Humans ; Epilepsy, Temporal Lobe/surgery* ; Mossy Fibers, Hippocampal ; Brain-Derived Neurotrophic Factor ; Dentate Gyrus

OBJECTIVETo investigate the functional role of hippocampal mossy fiber sprouting in the pathophysiologic mechanism of initiation and propagation of epilepsy.

METHODSThe authors examined hippocampal mossy fiber synaptic reorganization and the changes of hippocampal neurons in P77PMC rats at different stages in the course of recurrent seizures using Timm's method of silver sulfide staining and Nissl staining and observed the effects of recurrent audiogenic seizures (AGSs) on seizure behavior of P77PMC rats.

RESULTSFrequent recurrent AGSs could cause neuronal loss in CA(1) region of hippocampus and hippocampal mossy fiber sprouting got into the inner molecular layer of dentate gyrus in P77PMC rats, and could decrease the latency of IV/V grade of AGSs, increase the durations of AGSs. The mean A of CA(1) region of hippocampus in Nissl staining after 50 times of AGSs was 35.3 +/- 0.8, which was markedly lower than that of the control (44.1 +/- 0.5; F = 333.89, P < 0.001). The mean A of the inner molecular layer of dentate gyrus in Timm's staining after 50 times of AGSs was 49.3 +/- 4.6, which was markedly higher than that of the control (26.8 +/- 1.7; F = 76.83, P < 0.001). After 30 and 50 times of AGSs, the latent periods of IV/V grade of AGSs were 12 +/- 8 (t = 3.805; P < 0.02) and 17 +/- 7 (t = 5.927; P < 0.002) seconds shorter than the initial period of stimulation respectively on average, and the durations of AGSs were 19 +/- 18 (t = 2.644; P < 0.05) and 10 +/- 7 (t = 3.780; P < 0.02) seconds longer.

CONCLUSIONHippocampal mossy fiber sprouting and neuronal loss not only presents in limbic seizure, but also in AGS, the seizure can be initiated in brainstem but rapidly generalized;in AGS-prone rats, recurrent AGSs can cause mossy fiber synaptic reorganization and neuronal loss in hippocampus, and can also enhance seizure susceptibility of P77PMC rats. In the course of recurrent AGSs, enhanced seizure susceptibility happened before hippocampal mossy fiber sprouting. Their temporal relationships indicate that the anatomical changes may be preceded by functional changes of elevated excitability in the brain.

Acoustic Stimulation ; adverse effects ; Animals ; Epilepsy ; pathology ; Mossy Fibers, Hippocampal ; pathology ; Rats ; Rats, Wistar ; Seizures ; etiology ; pathology

PURPOSE: Reorganization of mossy fiber terminals in the supragranular layer of the dentate has been found in hippocampi of human epileptics and animal models by Timm staining. Many studies have provided evidence that mossy fiber sprouting is strongly associated with neuronal loss. But the question of whether cell loss is necessary for stimulation of mossy fiber sprouting is remained to be answered. In this present study, we evaluated whether hippocampal mossy fiber sprouting is induced in damaged hippocampus of the rats exposed to hypoxic-ischemic insults in juvenile and adult period. METHODS: At ages of 4-5 weeks and 2 months, the experimental rats were received procedure of right carotid artery unilateral ligation under anesthesia. After 3 hours of the recovery period, they were placed in an airtight 2000ml chamber and exposed to a 8% oxygen-92% nitrogen mixture delivered at 5 liter/min for 90 minutes (juvenile) and 50 minutes (adult). After the recovery period, The animals were returned to cages and housed with controls. 2 weeks later, rats of the control and hypoxic-ischemia group were anesthetised and then perfused with sodium sulfide solusion and fixed. 40micrometer (for Timm stain) and 5micrometer (for H & E stain) coronary brain sections were obtained, stained with Timm method and H < E stain for the observation of the neuronal loss and supragranular Timm granules in the hippocampi. RESULTS: Light microscopic examination of the brains from hypoxic-ischemic animals demonstrated ischemic changes of variable degrees in the hippocampal hilar and pyramidal cell layers. No supragranular mossy fiber sprouting were found in hippocampi of juvenile and adult rats with hypoxic-ischemic damages. CONCLUSION: These results implicated that hippocampal mossy fiber sprouting is not induced in the experimental hypoxic-ischemic encephalopathy of juvenile and adult rats, although cellular loss is found in hippocampus. Neuronal loss might be not necessary for the development of mossy fiber sprouting.
Adult ; Anesthesia ; Animals ; Brain* ; Carotid Arteries ; Hippocampus ; Humans ; Hypoxia-Ischemia, Brain ; Ligation ; Models, Animal ; Mossy Fibers, Hippocampal* ; Neurons* ; Nitrogen ; Pyramidal Cells ; Rats* ; Sodium

