【作者】 邓小华；

【导师】 罗学港； Marina Bentivoglio；

【作者基本信息】 中南大学 ， 神经生物学， 2006， 博士

【摘要】 目的：1．探讨小鼠脑实质，尤其是神经胶质细胞对于两种前炎症细胞因子丙种干扰素和肿瘤坏死因子(IFN-γ和TNF-α)的混合物诱导的急性神经炎症反应以及这种反应是否随着年龄的改变而变化；2．探讨前炎症细胞因子对神经元存活的影响；3．探讨神经元和神经胶质细胞在对抗炎症反应时二者的相互关系。方法：将不同年龄的动物C57BL／6J小鼠，分成三个组：2.3-3.5个月作为青年组，10-11个月作为中年组，19-22个月作为老年组。依赖立体定位技术，于小鼠右侧侧脑室内注射两种前炎症细胞因子的混合物(IFN-γ2μl+TNF-α2μl，生物活性均为500单位／每微升)4μl。PBS作为实验对照组。动物分别存活1、2和4天。使用GFAP免疫组织化学法检测小鼠脑实质星形胶质细胞GFAP的表达变化；用F4／80和CD11b两种小胶质细胞表面抗原的免疫组织化学法检测小胶质细胞F4／80和CD11b的表达变化；用抗凋亡蛋白Bcl-2的免疫组织化学法检测神经胶质细胞和神经元的Bcl-2的表达变化，并采用Western blot技术对其进行定量分析。Fluoro Jade B染色和Tunel染色，检测在前炎症细胞因子的作用下，神经元是否有凋亡或者坏死；免疫荧光双重标记技术，观察神经胶质细胞与Bcl-2阳性神经元之间的相互关系。结果：GFAP阳性的星形胶质细胞分布在梨状皮质和内嗅皮质、隔、整个海马结构、纹状体、杏仁核、皮质下白质及主要的纤维束。PBS注射的对照组动物，脑室周围的脑实质内GFAP阳性的星形胶质细胞展示出静息的形态特征：胞体小，分支细长，染色淡。细胞因子处理2天后，三个年龄组动物脑实质内GFAP阳性的星形胶质细胞明显被激活，表现出肥大的形态特征：细胞胞体相对增大，突起肥大，尤其在海马内明显。持续到注射后4天。与青年和中年组比较，老龄动物脑星形胶质细胞的这种变化尤为明显。定量分析表明，细胞因子处理2天后，老年组与中、青年组相比，海马CA1区、纹状体内GFAP阳性的星形胶质细胞数目和光密度比较，差异有显著性(P＜0.001)。PBS注射24小时后，三个年龄组小鼠脑室周围的脑实质内F4／80阳性的小胶质细胞展示出静息的形态特征：胞体较小，突起细长。细胞因子处理24小时后，与对照组比较，脑室周围的脑实质内有大量的F4／80阳性的小胶质细胞被激活，展示出肥大的(胞体增大，突起短粗)、阿米巴样或者杆形细胞等不同激活状态下的形态特征。激活的小胶质细胞主要分布于脑室周围的脑实质结构：海马、隔、纹状体、大脑皮质、脑室周围的丘脑结构和前丘脑，同时深入脑实质的深面。与青年和中年组动物比较，F4／80阳性小胶质细胞的激活在老龄动物脑中增强；老龄动物脑实质内有较多的阿米巴样小胶质细胞，CD11b的免疫组化结果与F4／80的相似。定量分析表明，细胞因子处理24小时后，在每个年龄组内，对于脑实质内小胶质细胞数目和密度进行Post-hoc检测，差异有显著性(P＜0.0001)；与青年和中年组动物比较，老龄动物脑实质内F4／80、CD11b阳性的小胶质细胞数目增加最为明显，染色密度最高，差异有显著性(P＜0.001)。FJB染色未发现FJB染色阳性的神经元；TUNEL染色未发现TUNEL染色阳性的神经元。Bcl-2阳性神经元在实验组和实验对照组均有所见，它们主要分布在第一、二运动皮质第ⅴ层和躯体感觉皮质、隔、斜角带核、海马和中脑红核等结构。Bcl-2免疫反应产物位于胞质及突起内，呈棕色，胞核不显色。Bcl-2阳性神经元在PBS注射后2天，与青年、中年组动物比较，老龄动物脑皮质、海马内Bcl-2表达明显上调(P＜0.01)。细胞因子注射2天后，与对照组比较，各年龄组动物皮质、海马内Bcl-2表达均有上调，表现为胞质及突起内Bcl-2免疫反应产物着色加深。这一变化可持续到注射后4天。与青年、中年组动物比较，老龄动物脑皮质内Bcl-2阳性细胞数目和光密度增加最为明显，定量分析结果表明其差异均有显著性(P＜0.01)。海马内Bcl-2的表达半定量结果与皮质的相似。此外，在皮质、海马和胼胝体内可见激活的星形胶质细胞表达抗凋亡蛋白Bcl-2；GFAP和Bcl-2免疫荧光双标记显示：Bcl-2免疫荧光阳性神经元周围被GFAP阳性星形胶质细胞的突起包绕或接触。实验对照组和细胞因子处理组动物皮质、海马内均有Bcl-2表达。Bcl-2为一分子量约为25-26KDa的单一蛋白带。定量的分析表明，PBS注射4天后，老年组与青年组和中年组比较，皮质和海马内Bcl-2蛋白质的表达均明显增加，差异有显著性(P＜0.01)；细胞因子注射4天后，中年组与青年组、老年组与青年组及老年组与中年组比较，皮质和海马内Bcl-2蛋白质的表达均明显增加，差异有显著性(P＜0.01)。结论：大脑脑室内注射联合细胞因子IFN-γ和TNF-α的混合物4μl，能够诱导脑实质急性神经炎症的发生。细胞因子注射后24小时，小胶质细胞明显被激活，F4／80和CD11b的表达上调；2天后，星形胶质细胞明显被激活，GFAP的表达上调；小胶质细胞的活化早于星形胶质细胞；神经胶质细胞的激活在老龄动物脑中最为明显。表明激活的神经胶质细胞可能参与脑内的免疫调节，老龄动物脑的神经胶质细胞对于炎症刺激的易感性增强。此剂量的联合细胞因子诱导的小鼠急性神经炎症反应，脑内没有发现已经死亡和正在经历死亡的神经元。细胞因子诱导的小鼠脑实质急性神经炎症反应，三个年龄组动物皮质、海马内抗凋亡蛋白Bcl-2表达均有上调，提示神经元内上调的Bcl-2可能参与脑内的免疫调节和保护性反应；Bcl-2的表达在老龄动物脑中强于中、青年组，提示老龄动物脑的神经元对于炎症刺激的易感性增强。激活的星形胶质细胞表达Bcl-2，可能参与其自身保护，Bcl-2阳性神经元周围被星形胶质细胞的突起包绕或接触，提示神经元和星形胶质细胞在参与脑内的免疫调节和保护性反应中关系密切。更多还原

【Abstract】 Objective This project was designed to investigate the mice brain response to an inflammatory challenge and whether parameters of this response vary in adulthood during lifetime. In particular, the project examined the features of activation of neuroglial cells in mice upon exposure to a mixture of proinflammatory cytokines (PICs), represented by interferon-γand tumor necrosis factor-α, and analyzed whether this activation varies with age. Furthermore, the project investigated the effect of PICs on neuronal survival in the periventricular parenchyma and its interaction with the activation of glial cells.Methods C57BL/6J mice