrant Supragranular Sprouting of Host Mossy Fibers

Selective lesion of the rat hippocampus using an intracerebroventricular administration of kainic acid (KA) represents an animal model for studying both lesion recovery and temporal lobe epilepsy. This KA lesion leads initially to loss of CA3 hippocampal neurons, the postsynaptic target of mossy fibers, and later results in aberrant mossy fiber sprouting into the dentate supragranular layer (DSGL). Because of the close association of this aberrant mossy fiber sprouting with an increase in the seizure susceptibility of the dentate gyrus, delayed therapeutic strategies capable of suppressing the sprouting of mossy fibers into the DSGL are of significant importance. We hypothesize that neural grafting can restore the disrupted hippocampal mossy fiber circuitry in this model through the establishment of appropriate mossy fiber projections onto grafted pyramidal neurons and that these appropriate projections will lead to reduced inappropriate sprouting into the DSGL. Large grafts of Embryonic Day 19 hippocampal cells were transplanted into adult hippocampus at 4 days post-KA lesion. Aberrant mossy fiber sprouting was quantified after 3–4 months survival using three different measures of Timm's staining density. Grafts located near the degenerated CA3 cell layer showed dense ingrowth of host mossy fibers compared to grafts elsewhere in the hippocampus. Aberrant mossy fiber sprouting throughout the dentate gyrus was dramatically and specifically reduced in animals with grafts near the degenerated CA3 cell layer compared to “lesion only” animals and those with ectopic grafts away from the CA3 region. These results reveal the capability of appropriately placed fetal hippocampal grafts to restore disrupted hippocampal mossy fiber circuitry by attracting sufficient host mossy fibers to suppress the development of aberrant circuitry in hippocampus. Thus, providing an appropriate postsynaptic target at early postlesion periods significantly facilitates lesion recovery. The graft-induced long-term suppression of aberrant sprouting shown here may provide a new avenue for amelioration of hyperexcitability that occurs following hippocampal lesions.     

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.     

Mossy fiber sprouting in the dentate gyrus in a newly developed epileptic mutant, Ihara epileptic rat

We examined the correlation between seizure activity and development of mossy fiber sprouting in the hippocampal formation using Timm staining in a newly developed Ihara epileptic rat (IER). The sprouting of mossy fibers were clearly shown in the inner molecular portion of the dentate gyrus and in the stratum oriens of CA3 pyramidal cell layer with repeated seizures. A positive correlation between the frequency of generalized tonic and clonic convulsions and the Timm staining score in molecular layer of dentate gyrus was revealed. Sprouting of mossy fiber in IER seems to be linked with seizure activities resulting from epileptic bursts, not to the genetic mutation.     

Stereological analysis of GluR2-immunoreactive hilar neurons in the pilocarpine model of temporal lobe epilepsy: correlation of cell loss with mossy fiber sprouting

Mossy fiber sprouting and the genesis of ectopic granule cells contribute to reverberating excitation in the dentate gyrus of epileptic brain. This study determined whether the extent of sprouting after status epilepticus in rats correlates with the seizure-induced degeneration of GluR2-immunoreactive (GluR2+) hilar neurons (presumptive mossy cells) and also quantitated granule cell-like GluR2-immunoreactive hilar neurons. Stereological cell counting indicated that GluR2+ neurons account for 57% of the total hilar neuron population. Prolonged pilocarpine-induced status epilepticus killed 95% of these cells. A smaller percentage of GluR2+ neurons (74%) was killed when status epilepticus was interrupted after 1-3.5 h with a single injection of phenobarbital, and the number of residual GluR2+ neurons varied among animals by a factor of 6.2. GluR2+ neurons were not necessarily more vulnerable than other hilar neurons. In rats administered phenobarbital, the extent of recurrent mossy fiber growth varied inversely and linearly with the number of GluR2+ hilar neurons that remained intact (P=0.0001). Thus the loss of each GluR2+ neuron was associated with roughly the same amount of sprouting. These findings support the hypothesis that mossy fiber sprouting is driven largely by the degeneration of and/or loss of innervation from mossy cells. Granule cell-like GluR2-immunoreactive neurons were rarely encountered in the hilus of control rats, but increased 6- to 140-fold after status epilepticus. Their number did not correlate with the extent of hilar cell death or mossy fiber sprouting in the same animal. The morphology, number, and distribution of these neurons suggested that they were hilar ectopic granule cells.     

NGF与癫痫后大鼠海马损伤修复及苔藓纤维发芽关系的研究

目的 探讨神经生长因子(NGF)与癫痫后大鼠海马损伤修复及与苔藓纤维发芽(MFS)的关系.方法 大鼠侧脑室内分别注入NGF.正、反义寡核苷酸和磷酸盐缓冲液,并使用红藻氨酸(KA)致痫后分别在48h、72h、1w和4w采用普通病理方法、免疫组化和Timm's染色法对鼠脑片进行研究.结果 KA致痫后48h大鼠海马NGF蛋白表达已可见显著升高,持续较高水平表达至少4w,并伴随大鼠海马明显MFS;给予NGF反义寡核苷酸能够减少NGF蛋白表达和MFS过程,但神经元损伤更为严重;大鼠行为学观察各癫痫组未见明显差异.结论 KA致痫后大鼠海马NGF蛋白表达升高并与MFS同时共存,但可被侧脑室内注入NGF反义寡核苷酸有效阻止,提示NGF可促进癫痫后大鼠海马损伤修复及MFS过程.

Abstract：

Objective To explore the correlation along nerve growth factor, the repairing of neuron damage and mossy fibers sprouting (MFS) in rat hippocampus after lcison. Methods Antisense or sense oligonucleotide to NGF and/ or PBS buffer was injected into the ventricles of rats of temporal lobe epilepsy model induced by KA and so as the control rats, then they were executed at 48h,72h, 1 w and 4w after lesion respectively. Immunohistochemical, pathobiology staining and Timm' s silver sulfide staining were used. Results The NGF-like protein expression increased at 48h after lesion, then the protein expression' s peak was at 72h and kept at a high level till about 4w, and with positive correlation with MFS.Timm' s staining showed that multiple aberrant Timm' s granules were found in the hippocampus of the KA model intracerebroventricularly injected with sense oligonucleotide to NGF and PBS. But antisense oligonucleotide to NGF alleviated these states. No obvious discrepancy of behavior between the epileptic rats injected antisense oligonucleotide and the sense oligonucleotide ones. Conclusions The NGF-like protein expression increased with time-dependence after lesion and coexisted with MFS, but antisense oligonucleotide to NGF could alleviate these states. It hinted that NGF can promote the repairing of neuron damage and mossy fibers sprouting (MFS) in rat hippocampus after leison.