BACKGROUND: Anterior thalamic nuclei (ATN) deep brain stimulation (DBS) is an effective method of controlling epilepsy, especially temporal lobe epilepsy. Mossy fiber sprouting (MFS) plays an indispensable role in the pathogenesis and progression of epilepsy, but the effect of ATN-DBS on MFS in the chronic stage of epilepsy and the potential underlying mechanisms are unknown. This study aimed to investigate the effect of ATN-DBS on MFS, as well as potential signaling pathways by a kainic acid (KA)-induced epileptic model. METHODS: Twenty-four rhesus monkeys were randomly assigned to control, epilepsy (EP), EP-sham-DBS, and EP-DBS groups. KA was injected to establish the chronic epileptic model. The left ATN was implanted with a DBS lead and stimulated for 8 weeks. Enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining were used to evaluate MFS and levels of potential molecular mediators in the hippocampus. One-way analysis of variance, followed by the Tukey post hoc correction, was used to analyze the statistical significance of differences among multiple groups. RESULTS: ATN-DBS is found to significantly reduce seizure frequency in the chronic stage of epilepsy. The number of ectopic granule cells was reduced in monkeys that received ATN stimulation (P < 0.0001). Levels of 3',5'-cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in the hippocampus, together with Akt phosphorylation, were noticeably reduced in monkeys that received ATN stimulation (P = 0.0030 and P = 0.0001, respectively). ATN-DBS also significantly reduced MFS scores in the hippocampal dentate gyrus and CA3 sub-regions (all P < 0.0001). CONCLUSION ATN-DBS is shown to down-regulate the cAMP/PKA signaling pathway and Akt phosphorylation and to reduce the number of ectopic granule cells, which may be associated with the reduced MFS in chronic epilepsy. The study provides further insights into the mechanism by which ATN-DBS reduces epileptic seizures.
Adenosine Monophosphate ; Anterior Thalamic Nuclei ; Cyclic AMP-Dependent Protein Kinases ; Deep Brain Stimulation ; Epilepsy/therapy* ; Epilepsy, Temporal Lobe/therapy* ; Hippocampus ; Humans ; Mossy Fibers, Hippocampal ; Signal Transduction

PURPOSE: Matrix metalloproteinases (MMPs) have been known to participate in various pathologic situations by modulating extracellular matrix. Although MMP-9 upregulation has been reported in some experimental seizure models, the exact role of MMP-9 in hippocampal cell death during epileptogenesis and subsequent mossy fiber sprouting (MFS) is not clear. Here, we investigated the role of MMP-9 on hippocampal cell death and MFS after pilocarpine-induced status epilepticus (SE) in mice, using highly specific hydroxamic MMP-9 inhibitor. METHODS: SE was induced by intraperitoneal pilocarpine administration in adult male C57BL/6 mice. MMP-9 specific inhibitor was administered intracerebroventrically 3 h after pilocarpine-induced SE. Expression and activation of MMP-9 were assessed by zymography and Western blot analysis. TdT-mediated UTP-biotin nick end labeling (TUNEL) and caspase-3 activity assay were also performed. MFS was investigated using Timm staining. RESULTS: Increased expression and activation of MMP-9 after pilocarpine-induced SE were observed in zymography and Western blot analysis. MMP-9 specific inhibitor decreased MMP-9 activity in in situ zymography and hippocampal cell death in cresyl violet staining. DNA fragmentation and caspase-3 activity were also attenuated by MMP-9 specific inhibitor. Four months after pilocarpine-induced SE, MFS was evident in vehicle-treated mice; in contrast, MFS was barely observed in MMP-9 specific inhibitor-treated mice. CONCLUSIONS: This study suggests MMP-9 is associated with hippocampal cell death and MFS after pilocarpine-induced SE. Furthermore, the findings that MMP-9 specific inhibitor ameliorates cell death and MFS offers the possibility of MMP-9 specific hydroxamic inhibitor as novel therapeutic strategy to reduce hippocampal damage and epileptogenesis.
Adult ; Animals ; Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Death* ; DNA Fragmentation ; Extracellular Matrix ; Humans ; Male ; Matrix Metalloproteinase 9 ; Matrix Metalloproteinases ; Mice* ; Mossy Fibers, Hippocampal ; Neurons* ; Pilocarpine ; Seizures ; Status Epilepticus* ; Up-Regulation ; Viola

OBJECTIVEData accumulated over the past years have led to widespread recognition that neurogenesis, the emergence of new neurons, persists in the hippocampal dentate gyrus of the adult mammalian brain, and can be increased by seizures in multiple models. Also, aberrant reorganization of dentate granule cell axons, the mossy fiber sprouting, occurs in human temporal lobe epilepsy and rodent epilepsy models. However a number of studies suggest that the immature brain is less vulnerable to the morphologic alteration of hippocampus after seizures. The goal of this study was to determine whether the seizures can induce dentate granule cell neurogenesis and mossy fiber sprouting in the immature rat.