of different ages were divided into three age groups, which consisted of young (2-3.5 months), middle-aged (10-11 months) and old (18-20 months) animals. A mixture of the PICs of 2μl recombinant murine interferon-γand of 2μl recombinant murine tumor necrosis factor-α(the biological activity of either cytokine was 500 units/perμl) was injected stereotaxically into the lateral cerebral ventricle of mice; vehicle (phosphate-buffered saline, PBS) was injected as control. After a survival of 1, 2 (all age groups) and 4 days (young and middle-aged animals) respectively, we examined the response of glial cells using glial fibrillary acidic protein (GFAP) as astrocyte marker, and the antibodies F4/80 and CD11b, which recognize microglial antigens, as markers of microglia. Expression of the anti-apoptotic protein Bcl-2 in the brain of mice of different ages was also investigated. Astrocytes and microglia were labeled with immunohistochemistry, and their number and immunosignal intensity were evaluated with quantitative image analysis. Neuronal cell death was examined in different brain structures using methods suited to detect necrotic or apoptotic phenomena, namely Fluoro-Jade B (FJB) histochemistry and the TUNEL technique, which were performed in adjacent sections. Furthermore, Bcl-2 expression was investigated with immunohistochemistry and Western blot in different brain regions after the intracerebroventricular (icv) injection of PICs. Double labeling with immunofluorescence was used to investigate whether Bcl-2 was expressed not only by neurons but also by glial cells.Results In both the PBS-injected control cases and the PICs-treated ones, GFAP-positive astrocytes in the brain of mice of the three age groups were observed. They were located mainly in the piriform and entorhinal cortex, the septum, throughout the hippocampal formation, amygdaloid complex, and in the white matter (subcortical white matter, as well as in the main fiber tracts). Qualitative differences in cell morphology were assessed between the PIC-injected and PBS-injected experimental groups. In the brain of PBS-injected control cases, glial cells did not display overt features of activation, consisting of smaller cell bodies and thinner processes. At 2 days following PIC icv treatment, in all age groups astrocytes were markedly activated in the periventricular regions, and especially in the hippocampus, exhibiting hypertrophy of cell bodies and processes and enhanced GFAP immunostaining. Such features of astrocytic activation persisted through the analyzed time course, i.e. up to 4 days following PIC treatment.Interestingly, PICs-induced astrocyte activation was significantly more marked in the brain of the aged animals, both in terms of cell hypertrophy and increased GFAP expression, than of younger age groups. This was documented by the quantitative analysis of the number and immunostaining intensity of GFAP positive astrocytes, by means of cell counts and densitometric evaluation, respectively. These analyses and their statistical evaluation showed a highly significant increase (P＜0.001) of these parameters in the old mice with respect to younger age groups