Anterior thalamic nuclei deep brain stimulation inhibits mossy fiber sprouting via 3',5'-cyclic adenosine monophosphate/protein kinase A signaling pathway in a chronic epileptic monkey model / 中华医学杂志(英文版)

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.

Effects of embryonic lead exposure on motor function and balance ability in offspring rats and possible mechanisms / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the effects of embryonic lead exposure on motor function and balance ability in offspring rats and the possible mechanisms.</p><p><b>METHODS</b>An 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSIONS</b>Embryonic 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.</p>

Effect of licorice flavonoids on kainic acid-induced seizure in mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of licorice flavonoid (LF) on kainic acid (KA)-induced seizure in mice and its mechanism.</p><p><b>METHODS</b>Male 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.</p><p><b>RESULTS</b>No 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).</p><p><b>CONCLUSION</b>LF has no significant anti-seizure effect. However, it can decrease epileptogenesis through inhibition of neurogenesis and mossy fiber sprouting.</p>

Activation of group II metabotropic glutamate receptors blocks zinc release from hippocampal mossy fibers

BACKGROUND: The hippocampal CA3 area contains large amounts of vesicular zinc in the mossy fiber terminals which is released during synaptic activity, depending on presynaptic calcium. Another characteristic of these synapses is the presynaptic localization of high concentrations of group II metabotropic glutamate receptors, specifically activated by DCG-IV. Previous work has shown that DCG-IV affects only mossy fiber-evoked responses but not the signals from associational-commissural afferents, blocking mossy fiber synaptic transmission. Since zinc is released from mossy fibers even for single stimuli and it is generally assumed to be co-released with glutamate, the aim of the work was to investigate the effect of DCG-IV on mossy fiber zinc signals. RESULTS: Studies were performed using the membrane-permeant fluorescent zinc probe TSQ, and indicate that DCG-IV almost completely abolishes mossy fiber zinc changes as it does with synaptic transmission. CONCLUSIONS: Zinc signaling is regulated by the activation of type II metabotropic receptors, as it has been previously shown for glutamate, further supporting the corelease of glutamate and zinc from mossy fibers.

Neurogenesis induced by seizures in the dentate gyrus is not related to mossy fiber sprouting but is age dependent in developing rats / A neurogênese induzida por crises no giro denteado não está relacionada ao brotamento de fibras musgosas, mas é dependente da idade, em ratos durante o desenvolvimento

Neurogenesis in the dentate gyrus (DG) has attracted attention since abnormal supragranular mossy fiber sprouting occurs in the same region, in temporal lobe epilepsy. Thus, we submitted developing rats to pilocarpine-induced status epilepticus (SE) to study the relationship between neurogenesis and mossy fiber sprouting. Groups were submitted to SE at: I-P9, II-P7, P8 and P9, III-P17 e IV-P21. Neurogenesis was quantified using BrdU protocol and confirmed through double staining, using neuronal pentraxin. Other animals were monitored by video system until P120 and their brain was studied (Timm and Nissl staining). The neurogenesis at P17 (p=0.007) and P21 (p=0.006) were increased. However, only P21 group showed recurrent seizures and the mossy fiber sprouting in the same region, during adult life, while P17 did not. Thus, our results suggest that neurogenesis is not related to mossy fiber sprouting neither to recurrent spontaneous seizures in pilocarpine model.

A neurogênese no giro dentado tem atraído atenção já que ela ocorre na mesma região do hipocampo que o brotamento das fibras musgosas, na epilepsia do lobo temporal. Assim, submetemos ratos em desenvolvimento ao status epilepticus induzido (SE) por pilocarpine. Grupos foram submetidos em I-P9, II-P7, P8, P9; III-P17 e IV-P21. A neurogênese foi observada usando o protocolo do BrdU e confirmada por dupla marcação com pentraxina neuronal. Outros animais foram monitorados até P120 e seus cérebros analisados (Nissl e Timm). A neurogênese nos grupos P17 (p=0,007) e P21 (p=0,006) aumentaram. Entretanto, o P21 apresentou crises espontâneas e brotamento de fibras musgosas, na mesma região onde ocorreu a neurogênese, enquanto o grupo P17 apresentou somente aumento na neurogênese. Assim, nossos resultados sugerem que o fenômeno da neurogênese não está relacionado com o brotamento de fibras musgosas nem com o aparecimento de crises espontâneas e recorrentes no modelo da pilocarpina.

