Noggin参与海人酸损伤后成年大鼠海马颗粒下区颗粒细胞增殖

目的 探讨侧脑室注射海人酸(KA)致大鼠海马损伤后Noggin的表达变化及其与颗粒细胞增殖的关系.方法 健康雄性SD大鼠32只采用随机数字表法分为实验组(16只)及对照组(16只).对照组又分为生理盐水对照组和空白对照组,各8只.实验组大鼠侧脑室注射KA,生理盐水对照组注射等剂量生理盐水.空白对照组不作处理.侧脑室注射KA 1周内,尼氏染色检测海马神经元的丢失.免疫荧光染色与原位杂交的方法检测海马齿状回BrdU标记细胞与Noggin mRNA阳性细胞的变化.结果 在侧脑室注射KA致海马损伤后1周,海马CA3、CA4区神经元丢失明显.与生理盐水对照组比较,实验组海马齿状回BrdU阳性细胞升高,差异有统计学意义(P=0.006),其中注射侧较对侧更为明显.海马Noggin mRNA阳性细胞在第3天时升高,第7天时下降.结论 侧脑室注射KA致海马损伤后.成年大鼠海马齿状回颗粒细胞异常增殖可能与Noggin表达波动有关.     

红藻氨酸致癫痫持续状态大鼠海马neuropilin-2 mRNA及其蛋白表达的研究

目的研究轴索导向分子NPN-2mRNA及其蛋白对癫痫持续状态(SE)后大鼠海马内神经纤维外向性生长和突触重建中的调控作用。方法采用侧脑室内注射红藻氨酸(KA)制作TLE大鼠模型,用Nissl染色、原位杂交和免疫组织化学的方法,分别检测致SE后1d、1w、2w、3w、4w大鼠海马齿状回(DG)、CA1区、CA3区、门区神经元丢失程度以及NPN-2mRNA及其蛋白的表达。结果 KA致SE后1d开始出现神经元丢失,至4w神经元丢失明显增多。KA致SE后1d,NPN-2mRNA及其蛋白在DG和CA1区表达明显下降,持续至3w(P0.01),4w恢复至正常(P0.05);NPN-2mRNA及其蛋白在门区、CA3区表达实验组与对照组无明显差别(P0.05)。结论 KA致SE后,海马DG及CA1区神经元下调NPN-2mRNA及其蛋白的表达,促进DG及CA1区神经纤维外向性生长和突触的重建。     

反义Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响

目的探讨Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响。方法反义寡核苷酸技术封闭内源性Noggin基因的表达,免疫组化法检测成年大鼠海马内Nestin与GFAP的表达。结果侧脑室连续4d注射Noggin基因的反义寡核苷酸后,可见海马齿状回(dentate gyrus,DG)内Nestin阳性细胞数与GFAP阳性细胞数较对照组显著增加;室下区GFAP阳性细胞数亦明显增加。结论Noggin对成年海马干细胞的分化有重要作用,内源性Noggin基因的表达可使神经干细胞向神经元方向分化。     

神经干细胞移植于慢性癫痫鼠海马CA3区对其癫痫发作影响的研究

目的 研究神经干细胞(neural stem cells,NSCs)移植到慢性海人酸(kainic acid,KA)癫痫鼠海马CA3区后对大鼠癫痫发作的影响.方法 用KA脑审注射制作慢性癫痫模型.将原代培养的、EGFP标记的NSCs移植到慢件癫痫鼠的海马CA3区.分别在移植后第2周、第4周、第8周和第12周连续进行7天观察大鼠癫痫发作频率和程度,在移植后第10周进行发作间期右侧海屿深部脑电监测.然后取脑冰冻切片,在倒置荧光显微镜下直接观察移植细胞的存活和迁移,用免疫荧光染色观察移植细胞分化情况,Timm's染色观察海马齿状回异常苔状纤维发芽.结果 移植后12周仍有大量移植细胞存活(65,045.00±881.72).NSCs在移植区以胶质细胞分化为主,在齿状回和海马各区以神经元分化为主,γ-氨基丁酸(GABA)能神经元在齿状回门区和海马CA3区分化比率较高.NSCs移植后第4周移植组癫痫鼠的发作次数与对照组相比开始减少,Timm's染色计分和发作程度也有明显改善,两组发作问期脑电图尖、棘波在每个观察期的发放次数分别是3.83±4.96和27.16±21.08,,结论 将NSCs移植到慢性KA癫痫鼠的海马CA3区,移植细胞不仪能够长期存活、迁移到海马齿状回的各区,而且能够分化为神经元和神经胶质细胞,特别是GABA能神经元,同时还能够抑制齿状回颗粒细胞的苔状纤维发芽,从而减少癫痫发作次数,减轻癫痫发作程度.     