METHODSSeizures was elicited by unilateral microinfusion of kainic acid (KA, 1 micro g) into the amygdula at postnatal day 15 (P15). Rat pups were given bromodeoxyuridine (BrdU) intraperitoneally on day 5 after KA administration and killed 7 d or 21 d later. The brains were processed for BrdU mitotic labeling combined with double-label immunohistochemistry using neuron-specific, early differentiation marker TuJ1 (betaIII tubulin) or granule-specific marker CaBP (calcium-binding protein calbindin D28k) as well as glia-specific marker GFAP (glial fibrillary acidic protein). Mossy fiber sprouting in intermolecular layer and CA3 subfield was assessed in Timm-stained sections both 1 month and 3 months after KA administration by using a rating scale and density measurement.

RESULTSThe dentate BrdU-immunoreactive cells of the KA-treated rats increased significantly compared with those of control rats on day 7 and 21 after BrdU administration (7 d: 244 +/- 15 vs. 190 +/- 10; 21 d: 218 +/- 19 vs. 133 +/- 12, P < 0.05). Approximately 80.2% and 78.7% of BrdU-labeled cells coexpressed TuJ1 in KA-treated rats and control rats on day 7 after BrdU respectively (P > 0.05). On 21 d after BrdU, 60.2% and 58.2% of dentate BrdU-labeled cells coexpressed GaBP in KA-treated rats and control rats respectively (P > 0.05). GFAP colocalized with 3%-5% dentate BrdU-labeled cells in the rats of both groups on day 7 and 21 after BrdU. It was also demonstrated that status epilepticus at P15 did not result in any detectable mossy fiber sprouting within the hippocampus both 1 month and 3 months after KA administration.

CONCLUSIONSKA induced seizures can increase granule cell neurogenesis in the immature rat. Most of newly appeared cells migrate from subgranular proliferation zone (SGZ) into granule cell layer, the hilus as well as the molecular layer, and there they can differentiate into granule neurons. These observations also indicate that there is an early developmental resistance to seizure-induced mossy fiber sprouting in the immature brain.

Animals ; Cell Differentiation ; Cell Proliferation ; Dentate Gyrus ; cytology ; physiopathology ; Disease Models, Animal ; Excitatory Amino Acid Agonists ; adverse effects ; Kainic Acid ; adverse effects ; Mossy Fibers, Hippocampal ; physiopathology ; Neurogenesis ; physiology ; Rats ; Seizures ; chemically induced ; physiopathology

OBJECTIVETo explore the effects of embryonic lead exposure on motor function and balance ability in offspring rats and the possible mechanisms.

METHODSAn animal model of embryonic lead exposure was prepared with the use of pregnant Sprague-Dawley rats freely drinking 0.1% (low-dose group, LG) or 0.2% (high-dose group, HG) lead acetate solution. A normal control group (NG) was also set. The male offspring rats of these pregnant rats were included in the study, consisting of 12 rats in the NG group, 10 rats in the LG group, and 9 rats in the HG group. The offspring rats' motor function and balance ability were evaluated using body turning test and coat hanger test. Eight rats were randomly selected from each group, and immunohistochemistry and Timm's staining were employed to measure the expression of c-Fos and mossy fiber sprouting (MFS) in the hippocampus.

RESULTSThe HG group had a significantly longer body turning time than the NG and LG groups (P<0.05), and the LG group had a significantly longer body turning time than the NG group (P<0.05). The HG group had a significantly lower score of balance ability than the NG and LG groups (P<0.05), and the LG group had a significantly lower score of balance ability than the NG group (P<0.05). The area percentage of c-Fos-positive neurons in the hippocampal CA1 region was significantly higher in the HG group than in the other two groups (P<0.05), and it was significantly higher in the LG group than in the NG group (P<0.05). The semi-quantitative scores of MFS in the hippocampal CA3 region and dentate gyrus were significantly higher in the HG group than in the other two groups (P<0.05), and they were significantly higher in the LG group than in the NG group (P<0.05).

CONCLUSIONSEmbryonic lead exposure could impair the offspring rats' motor function and balance ability. These changes may be related to increased c-Fos expression in the hippocampal CA3 region and abnormal MFS in the hippocampal CA3 region and dentate gyrus.

Animals ; Female ; Fetus ; drug effects ; Hippocampus ; chemistry ; drug effects ; Lead ; toxicity ; Male ; Mossy Fibers, Hippocampal ; drug effects ; Motor Activity ; drug effects ; Postural Balance ; drug effects ; Pregnancy ; Proto-Oncogene Proteins c-fos ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of licorice flavonoid (LF) on kainic acid (KA)-induced seizure in mice and its mechanism.