Protein tyrosine kinase inhibitors modify kainic acid-induced epileptiform activity and mossy fiber sprouting but do not protect against limbic cell death

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 ± 8 min in saline controls (N = 4) which increased to 369 ± 71 and 322 ± 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90 percent of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Hippocampal mossy fiber sprouting and Cdk5/p35 expressions in the pentylenetetrazole kindling rat model / 中南大学学报(医学版)

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.

Changes of mossy fiber sprouting in hippocampus of pentylenetetrazole kindling rats / 中南大学学报(医学版)

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.

Kindling & mossy fibre sprouting in the rat hippocampus following hot water induced hyperthermic seizures.

BACKGROUND & OBJECTIVES: Hot water epilepsy (HWE) is well recognized reflex epilepsy with possible genetic susceptibility. Rat model and human experimentation had proven that HWE is a type of hyperthermic seizure with possible kindling on repeated stimulation in animals. The present study was undertaken to investigate kindling associated with hyperthermic seizures induced by repeated hot water stimulation in the rat model and to prove hyperthermic kindling. METHODS: Epileptic seizures were induced in 36 male Wistar albino rats by means of hot water sprays at 48 h time intervals. Progression of seizure activity was investigated by studying the behaviour, severity and duration of the seizure. Threshold of rectal temperatures and timed latency for seizure induction were studied. Seizure discharges (EEG) were recorded from ventral hippocampus in six of these rats. Timm's staining was used to study the neuronal sprouting as a consequence of kindling. Studying the seizure threshold, latency, duration of seizure discharge and behavioural seizure following a stimulus-free interval of 30 days tested permanence of kindling. RESULTS: Following 8-12 episodes of hot water stimulations there was progressive epileptic activity manifested in the form of lowering of rectal temperature thresholds from 41.5 to 40.0 degrees C, drop in latency for developing seizures from 185 to 118 sec, increase in duration of hippocampal seizure discharge from 15 to 140 sec, along with progressive increase in complexity of EEG after discharges, increase in behavioural seizure severity from Grade 1 to 5 in all the rats, and neuronal sprouting observed in supragranular molecular layer and in stratum lacunosum. INTERPRETATION & CONCLUSION: Our study covered all aspects of kindling and provided a useful animal model for human hot water epilepsy. Hyperthermic seizures induced by hot water in the rat model kindle as demonstrated by Timm's staining.

Effect of the zinc chelator N, N, N',N'- tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butiric acid (GABA A) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 ìM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 ìM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.

Effect of ketogenic diet on hippocampus synaptic reorganization and GluR5 expression in kainic acid induced rat model of epilepsy / 中华儿科杂志

<p><b>OBJECTIVE</b>Ketogenic diet (KD) is a high fat, low protein, low carbohydrate diet. Its antiepileptic effect is certain but the underlying mechanism is unknown. The aim of the study was to reveal the possible mechanism from the view points of synaptic reorganization and GluR(5) expression in hippocampus.</p><p><b>METHODS</b>Epilepsy was induced in Sprague-Dawley rats by kainic acid at postnatal day 28, all control animals were fed with normal rodent chow, whereas experimental rats were fed with ketogenic feed for 8 weeks. Spontaneous recurrent seizures were recorded. Mossy fiber sprouting and neuron damage in hippocampus were investigated by Timm staining and Nissl staining. Western blot and RT-PCR methods were applied to detect the expression of GluR(5) and GluR(5) mRNA in hippocampus.</p><p><b>RESULTS</b>KD-fed rats (1.40 +/- 1.03) had significantly fewer spontaneous recurrent seizures than control diet-fed rats (7.36 +/- 3.75). The mean A of mossy fiber sprouting in the inner molecular layer of dentate gyrus was markedly higher in KA induced animals than that in saline control animals but it was similar in different diet fed groups. No significant differences were found in the mean A of Timm staining in CA(3) area and Nissl staining of neuron in hilus, CA(3) and CA(1) area. After KA kindling, KD-fed animals [(189.38 +/- 40.03)/mg pro] had significantly higher GluR(5) expression in hippocampus than control diet-fed animals [(128.79 +/- 46.51)/mg pro] although their GluR(5) mRNA was the same.</p><p><b>CONCLUSION</b>Mossy fiber sprouting may be responsible for epileptogenesis in KA induced model and KD can suppress seizures in these animals. KD may upregulate young rat GluR(5) in inhibitory interneurons of CA(1) thus lead to an increased inhibition to prevent the propagation of seizure.</p>