红藻氨酸致颞叶癫痫大鼠海马内Semaphorin3C、Semaphorin3F基因表达的研究

目的研究神经轴索导向分子Sem aphorin3C(Sem a3C),Sem aphorin3F(Sem a3F)mRNA对颞叶癫痫(TLE)大鼠海马神经轴索环路重建的调控作用。方法采用侧脑室内注射红藻氨酸(KA)制作TLE大鼠模型,用N issl染色及原位杂交的方法,分别检测致痫后1d、1w、2w、3w、4w大鼠海马的齿状回(DG),CA1区、CA3区神经细胞丢失程度以及Sem a3C、Sem a3F mRNA的表达。结果KA致痫后1d始出现神经元丢失,至4w神经元丢失明显增多。KA致痫后1w,Sem a3C、Sem a3F mRNA在海马的CA1区、Sem a3F mRNA在海马的CA3区表达明显下降,持续至3w(P<0.01),4w时恢复至正常(P>0.05);Sem a3C、Sem a3F mRNA在DG的表达,Sem a3C在CA3区的表达,实验组与对照组均无明显差别(P>0.05)。结论KA致痫后海马CA1区神经元下调Sem a3C、Sem a3F mRNA的表达,CA3区神经元下调Sem a3F mRNA的表达,可能促进TLE大鼠海马神经轴索环路重建。     

戊四氮点燃过程中大鼠海马 BMP4表达与神经增殖关系

目的探讨戊四氮(PTZ)点燃过程中大鼠海马骨形成蛋白4(bone morphogenetic protein-4,BMP4)的表达变化与神经增殖的关系。方法将成年大鼠分为对照组与模型组,模型组大鼠又根据在点燃中的不同时相点分为9组。用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA与BrdU阳性细胞数的变化。结果正常成年大鼠BMP4阳性细胞主要分布于齿状回的门区、颗粒下层、CA3、CA1区。BrdU阳性细胞主要分布在齿状回颗粒下层。BMP4阳性细胞与BrdU阳性细胞在点燃过程中均明显增加,呈明显正相关,点燃后2月降至基线水平。结论BMP4可能通过影响成年大鼠海马神经发生在PTZ点燃过程中起重要作用。     

红藻氨酸诱导癫痫发作大鼠海马EphA5受体及其配体ephrinA3基因表达变化的研究

目的研究在红藻氨酸(Kainic acid,KA)诱导的损伤型颞叶癫痫(Mesial temporal lobe epilepsy,MTLE)的大鼠海马中,轴突导向因子EphA5受体及其配体ephrinA3基因表达的变化,探讨EphA5/ephrinA3与癫痫后海马兴奋性神经网络形成的作用和关系。方法侧脑室内微量注射KA,建立KA诱导的成年大鼠MTLE模型,用原位杂交法检测癫痫发作1d、1周、2周、3周、4周大鼠海马内EphA5/ephrinA3 mRNA的表达,定量分析表达的动态变化。结果EphA5/ephrinA3 mRNA于癫痫发作后1周,在海马齿状回颗粒细胞层和CA_3区锥体细胞层开始增强,2周达到高峰,4周恢复接近对照组水平。结论在KA所致的癫痫持续状态(Status epilepsy,SE)中,海马神经元通过增强EphA5/ephrinA3 mRNA的表达。调控MTLE大鼠海马内苔藓纤维和突触的重建,是癫痫后海马新的稳定的异常兴奋性神经网络形成的可能机制。     