METHODSMale adult ICR mice were injected with 25 mg/kg KA to induce temporal lobe seizure. LF was administrated 7 d before seizure induction (pre-treatment) or 24 h after seizure induction (post-treatment) for 7 d. Acute seizure latency, seizure stage and duration were observed and compared between LF- and vehicle-treated mice. From d2 on, mice with status epilepticus were video-monitored for spontaneous seizures, 10 h/d for 6 w. Immunohistochemical analysis of BrdU and Timm staining was conducted to detect the neurogenesis and mossy fiber sprouting, respectively.

RESULTSNo significant difference was observed in acute seizure latency, seizure stage and duration between LF-and vehicle-treated mice. KA-induced acute seizure resulted in spontaneous seizure in mice, and the seizure frequency was increased with time. Pre- and post-treatment with LF decreased seizure frequency from w3 after modeling [(0.58±0.15)/d, (0.38±0.38)/d vs (1.23±0.23)/d, P <0.05]. Furthermore, KA-induced seizure resulted in robust neurogenesis and mossy fiber sprouting, while treatment with LF both pre- and post- KA injection significantly inhibited neurogenesis (15.6±2.6, 17.1±3.1 vs 28.9±3.5, P <0.05) and mossy fiber sprouting (1.33±0.31, 1.56±0.42 vs 3.0±0.37, P <0.05).

CONCLUSIONLF has no significant anti-seizure effect. However, it can decrease epileptogenesis through inhibition of neurogenesis and mossy fiber sprouting.

Animals ; Disease Models, Animal ; Flavonoids ; pharmacology ; Glycyrrhiza ; chemistry ; Kainic Acid ; adverse effects ; Male ; Mice ; Mice, Inbred ICR ; Mossy Fibers, Hippocampal ; drug effects ; Neurogenesis ; drug effects ; Seizures ; chemically induced ; drug therapy ; Status Epilepticus ; drug therapy

OBJECTIVE: To determine the changes of mossy fiber sprouting in hippocampus of pre-kindling and post-kindling rats of chronic epilepsy induced by pentylenetetrazole (PTZ). METHODS: Sixty rats were randomly divided into a control group and a PTZ group (PTZ 30 mg/kg, intraperitoneal injection, once daily). The changes of mossy fiber sprouting in hippocampus of pre-kindling and post-kindling rats were examined by Timm staining. RESULTS: Before the occurrence of convulsion confirmed by behavior and EEG, the mossy fiber sprouting was found in the PTZ group. The grade of the mossy fiber sprouting increased with the gradual establishment of kindling effect. CONCLUSION Mossy fiber sprouting may play an important role in the onset and development of epilepsy.
Animals ; Disease Models, Animal ; Epilepsy ; chemically induced ; pathology ; Hippocampus ; drug effects ; pathology ; Kindling, Neurologic ; drug effects ; Male ; Mossy Fibers, Hippocampal ; growth & development ; Neurons ; metabolism ; Pentylenetetrazole ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To observe the expression of cyclin-dependent kinase 5 (Cdk5) and p35 in rat hippocampus during pentetrazole kindling process and their relation with mossy fiber sprouting (MFS), and to investigate the role of Cdk5/p35 in epileptogenesis. METHODS: Altogether 120 healthy male SD rats were randomly divided into a control group and a pentylenetetrazole (PTZ) treated group. The epileptic models were established by the injection of PTZ intraperitoneally while the control rats were injected with an equal dose of saline. At the 3rd day, 1st week, 2nd week, 4th week, and 6th week after daily injection, Timm staining was performed in area CA3 and dentate gyrus, and the mRNA and protein of Cdk5 and p35 were analyzed in the hilus and stratum granulosum of dentate gyrus and area CA1 and CA3 of hippocampus, by in situ hybridization and immunohistochemistry, respectively. RESULTS: The expression levels of Cdk5 and p35 mRNA were significantly higher in the PTZ treated subgroups of the 3rd day, 1st week, 2nd week, and 4th week than those in the controls. Thereafter, the expression decreased to the level of controls. The expression level of Cdk5 and p35 protein increased from the 3rd day to 2nd week, and then gradually decreased to the level of the controls. Timm scores for PTZ groups were 1 to approximately 4 before kindling and 4~5 after kindling in area CA3. CONCLUSION Change of Cdk5/p35 expression in the hippocampus may play a role in epileptogenesis by influencing the process of mossy fiber sprouting.
Animals ; Cyclin-Dependent Kinase 5 ; genetics ; metabolism ; Epilepsy ; chemically induced ; metabolism ; Kindling, Neurologic ; drug effects ; metabolism ; Male ; Mossy Fibers, Hippocampal ; metabolism ; Pentylenetetrazole ; toxicity ; Phosphotransferases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley