构建海人酸诱导的内侧颞叶癫痫小鼠模型

目的 建立C57BL/6颞叶癫痫小鼠模型,观察其在行为学和病理学上的变化.方法 将C57BL/6小鼠随机分为空白对照组、0.9%氯化钠注射组(35μL/g)和海人酸注射组(12 mg/kg),进行单侧海马注射.注射5 d或5周后,取小鼠脑进行HE染色,观察海马病理结构改变;检测海马组织中mTOR通路标志物P-S6蛋白质...     

海人酸诱导大鼠海马nestin的表达

为了探讨海人酸(kainicacid,KA)侧脑室注射后大鼠海马内nestin的表达变化,本文选用成年雄性SD大鼠,并随机分成实验组和对照组。实验组大鼠侧脑室注射1μl(0.5μg/μl)KA,分别于术后1、3、7、14、30、60、90d处死动物,并应用免疫荧光方法观察神经前体细胞标记物nestin、神经元特异烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)的表达。实验组大鼠海马的nestin阳性细胞在KA注射后3d开始出现于齿状回、CA3和CA1区,到7d达到高峰,然后逐渐减少。注射侧的nestin阳性细胞数均较非注射侧多(P<0.01);对照组仅有散在阳性细胞。本结果提示,海人酸可诱导和增强大鼠海马nestin的表达,而这些表达nestin的细胞主要为星形胶质细胞。     

海人酸致癫痫大鼠海马结构中noggin表达变化

目的:研究海人酸(kainic acid,KA)侧脑室注射并诱发癫痫持续状态(status epilepticus,SE)后大鼠海马结构中noggin基因在大鼠海马的表达变化。方法:大鼠在侧脑室注射KA后1、3、7、14、30和60d等不同时间,采用RT-PCR研究noggin mRNA含量变化,采用免疫组化观察noggin蛋白表达变化。结果:在正常大鼠海马结构中noggin mRNA有少量表达。noggin阳性细胞主要位于齿状回及CA3、CA1区,数量较少。侧脑室注射KA诱发SE后,noggin表达持续升高,3d达到高峰;7d在脑内的表达开始降低;注射后2个月,noggin表达降至术前水平,仅见散在的阳性细胞。结论:侧脑室注射KA并诱发SE后,大鼠海马结构中noggin表达明显增加。     

海人酸致痫大鼠的海马微血管构筑的改变

目的观察海人酸(KA)癫痫模型中海马微血管构筑的改变。方法采用KA癫痫模型,在造模后第7天应用碱性磷酸酶法显示海马脑片的微血管,光镜观察,定量分析。结果海马内的微血管呈层分布,构筑模式与神经元的构筑模式相一致;KA组的微血管数目明显多于对照组(P=0.001),血管平均直径明显低于对照组(P=0.030),血管总长度明显高于对照组(P=0.000),血管的平均长度略高于对照组(P=0.085)。结论癫痫大鼠海马微血管构筑的改变是癫痫发病的形态学基础之一。     

癫痫大鼠海马内凋亡神经元超微结构的研究

目的　为揭示海人酸癫痫模型海马内神经元的死亡机制。　方法　选用海人酸诱导的癫痫大鼠模型,对海马内凋亡神经元的超微结构进行观察。　结果　电镜下,实验组大鼠海马齿状回门区和ＣＡ１区内可见散在的凋亡神经元,凋亡神经元主要表现为核周染色体的聚集和凝结成块,其核膜表现为皱缩和扭曲,在晚期凋亡的神经元有时可见核膜破裂。凋亡神经元胞浆内的细胞器保持完整。　结论　凋亡参与了海人酸诱发癫痫发作后海马内神经元的死亡过程。     

CCK与海人酸诱发癫痫敏感性形成的关系

用海人酸 (Kainic acid,KA) 10 mg/ kg给 Sprague- Dawley (SD)大鼠颈部皮下注射 ,诱发急性癫痫发作 ,在急性癫痫发作后一周 ,用阈下剂量 (5 mg/ kg)的 KA检测癫痫敏感性。同时分别用原位杂交和免疫组化技术检测并发现癫痫敏感性形成大鼠额叶皮层胆囊收缩素原 (PCCK) m RNA明显增加 ;海马结构中 ,靠近海马齿状回颗粒细胞 (DGCs)基底部锥体样胆囊收缩素 (Cholecystokinin,CCK)免疫反应阳性神经元染色明显增强 ,但海马门尖部 CCK免疫反应阳性神经元明显减少。而癫痫敏感性未形成大鼠未见上述变化 ,提示上述变化与癫痫敏感性形成有关     