Brotamento das fibras musgosas hipocampais em epilepsia do lobo temporal: relação com aprendizagem e memória / Mossy fiber sprouting im temporal lobe epilepsy: relation to learning and memory

A ciclohexamida influencia a reorganização sináptica resultante do status epilepticus induzido por pilocarpina (SE). Para investigar possíveis conseqüências funcionais deste efeito, foi induzido SE em animais na ausência (grupo Pilo) ou presença de ciclohexamida (grupo Chx). Passados 5 ou 60 dias da indução do SE, os animais foram submetidos a diferentes testes comportamentais. Posteriormente os mesmos foram perfundidos e os encéfalos foram processados para as colorações de Neo-Timm e Cresil. Os dados apresentados neste trabalho mostraram que a administração de ciclohexamida diminuiu drasticamente a intensidade de brotamento das fibras musgosas, além de apresentar efeito neuroprotetor em algumas estruturas do lobo temporal (hilo, córtex entorrinal e córtex piriforme). Do ponto de vista comportamental, os animais epilépticos que receberam ciclohexamida mantiveram a emocional idade inalterada, enquanto que os animais epilépticos que não receberam a droga mostraram-se hiporeativos às situações estressantes (labirinto em cruz elevado e campo aberto). Contudo, não houve diferenças entre estes animais nos testes de aprendizagem e memória (labirinto aquático de Morris, condicionamento de medo ao contexto e ao som). Portanto, os resultados apresentados sugerem que a integridade de certas estruturas do lobo temporal (hilo, córtex entorrinal e córtex piriforme) é crucial para os aspectos emocionais no modelo de EL T induzido por pilocarpina. Além disso, o BFM não minimiza e nem potencializa os déficits cognitivos proporcionados pela lesão neuronal decorrente do SE induzido por pilocarpina.

Role of gamma-aminobutyric acid B (GABA B) receptors in the regulation of kainic acid-induced cell death in mouse hippocampus

Kainic acid (KA) is well-known as an excitatory, neurotoxic substance. In mice, KA administered intracerebroventricularly (i.c.v.) lead to morphological damage of hippocampus expecially concentrated on the CA3 pyramidal neurons. In the present study, the possible role of gamma-aminobutyric acid B (GABA B) receptors in hippocampal cell death induced by KA (0.1 microgram) administered i.c.v. was examined. 5-Aminovaleric acid (5-AV; GABA B receptors antagonist, 20 microgram) reduced KA-induced CA3 pyramidal cell death. KA increased the phosphorylated extracellular signal-regulated kinase (p-ERK) and Ca2+ /calmodulin-dependent protein kinase II (p-CaMK II) immunoreactivities (IRs) 30 min after KA treatment, and c-Fos, c-Jun IR 2 h, and glial fibrillary acidic protein (GFAP), complement receptor type 3 (OX-42) IR 1 day in hippocampal area in KA-injected mice. 5-AV attenuated KA-induced p-CaMK II, GFAP and OX-42 IR in the hippocampal CA3 region. These results suggest that p-CaMK II may play as an important regulator on hippocampal cell death induced by KA administered i.c.v. in mice. Activated astrocytes, which was presented by GFAP IR, and activated microglia, which was presented by the OX-42 IR, may be a good indicator for measuring the cell death in hippocampal regions by KA excitotoxicity. Furthermore, it showed that GABA B receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.

The Role of MMP-9 on the Hippocampal Neuronal Cell Death and Mossy Fiber Sprouting due to Pilocarpine-Induced Status Epilepticus in Mice / 대한간질학회지

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.

