Long-term deficits following cerebral hypoxia-ischemia in four-week-old rats: correspondence between behavioral, histological, and magnetic resonance imaging assessments

We examined whether following a hypoxic-ischemic insult in young animals there are long-lasting functional deficits that correlate either to histological tissue damage or to potential compensatory plasticity changes. Four-week-old rats were subjected to an episode of cerebral hypoxia-ischemia (right carotid artery occlusion + 30 min of hypoxia) or a sham operation. In hypoxic-ischemic animals there were gross neurological deficits 1, 24, and 48 h postinsult with recovery by 1 week. Behavioral deficits were observed in both the acquisition and the performance of a response duration differentiation test and a fine motor control test (staircase test) 3 months after the hypoxia-ischemia. Functional magnetic resonance imaging studies demonstrated less activation in the sensory-motor cortex of hypoxic-ischemic animals in response to left vs right forepaw stimulation 4 months postinsult. Histological assessment delineated striatal, cortical, and hippocampal damage in the hypoxic-ischemic hemisphere and a reduction in cortical thickness, bilaterally. GFAP immunoreactivity was increased in injured striatum and cortex. Neurofilament heavy chain (NF200) immunoreactivity was normally most intense in white matter and decreased in areas of ipsilateral cortical damage. Synaptophysin immunoreactivity was reduced around areas of infarction and somewhat increased in adjacent undamaged striatum and in layer IV of parietal cortex. The histological damage or chronic degenerative changes could account for much of the variance in functional outcome detected with sensitive behavioral tests so that overall the compensatory or plasticity changes evident within the juvenile brain are rather modest.     

脑发育不同阶段丰富环境刺激对大鼠海马突触素表达的影响

【目的】探讨脑发育不同阶段丰富环境刺激对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)新生鼠神经可塑性的影响及机制。【方法】7日龄SD大鼠通过结扎左侧颈总动脉,吸入8%氧氮混合气,制成HIBD模型,分为早期干预组、晚期干预组、非干预组,另设假手术组。早期干预组于脑发育关键期内,即建模后第2 d开始进行丰富环境(environmental enrichment,EE)干预。晚期干预组于脑发育关键期后,即建模后第23 d(日龄30 d)开始进行EE干预。两组干预条件一致,总干预时间为20 d。各组大鼠饲养至日龄100 d时用免疫组织化学法检测患侧海马突触素(synaptophysin,p38)的表达水平。【结果】早期干预组患侧海马p38的表达明显高于晚期干预组和非干预组(P<0.01),早期干预组与假手术组差异无显著性(P>0.05),晚期干预组p38的表达高于非干预组(P<0.05)。【结论】EE干预可增强神经可塑性。p38在海马表达的变化,可能参与了脑发育不同阶段EE对HIBD神经可塑性的影响机制。     

Effect of memantine on the α7 neuronal nicotinic receptors, synaptophysin- and low molecular weight MAP-2 levels in the brain of transgenic mice over-expressing human acetylcholinesterase

Summary. Transgenic mice over-expressing human acetylcholinesterase (hAChE-Tg) display memory impairments, cholinergic deficits and reduced dendritic branching. In this study, we found a reduced number of N-Methyl-D-Aspartate (NMDA) binding sites and reduced levels of low molecular weight (LMW) microtubule associated protein 2 (MAP-2), in addition to an increased number of 4 and 7 nicotinic receptor (nAChR) binding sites in the brain of hAChE-Tg mice. Treatment with memantine, 20mg/kg/day during 14 days, significantly increased the number of [¹²⁵I]bungarotoxin (7 nAChR) binding sites in the frontal- and retrosplenial cortex of hAChE-Tg mice and synaptophysin- and LMW MAP-2 levels in the cortex of both hAChE-Tg and FVB/N controls. The findings reveal an alteration of the glutamatergic system in hAChE-Tg mice. Whether the effect of memantine on 7 nAChRs, synaptophysin- and LMW MAP-2 levels is a direct effect, or an indirect effect via the NMDA receptors, has to be further evaluated.     