Akt/mTOR信号通路在海人酸损伤大鼠海马组织中的激活

目的 研究Akt/mTOR信号通路在海人酸诱导的大鼠海马神经元损伤中的激活和HIF1α的表达情况。方法 成年SD大鼠随机分为3组,其中2组接受脑室注射,一组为假手术组。大鼠麻醉后颅骨钻孔,侧脑室注射KA 1.0 ?g（KA组）或等体积生理盐水（NS组）或相应位置颅骨钻孔,不予注射（假手术组）。8 和16 h及1 、3 和5 d后,用Western blot观察Akt和mTOR的表达及其磷酸化水平,用免疫组化观察HIF1α的表达情况。结果 尼氏染色显示KA组大鼠海马CA3区1 d时即开始有死亡,5 d时累及CA1区。Akt在KA损伤16 h后就开始激活并持续至第5 d,mTOR于第1 d开始激活,第3 d时达峰值。CA3区的HIF1α于第1 d明显升高,CA1区的HIF1α表达则于3 d时明显升高。NS组和假手术组上述诸指标变化均不明显。结论 KA诱导大鼠海马中Akt/mTOR通路有激活,可能调节神经元的存活。     

神经干细胞在海人酸毁损大鼠海马中的迁移和分化

本研究旨在观察胎鼠神经干细胞移植入海人酸毁损成年大鼠海马中的迁移和分化情况。立体定位注射海人酸毁损大鼠海马CA1区锥体细胞,毁损一周后,将Hoechst33342标记的神经干细胞移植毁损区,分别于术后1、2、4、8周取材,利用荧光技术和免疫组织化学方法,追踪移植的神经干细胞在毁损侧海马中的存活、迁移和分化情况。结果显示,移植的神经干细胞在海马锥体层呈链状迁移,并分化为MAP2阳性细胞和GFAP阳性细胞。这些结果提示移植的神经干细胞在海马锥体层呈链状迁移,大部分分化为胶质细胞,部分分化为神经元。     

BMP4参与海人酸损伤后成年大鼠海马齿状回颗粒细胞增殖及胶质增生

目的探讨海人酸（KA）侧脑室注射致大鼠海马损伤后骨形成蛋白-4（BMP4）的表达变化及其与颗粒细胞增殖和胶质细胞增生的关系。方法将成年大鼠分为对照组与实验组。侧脑室注射KA7d后,用尼氏染色检测海马神经元丢失,用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA阳性细胞与BrdU标记细胞、GFAP阳性细胞数的变化。结果正常成年大鼠BMP4mRNA阳性细胞主要分布于海马齿状回的门区、颗粒下层、CA3、CAI区。BrdU标记细胞主要分布在齿状回颗粒下层。GFAP阳性细胞主要分布在齿状回、CA3区。在KA侧脑室注射致海马损伤后7d,海马CA3、CA4区神经元丢失明显,BMP4mRNA阳性细胞与BrdU、GFAP阳性细胞均明显增加。结论KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒细胞增殖增强和胶质增生可能与BMP4表达增加有关。     

海人酸对家兔海马和尾核神经元的作用

本文比较了成年兔和幼年兔神经元对海人酸的敏感性,并观察了海人酸对家兔海马和尾核神经元的作用。主要结果为: 1.成年兔侧脑室内注射不同剂量的海人酸后,海马不同部位的神经元有不同程度的损伤。注射剂量为0.8μg时,CA_3和CA_4区的大部分锥体细胞有明显的溃变,细胞皱缩,着色深,并有胶质细胞增生;同时尾核神经元也有严重损伤,胶质细胞大量增生。剂量为0.5μg或0.3μg时,CA_3和CA_4的锥体细胞约有1/2被破坏。剂量增至1μg时,锥体细胞损伤范围扩大,溃变更严重。 2.幼兔侧脑室内注射0.1μg或0.2μg海人酸后,海马的锥体细胞有一定的损伤,但仪限于CA_3a区。 3.向成年兔海马CA_2区注射0.3μg海人酸后,海马神经元即有明显的损伤,但只局限于注射的部位。成年兔尾核头部注射0.3μ海人酸后,可在注射部位观察到损伤的神经元和数在的胶质细胞。成年家兔海马锥体细胞对海人酸的敏感性较幼兔为强。海人酸对家兔海马锥体细胞的作用比较强,同时尾核神经元对海人酸也有一定的敏感性。     

The prodynorphin system in the rat hippocampus is differentially influenced by kainic acid and pentetrazole.