下丘脑内基因转染对大鼠颞叶癫痫海马病理变化的影响

目的 探讨下丘脑过度兴奋对于颞叶癫痫海马病理变化的影响。方法 利用仙台病毒(HVJ)-脂质体转染法在下丘脑的乳头体内转染兴奋性氨基酸受体亚基GluR2Q，研究其对于海人酸(kainic acid，KA)致痫鼠海马病理变化的影响。结果 GluR2Q能加重KA引起的海马齿状回损伤，但是对CA1及CA3区神经元损伤有一定程度的改善作用。结论 GluR2Q可以使下丘脑兴奋性增强，通过对海马齿状回的抑制解除作用促进癫痫波在海马内的传播。     

神经营养因子-3在颞叶癫(癎)后的表达与海马苔藓纤维发芽的关系

目的 探讨神经营养因子-3(NT-3)在颞叶癫(癎)大鼠海马内表达与苔藓纤维发芽(MFS)的可能关系.方法 大鼠侧脑室注射红藻氨酸(KA)建寺颞叶癫(癎)模型后,侧脑室注射NT-3反义寡核苷酸(ASODN)及正义寡核苷酸(SODN);应用免疫组化法观察海马NT-3表达;应用Timm银染方法,光镜和电镜观察海马MFS,并与空白对照组及脂质体对照组进行比较.结果 致(癎)后大鼠海马齿状回NT-3表达下降;海马苔藓纤维明显粗乱,侧支发芽;齿状回内分子层可见银标记突触末端,主要为轴-树型非对称突触,其中ASODN组海马NT-3蛋白水平降低及MFS程度更明显.结论 KA致痢和NT-3 ASODN均能降低大鼠海马齿状回NT-3表达,增加MFS程度.提示NT-3可能通过对MFS及突触重组作用来影响颞叶癫(癎)的发生和发展.     

雌激素和姜黄素对海人酸杏仁核点燃大鼠行为学、脑电图及海马神经元损伤的影响

目的了解雌激素和姜黄素对海人酸(kainic acid,KA)杏仁核点燃大鼠癫痫发作的影响。方法给去势的雌性大鼠添加雌激素治疗,添加姜黄素治疗,或添加雌激素和姜黄素治疗,比较各组大鼠致痫后癫痫发作的行为学、脑电图和海马神经元损伤的变化。结果给雌激素治疗的大鼠重型发作(Racine 4/5级)评分最高,而雌激素加姜黄素治疗组评分最低(P<0.05)。脑电图的变化与行为学的改变基本一致。致痫后大鼠注射KA侧海马CA3区、CA4区可见到明显的细胞损伤,而该侧海马CA1区、齿状回区(DG)及对侧海马CA3区、CA1区及DG区神经元损害不明显。雌激素组大鼠双侧海马CA3区均出现加重的神经元损害,姜黄素组及雌激素加姜黄素组大鼠海马注射对侧CA3区存活神经元较雌激素组明显增加(P<0.01)。结论高水平的雌激素可以加重癫痫的发作,给姜黄素治疗可以减轻大鼠海马CA3区神经元损害。     

Differential Regulation of Vasoactive Intestinal Peptide (VIP) in the Dentate Gyrus and Hippocampus via the NO-cGMP Pathway Following Kainic Acid-Induced Seizure in the Rat

We have previously shown that kainic acid (KA) increases nitric oxide (NO) synthase (NOS) production in the rat dentate gyrus (DG) and hippocampus (CA3), and NOS inhibition [(by N^G-nitro-L-arginine methylester (L-NAME)] modulates the vasoactive intestinal peptide (VIP)-responsive gene, activity-dependent neuroprotective protein, and alters neuro- and astrogliogenesis (Cosgrave et al. in Neurobiol Dis 30(3):281–292 2008, J Mol Neurosci 39(1–2):9–21, 2009, 2010). In the present study, using the same model we demonstrate that VIP synthesis is differentially regulated by the NO-cyclic guanosine monophosphate (cGMP) pathway in the DG and CA3 at 3 h and 3 days post-KA. At 3 h post-KA: In L-NAME+KA/7-nitroindazole (7-NI)+KA, stratum granulosum (SG) and subgranular zone (SGZ) cells were intensely stained for VIP when compared with L-NAME/7-NI/KA alone. Soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, blocks cGMP production), suppressed astrocytic activation (glial fibrillary acidic protein) but other cell types were VIP⁺; however, ODQ+KA suppressed overall VIP synthesis in the DG. At 3 days post-KA: In L-NAME+KA/7-NI+KA, SGZ and SG cells continued to express VIP, while in the KA alone, only SGZ cells were VIP⁺. ODQ increased VIP⁺ cells in the SG, and in contrast to 3 h, VIP-containing nNOS⁺ cells increased in ODQ+KA when compared to vehicle+KA. In the hippocampus, 7-NI/ODQ had no effect on VIP at 3 h/3 days, while L-NAME+KA at 3 days increased VIP⁺ cells, but reduced VIP-like immunoreactivity in astrocytes. These results suggest that the NO-cGMP pathway differentially regulates VIP in the DG and hippocampus during seizure.     