Loss of synaptophysin-positive boutons on lumbar motor neurons innervating the medial gastrocnemius muscle of the SOD1G93A G1H transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a common form of motor neuron disease (MND) that involves both upper and lower nervous systems. In the SOD1G93A G1H transgenic mouse, a widely used animal model of human ALS, a significant pathology is linked to the degeneration of lower motor neurons in the lumbar spinal cord and brainstem. In the current study, the number of presynaptic boutons immunoreactive for synaptophysin was estimated on retrogradely labeled soma and proximal dendrites of alpha and gamma motor neurons innervating the medial gastrocnemius muscle. No changes were detected on both soma and proximal dendrites at postnatal day 60 (P60) of alpha and gamma motor neurons. By P90 and P120, however, alpha motor neuron soma had a reduction of 14 and 33% and a dendritic reduction of 19 and 36%, respectively. By P90 and P120, gamma motor neuron soma had a reduction of 17 and 41% and a dendritic reduction of 19 and 35%, respectively. This study shows that levels of afferent innervation significantly decreased on surviving alpha and gamma motor neurons that innervate the medial gastrocnemius muscle. This finding suggests that the loss of motor neurons and the decrease of synaptophysin in the remaining motor neurons could lead to functional motor deficits, which may contribute significantly to the progression of ALS/MND.     

甲状腺激素对大鼠脑皮质发育期突触体素表达的影响

目的探讨甲状腺激素（TH）对发育关键期大鼠大脑皮质突触体素（syn）表达的作用和影响。方法选用甲巯咪唑（MM）复制甲状腺机能减退大鼠模型,实验分成正常对照组（n=42）和甲减组两组（n=36）,收集出生后第0,4,7,14,21,30,120天的正常、甲减组仔鼠脑组织。采用原位杂交组织化学法检测发育关键期仔鼠大脑皮质突触体素的表达变化情况。结果正常对照组syn mRNA在生后不同发育阶段的表达变化明显且具有层状分布的特点。生后第0天表达很低,在第4天表达最高。以后表达逐渐下降,至第30天表达水平基本接近于成年鼠水平（P120d）。甲减组synmRNA表达在各时点均明显低于正常对照组（P〈0.001）。甲减组syn mRNA表达高峰延迟3d,在出生后第7天达到高峰。结论在脑发育关键期,甲状腺激素水平的异常改变了大脑皮质突触体素的表达,进一步影响了其突触的发生及相关神经回路的建立,出现神经元的旷置或错误神经通路,阻碍了脑发育与其功能的成熟。     

Cell lines derived from hippocampal neurons of the normal and trisomy 16 mouse fetus (a model for Down syndrome) exhibit neuronal markers,cholinergic function,and functional neurotransmitter receptors

We have established hippocampal cell lines from normal and trisomy 16 fetal mice, a model of human trisomy 21. Both cell lines, named H1b (derived from a normal animal) and HTk (trisomic) possess neuronal markers by immunohistochemistry (enolase, synaptophysin, microtubule associated protein-2, and choline acetyltransferase) and lack glial markers (glial fibrillary acidic protein and S-100). Also, we evaluated intracellular Ca(2+) levels ([Ca(2+)](i)) in response to neurotransmitter agonists, in cells loaded with the fluorescent Ca(2+) indicators Indo-1 and Fluo-3. Both cell lines responded to glutamatergic stimuli induced by glutamate, N-methyl-D-aspartate, I-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate. Glutamate responses were only partially prevented by addition of 5 mM EGTA and the metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), increased [Ca(2+)](i) in both cell types. These results confirm the presence of glutamatergic metabotropic receptors. In glutamate-induced responses, HTk cells exhibited slower time-dependent decay kinetics than H1b cells. Cholinergic agonists (nicotine and muscarine) induced a rapid, transient increase in [Ca(2+)](i) in both cell types. Furthermore, some cells were sensitive to histamine and norepinephrine. All responses to the aforementioned agonists were prevented by addition of specific antagonists. We also studied incorporation and release of [(3)H]choline in the cells, and observed no differences in uptake parameters. However, release induced by K(+) and nicotine depolarization was greatly reduced in HTk cells. The results show that H1b and HTk cells retain neuronal characteristics and respond to specific neurotransmitter stimuli. The HTk differences could be related to neuronal pathophysiology in Down syndrome.     