Administration of kainic acid (15 mg/kg, i.p.) or pentetrazole (75 mg/kg, i.p.) to rats evoked recurrent limbic or tonic-clonic seizures, respectively. Radioimmunoassay showed that the level of alpha-neoendorphin (prodynorphin-derived peptide) in the hippocampus was decreased after 3 h (by c. 60%) and 72 h (by c. 40%), but was not changed after 24 h following kainic acid administration. The basal release of alpha-neoendorphin from hippocampal slices of kainic acid-treated rats was decreased after 3, 24 and 72 h following the drug injection by c. 50%. The K(+)-stimulated release was decreased after 3 and 24 h (by c. 300 and 200%, respectively) and was back to the control level after 72 h. An in situ hybridization study showed that kainic acid strongly enhanced the prodynorphin messenger RNA levels in the dentate gyrus after 3 and 24 h (by c. 200%), whereas after 72 h it tended to decrease. Twenty four hours after pentetrazole injection the hippocampal level of alpha-neoendorphin was elevated (by c. 33%) and remained unchanged after 3 and 72 h. No significant changes in the basal or K(+)-stimulated alpha-neoendorphin release from hippocampal slices of pentetrazole-treated rats were found at any time points measured. Three and 24 h after pentetrazole administration the level of prodynorphin mRNA in the dentate gyrus was slightly decreased (by c. 30%), but was back to the control values after 72 h. Hence seizure-related changes in hippocampal prodynorphin neuron activity seem to depend on the experimental model of epilepsy.(ABSTRACT TRUNCATED AT 250 WORDS)     

脑缺血后神经发生及其机制的研究进展

脑缺血是严重影响人类健康和生活质量的脑血管疾病,目前尚不能有效地改善其长期病理状态。研究表明,成年哺乳动物包括人类“和灵长类“动物的中枢神经系统（CNS）存在神经发生（neurogenesis）,仍能不断产生新的神经元,此现象持续存在于侧脑室室下区吻侧（the rostral subventricular zone,SVZ）-吻侧迁移流（the rostral migratory stream,RMS）-嗅球（olfactory bulb）通路和海马齿状回（dentate gyrus,DG）的颗粒下区（the subgranular zone,SGZ）,这为脑缺血或神经退行性病变后自体修复提供了依据？本文就脑缺血后神经发生及其调控机制的研究进展进行综述？     

Sex differences in response to kainic acid and estradiol in the hippocampus of newborn rats

Premature and full-term human infants are at considerable risk of excitotoxic-mediated brain damage due to hypoxia-ischemia, infection or other trauma. Glutamate receptor activation is a major source of excitoxicity in the adult and developing brain, and the hippocampus is particularly vulnerable to damage. The seven-day-old rat is a widely used model of pediatric brain damage, in large part due to the relative insensitivity of the brain to exogenous glutamate treatment prior to this age. We have reexamined the possible role of glutamate in pediatric brain damage in the newborn rat using kainic acid treatment and attending to the sex of the animal as well as the effects of pretreatment with the gonadal steroid estradiol. Consistent with previous studies, we found no evidence of damage 7 days posttreatment in the CA1 region of the hippocampus in males or females. There was also little to no damage in the CA2/3 or dentate gyrus of males. In females, however, kainic-acid treatment induced substantial damage in the dentate gyrus and moderate damage in CA2/3, as assessed by neuron number and regional volume. Pretreatment with estradiol was protective against kainic acid-induced damage in females but was permissive for damage in the dentate gyrus of males. Estradiol treatment in the absence of kainic acid treatment was also neuroprotective in females in that it increased neuron number and volume throughout the hippocampal formation, suggesting that the basis of the sex difference observed in hippocampal volume was hormonally mediated. There was no effect of exogenous estradiol given to males in the absence of kainic acid. We conclude that the newborn female rat brain, but not the male, is sensitive to glutamate-mediated toxicity and that gonadal steroids play a complex role in both naturally occurring sex differences in hippocampal volume and response to injury.     

Reduction of NGF protein level in rat dorsal hippocampus following administration of kainic acid.

Intraperitoneal administration of kainic acid (KA) into adult rats caused a profound increase in nerve growth factor (NGF) mRNA and a significant reduction of NGF protein level in the hippocampus. Diazepam pretreatment suppressed both. The reduction of NGF level was apparent in the dorsal hippocampus at 2 h after KA administration, but a marked elevation of NGF protein was observed in the ventral hippocampus at 4 h. These results suggest that non-N-methyl-D-aspartate (NMDA) receptor agonists negatively influence NGF synthesis or stimulate NGF protein degradation in the dorsal hippocampus involving the CA1 sector.     

神经调控技术对海马齿状回神经发生的影响

大多数神经发生出现在最初的发育过程中,但大脑的某些区域在一生中都保持着神经发生.在成年海马齿状回中,每天都不断有新生的神经元产生,并逐渐发育、整合到原有的齿状回神经网络中去.目前观念认为海马相关的认知功能与海马齿状回神经发生有关.神经调控技术作为一种不破坏神经组织的可逆性神经外科疗法,在癫痫、疼痛、帕金森病、抑郁症、强...     