Chronic temporal lobe epilepsy is associated with severely declined dentate neurogenesis in the adult hippocampus

While it is clear that acute hippocampal injury or status epilepticus increases the production of new neurons in the adult dentate gyrus (DG), the effects of chronic epilepsy on dentate neurogenesis are unknown. We hypothesize that epileptogenic changes and spontaneous recurrent motor seizures (SRMS) that ensue after hippocampal injury or status epilepticus considerably decrease dentate neurogenesis. We addressed this issue by quantifying the number of cells that are positive for doublecortin (DCX, a marker of new neurons) in the DG of adult F344 rats at 16 days and 5 months after an intracerebroventricular kainic acid (ICV KA) administration or after graded intraperitoneal KA (IP KA) injections, models of temporal lobe epilepsy (TLE). At early post-KA administration, the injured hippocampus exhibited increased dentate neurogenesis in both models. Conversely, at 5 months post-KA administration, the chronically epileptic hippocampus demonstrated severely declined neurogenesis, which was associated with considerable SRMS in both KA models. Additionally, stem/progenitor cell proliferation factors, FGF-2 and IGF-1, were decreased in the chronically epileptic hippocampus. Interestingly, the overall decrease in neurogenesis and the extent of SRMS were greater in rats receiving IP KA than rats receiving ICV KA, suggesting that the extent of neurogenesis during chronic TLE exhibits an inverse relationship with SRMS. These results provide novel evidence that chronic TLE is associated with extremely declined dentate neurogenesis. As fraction of newly born neurons become GABA-ergic interneurons, declined neurogenesis may contribute to the increased seizure-susceptibility of the DG in chronic TLE. Likewise, the hippocampal-dependent learning and memory deficits observed in chronic TLE could be linked at least partially to the declined neurogenesis.     

Mitochondrial dysfunction and ultrastructural damage in the hippocampus during kainic acid-induced status epilepticus in the rat

PURPOSE: Prolonged and continuous epileptic seizure (status epilepticus) results in cellular changes that lead to neuronal damage. We investigated whether these cellular changes entail mitochondrial dysfunction and ultrastructural damage in the hippocampus, by using a kainic acid (KA)-induced experimental status epilepticus model. METHODS: In Sprague-Dawley rats maintained under chloral hydrate anesthesia, KA (0.5 nmol) was microinjected unilaterally into the CA3 subfield of the hippocampus to induce seizure-like hippocampal EEG activity. The activity of key mitochondrial respiratory chain enzymes in the dentate gyrus (DG), or CA1 or CA3 subfield of the hippocampus was measured 30 or 180 min after application of KA. Ultrastructure of mitochondria in those three hippocampal subfields during KA-induced status epilepticus also was examined with electron microscopy. RESULTS: Microinjection of KA into the CA3 subfield of the hippocampus elicited progressive build-up of seizure-like hippocampal EEG activity. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (marker for Complexes I+III) in the DG, or CA1 or CA3 subfields 180 min after KA-elicited temporal lobe status epilepticus. Conversely, the activities of succinate cytochrome c reductase (marker for Complexes II+III) and cytochrome c oxidase (marker for Complex IV) remained unaltered. Discernible mitochondrial ultrastructural damage, varying from swelling to disruption of membrane integrity, also was observed in the hippocampus 180 min after hippocampal application of KA. CONCLUSIONS: Our results demonstrated that dysfunction of Complex I respiratory chain enzyme and mitochondrial ultrastructural damage in the hippocampus are associated with prolonged seizure during experimental temporal lobe status epilepticus.     