人胚胎视网膜发育过程中突触囊泡蛋白的表达

用光、电镜免疫组织化学方法研究了６～３８周人胚胎视网膜突触囊泡蛋白的表达及超微定位。结果显示：１．人胎１４周突触囊泡蛋白才出现在早期视细胞的胞质中，随后，内网层及节细胞的胞体也相继出现，且由视网膜的中央区向周边区发展；２．随着胎龄增长，神经细胞发育长大，突触囊泡蛋白的含量也逐渐增加，并从核周质中转移至突触前末梢，３２周时基本与成人相似；３．突触囊泡蛋白主要定位于突触前终末的突触小泡，核周质及轴突中     

缬沙坦对血管性痴呆大鼠突触素表达的影响

目的 探讨缬沙坦对血管性痴呆大鼠突触素表达的影响。方法 随机将30只大鼠分为对照组、模型组、治疗组，每组10只。采用双侧颈总动脉永久性结扎（2VO）制作血管性痴呆动物模型，术后24h治疗组以缬沙坦（15gm／kg）灌胃，另两组用蒸馏水灌胃，每日一次。术后8周，采用Y型电迷宫观察血管性痴呆大鼠的认知功能，免疫组化法观察突触素的表达情况。结果 治疗组大鼠海马CAI区突触素免疫活性明显高于模型组，认知功能明显优于模型组。结论 缬沙坦改善血管性痴呆大鼠的认知功能可能与其使海马CAI区的突触素表达增加有关。     

黄精口服液对血管性痴呆大鼠学习记忆与海马突触可塑性的影响

应用Morris水迷宫、免疫组织化学法、透射电镜、图像分析和细胞形态计量学等技术,观察黄精口服液对血管性痴呆大鼠学习记忆能力和突触可塑性的影响。结果显示: (1)术后3. 5个月(即给药2. 5个月)痴呆+黄精口服液组大鼠平均逃避潜伏期较血管性痴呆组和痴呆+生理盐水组大鼠明显缩短(P<0. 05); (2)血管性痴呆组和痴呆+生理盐水组大鼠海马结构突触膜糖蛋白免疫反应产物校正灰度值比空白对照组明显降低;痴呆+黄精口服液组大鼠海马结构突触膜糖蛋白免疫反应产物校正灰度值高于血管性痴呆组和痴呆+生理盐水组大鼠(P<0. 05);大鼠平均逃避潜伏期与海马结构突触膜糖蛋白免疫反应产物校正灰度值呈负相关。(3)痴呆+黄精口服液组大鼠海马CA1区突触界面曲率增大、突触后致密物增厚、突触活性区增长(P<0.05)。这些结果表明:黄精口服液具有重塑突触结构与功能、改善血管性痴呆SD雌性大鼠学习记忆能力的作用。     

Genistein对体外培养的大鼠多巴胺能神经元的保护作用

为了探讨genistein(GST)对离体培养的Parkinson模型多巴胺能神经元的保护作用,本实验取孕14～16 d的SD大鼠胚胎中脑腹侧,常规体外培养。将培养细胞分为四组:正常对照组、E2+MPP+组、GST+MPP+组、MPP+组。用免疫细胞化学染色法观察培养物中TH阳性神经元的生长状况、观察神经元中微管相关蛋白-2(MAP-2)的染色情况。通过突触素(SYN)免疫荧光染色,观察各实验组突触数量的变化。结果显示:与正常对照组、E2+MPP+组以及GST+MPP+组相比,MPP+组TH阳性神经元数量少,SYN免疫阳性产物表达亦减少(P<0.05);MAP-2阳性染色的平均灰度减少约40%左右。而E2+MPP+组、GST+MPP+组中上述指标与正常对照组相比,差异无统计学意义(P>0.05)。本研究结果表明genistein对体外培养的多巴胺能神经元具有类似雌激素样的神经保护作用。