Effects of focal injection of kainic acid into the mouse hippocampus in vitro and ex vivo

Intra-hippocampal kainate injection induces an epileptiform activity termed status epilepticus. We examined the emergence of this activity with extracellular and intracellular records of responses (1) to focal kainate (KA) application in slices of mouse hippocampus and (2) of slices from mice injected with KA. The effects varied with distance from the injection site of KA. At distances less than ∼800 μm, KA injection induced a strong increase in extracellular firing which ceased after 2–4 min. Pyramidal cells in this zone fired and depolarized to a potential at which action potentials were no longer evoked. No further activity was detected near the injection site for 3–5 h. In longitudinal slices of the CA3 region, firing induced by KA injection spread at a velocity close to 1 × 10⁻⁴ mm ms⁻¹. The velocity increased to ∼1 × 10⁻¹ mm ms⁻¹ when synaptic inhibition was blocked, suggesting that inhibitory processes normally restrict the spread of firing. At distances of 1.5–2.5 mm, KA injection induced a short-term increase in firing which was maintained, and often increased and rhythmic at gamma frequencies at 2–5 h after injection. We also examined slices prepared from animals injected with KA, at a delay of 2–5 h corresponding to the expression of status epilepticus. Near the injection site, Gallyas silver staining revealed cellular degeneration, and no activity was recorded. Interictal-like activity was generated by ipsilateral slices distant from KA injection. Contralateral slices also generated an interictal-like activity, but no cell death was detected. Hippocampal oscillations generated at distant sites may be associated with status epilepticus.     

Some behavioral effects of selective neuronal depletion by kainic acid in the dorsal hippocampus of rats

Iontophoretic injections of the neurotoxin kainic acid (KA) into the dorsal hippocampus resulted in an essentially complete attrition of neurons throughout the dorsal hippocampus but spared the fimbria/fornix system. The ventral hippocampus, subiculum, and associated aspects of the entorhinal area appeared completely intact. There was also no detectable damage to neocortex or thalamus adjacent to the dorsal hippocampus or to such KA-sensitive distant regions of the brain as the piriform cortex or striatum. Bilateral depletion of neurons from the dorsal hippocampus retarded the acquisition of a brightness discrimination (because the animals developed position habits during the early phases of acquisition) but had no deleterious effects on brightness discrimination reversal. The KA injections also sharply reduced spontaneous alternation in a T maze and increased locomotor activity in an open field. Food and water intake as well as saline and sucrose preferences were normal after the initial post-operative period but the animals continued to spill more of their food than controls.     

Role of the hippocampus in the sex-dependent regulation of eating behavior: studies with kainic acid

Marked hyperphagia with an increase in the rate of body weight gain was noted in adult female rats 4 days after injections of 2 nmoles of kainic acid into the dorsal and ventral parts of hippocampus. The effect was still present 70 days later. At this time the increase in daily food intake and body weight gain amounted, respectively, to 39% and 93% over the control value. There was no change in water intake. The injection of kainic acid into only one part of the hippocampus--either dorsal or ventral--did not induce hyperphagia. Male rats with kainic acid lesion did not show changes in food intake or body weight gain as compared to vehicle-treated controls. In both sexes the degeneration of hippocampal perikarya induced by kainic acid was associated with a 50-60% decrease in glutamic acid decarboxylase activity and [3H]glutamate uptake, as well as with a small decrease in [3H]glutamate uptake in the hypothalamus, an area that receives glutamatergic fibers from the hippocampus. The results show that the hippocampus appears to play an important role in appetite motivation control by a mechanism which is sex-related.     

Induction of somatostatin by kainic acid in pyramidal and granule cells of the rat hippocampus.

Seizures were induced in rats by systemic administration of kainic acid and, 1.5-12 h after, expression of preprosomatostatin and c-fos mRNAs in 9 hippocampal areas and in the cerebral perirhinal cortex was investigated using in situ hybridization histochemistry. Immunohistochemistry was also performed to study somatostatin peptide. In the control animals preprosomatostatin mRNA was expressed in some cells in the dentate hilus, the stratum oriens and the stratum radiatum of Ammon's horn, the subiculum and the cortex. Starting 3 h after kainic acid administration preprosomatostatin mRNA was expressed in a subpopulation of granule and pyramidal cells which did not normally express it. Preprosomatostatin mRNA-positive cells were markedly increased in the subiculum. Immunohistochemical examination confirmed that preprosomatostatin mRNA in granule and pyramidal cells was translated into peptide. In contrast, c-fos mRNA was induced in most hippocampal and cortical neurons starting 1.5 h after the kainic acid injection. When diazepam was injected to suppress the generalized seizures, preprosomatostatin mRNA was still expressed in pyramidal and subicular cells but not in granule cells.