Integration of the CA2 region in the hippocampal network during epileptogenesis

The CA2 pyramidal cells are mostly resistant to cell death in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis, but they are aberrantly integrated into the epileptic hippocampal network via mossy fiber sprouting. Furthermore, they show increased excitability in vitro in hippocampal slices obtained from human MTLE specimens or animal epilepsy models. Although these changes promote CA2 to contribute to epileptic activity (EA) in vivo, the role of CA2 in the epileptic network within and beyond the sclerotic hippocampus is still unclear. We used the intrahippocampal kainate mouse model for MTLE, which recapitulates most features of the human disease including pharmacoresistant epileptic seizures and hippocampal sclerosis, with preservation of dentate gyrus (DG) granule cells and CA2 pyramidal cells. In vivo recordings with electrodes in CA2 and the DG showed that EA occurs at high coincidence between the ipsilateral DG and CA2 and current source density analysis of silicon probe recordings in dorsal ipsilateral CA2 revealed CA2 as a local source of EA. Cell-specific viral tracing in Amigo2-icreERT2 mice confirmed the preservation of the axonal projection from ipsilateral CA2 pyramidal cells to contralateral CA2 under epileptic conditions and indeed, EA propagated from ipsi- to contralateral CA2 with increasing likelihood with time after KA injection, but always at lower intensity than within the ipsilateral hippocampus. Furthermore, we show that CA2 presents with local theta oscillations and like the DG, shows a pathological reduction of theta frequency already from 2 days after KA onward. The early changes in activity might be facilitated by the loss of glutamic acid decarboxylase 67 (Gad67) mRNA-expressing interneurons directly after the initial status epilepticus in ipsi- but not contralateral CA2. Together, our data highlight CA2 as an active player in the epileptic network and with its contralateral connections as one possible router of aberrant activity.     

Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy

Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABA(A)-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice.     

Functional impairment of the rat superior colliculus after kainic acid intraocular injection: a 2-deoxyglucose study.

Long Evans rats monocularly injected with the kainic acid (KA), were exposed to "tonic" (diffuse steady light, stationary pattern, total darkness) and "phasic" (flashing, moving pattern) stimulations. By means of the autoradiographic 2-deoxyglucose (2DG) technique we assessed the functional activity of the Superior Colliculus (SC) contralateral to the injected eye as compared to the normal eye SC. In the control SC all "tonic" stimulations determined low 2DG uptake not modified by the intraocular KA injection. On the contrary, "phasic" stimulations elicited a strong 2DG consumption in the normal SC, with a peculiar pattern of distribution depending on the kind of stimulus. Considering the total 2DG uptake as the added intrinsic and afferent metabolism, KA was able to affect only the latter, decreasing two-fold that expected for the afferent input loss. These findings can suggest a possible KA effect on off-line ganglion cells and, on the other side, they confirm the role of the SC in discriminating "phasic" and sudden phenomena from "tonic" and continuous ones.     

Time-dependent changes in distribution of basic fibroblast growth factor immunoreactive cells in hippocampus after kainic acid injection in rat pups

Five-day-old Wistar albino rats were injected with kainic acid (KA) or saline i.p. to investigate time-dependent alterations in morphology and number of basic fibroblast growth factor (bFGF) immunoreactive (-ir) astrocytes and neurons in hippocampus at 15, 30, and 90 days after the injections. Sections were stained with cresyl violet for morphological evaluation and bFGF immunohistochemistry was used for quantitative evaluation of bFGF-ir cell density. Fifteen days after KA injection, there was gliosis but no neuronal loss although disorganization in CA1, CA3, CA4 pyramidal layers and neuronal loss were evident 30 and 90 days after the injection. KA injected rats demonstrated significantly increased number of bFGF-ir astrocytes throughout the hippocampus and pyramidal neurons in CA2 after 15 days and decreased number of bFGF-ir cells after 30 and 90 days. The decrease in the number of bFGF-ir astroglia and neurons in long term after KA injection may indicate a decrease in the production of bFGF and/or number of bFGF-ir cells suggesting that protective effects of bFGF may be altered during epileptogenesis in hippocampus.