Neurogenesis of dentate granule cells following kainic acid induced seizures in immature rats / 中华儿科杂志

<p><b>OBJECTIVE</b>Data 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.</p><p><b>METHODS</b>Seizures 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSIONS</b>KA 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.</p>

Effects of recurrent audiogenic seizures on hippocampal structure and seizure behavior of P77PMC rats / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the functional role of hippocampal mossy fiber sprouting in the pathophysiologic mechanism of initiation and propagation of epilepsy.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Frequent 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.</p><p><b>CONCLUSION</b>Hippocampal 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.</p>

Epilepsia e brotamento das fibras musgosas hipocampais: condiçäo mecessária e suficiente? / Epilepsy and mossy fiber sprouting: necessary and suficient condition?

Uma das alterações plásticas mais estudadas na epilepsia do lobo temporal é o brotamento das fibras musgosas (BFM), observado no hipocampo de pacientes epilépticos e de animais de laboratório. O aparecimento deste fenômeno tem sido associado ao surgimento das crises epilépticas. No entanto, nossos trabalhos indicam que o padrão e a freqüência das crises epilépticas espontâneas em ratos crônicos não são alterados pelo bloqueio do BFM através do uso de um inibidor de síntese proteica, a cicloheximida (CHX). Direcionamos nossas investigações sobre o BFM de acordo com diferentes perspectivas. Utilizamos protocolos agudos e crônicos com diferentes tempos de sobrevivência dos animais após o status epilepticus (SE) e avaliamos suas respostas ao bloqueio do BFM e ao emprego da CHX. Os agentes epileptogênicos utilizados foram a pilocarpina e o ácido caíníco, injetados por vias sistêmicas e intracerebrais, respectivamente. Em termos metodológicos, recorremos às seguintes estratégias: análise comportamental, eletrográfica, e histológica dos animais epilépticos. A técnica de Timm foi usada para verificar o bloqueio do BFM na camada supragranular, a imunohistiquímica foi usada para marcar a expressão de CGRP (calcitonin gene related peptide), e a impregnação por prata (dark cells) para avaliar a degeneração neuronal sob o bloqueio do BFM. O bloqueio do BFM, por meio de inibidores de síntese proteica, mostrou ser uma excelente estratégia de estudo do hipocampo epiléptico, pois permitiu a investigação deste fenômeno "isolado" de outras alterações que acompanham os eventos epilépticos. Baseado nesta idéia, nosso trabalho sugere um novo modelo para o estudo das epilepsias do lobo temporal, complementar aos já existentes. A utilização desta estratégia em nossos experimentos ajudou a esclarecer aspectos importantes do BFM e de sua relação com as crises epilépticas. Indicamos, através dos experimentos descritos em nossos artigos, que a reorganização sináptica (BFM) decorrente de um estímulo epíleptogênico (indução do SE) não constitui a principal causa das manifestações epilépticas (crises espontâneas). Em outras palavras, sugerimos que o BFM do hipocampo não constitui um fator necessário, nem suficiente, I para a geração das crises epilépticas na epilepsia do lobo temporal

Frequencia da mutacao delta-mtDNA 4977 e acumulo de placas neuriticas e emaranhados neurofibrilares no giro para-hipocampal humano, no envelhecimento normal e na doenca de Alzheimer / Delta mtDNA 4977 mutational frequence and neuritic plaques and neurofibrillary tangles in human parahippocampal gyrus, in normal aging and Alzheimer's disease

A presenca da mutacao-delecao mtDNA no giro para-hipocampal humano foi investigada em 95 pacientes autopsiados de tres series de origens geograficas distintas, Alemanha, Brasil e Japao, incluindo 70 pacientes sem doencas neuropsiquiatricas e 25 pacientes portadores da doenca de Alzheimer. Somente a serie alema, caracterizada por maiores proporcoes de neuronios medios e grandes, e alta incidencia de placas neuriticas e emaranhados neurofibrilares no giro para-hipocampal, apresentou a delta-mtDNA em niveis detectaveis pela reacao de cadeia da polimerase (PCR). As series brasileira e japonesa, caracterizadas por menores proporcoes de neuronios medios e grandes e baixa incidencia de placas e emaranhados, nao apresentaram niveis detectaveis da alfa-mtDNA. A frequencia f da alfa-mtDNA foi tres vezes menor no grupo de pacientes portadores da doenca de Alzheimer (f=0,12) que no grupo controle (f=0,37) (p=0,03)...

Observation on the Hippocampal Mossy Fiber Sprouting After Hypoxic-Ischemic Neuronal Loss in Juvenile and Mature Rat Brain

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.