in the CA1 field of the hippocampus and in the striatum at 2 days after PIC icv injections.Concerning microglia, in control brains of the three age groups most of the F4/80-positive elements exhibited the phenotype of resting microglia, with relatively small cell bodies and thin, long and ramified cell processes. After PIC icv injections, F4/80-positive cells within the brain parenchyma exhibited phenotypic changes indicative of microglial activation, which were well evident already at 24 h. These immunostained cells were characterized by hypertrophic cell bodies with thicker and stouter processes, as well as an amoeboid or rod shape, and intense immunostaining. The most prominent changes in F4/80-positive cell reactivity were detected in periventricular regions as well as in deeper brain areas, including the hippocampal formation, septum, striatum, cerebral cortex, periventricular thalamic regions, and anterior hypothalamus. As observed for astrocytes, the features of activation of F4/80-immunopositive cells were more marked in the brain of the old mice than in those of the young and middle-aged ones. Among other features, in the brain of PIC-treated old mice numerous ameboid microglial cells were observed. Quantitative analysis and statistical evaluation implicating pairwise post hoc comparison documented in the analyzed structures in each age group highly significant increases (P＜0.0001, in all groups) of the number of cells and their immunostaining intensity after PIC treatment with respect to the matched control experiments. Quantitative analysis of microglia activation also revealed significant age-by-treatment interactions in both cell counts and densitometric evaluation. In particular, PIC-dependent increases in the number of activated microglial cells were selectively larger in old mice than in young or middle-aged mice. Likewise, the immunostaining intensity of microglial cells (F4/80 optical density, OD; values) exhibited a significantly higher increase in the old mice than in the younger ones in all the analyzed structures. The results observed with CD11b immunohistochemistry were similar to those obtained with F4/80 antibodies. Neither FJB-stained neurons nor TUNEL-positive cell nuclei were found in neurons in the brain of the young and old mice after PIC icv injection, indicating that the observed features of glial activation were non secondary to neurodegenerative phenomena.Bcl-2-immunopositive cells were distributed widely in the brain of mice, in the cortex, hypothalamic parenchyma surrounding the third ventricle, in the diagonal band, in the hippocampus, septum and in the red nucleus of midbrain. Bcl-2 staining consisted of brownish reaction products mostly localized in the cytoplasm and processes of the neurons. In control brains of the three age groups, the density of Bcl-2-immunopositive elements was higher in the old mice than in the younger animals, especially in the hippocampus and cortex. At 2 days after PIC injections, the density of Bcl-2-positive neurons increased in the cortex and hippocampus with the matched control groups (P＜0.01, in all groups), and this persisted for 4 days. Thus, quantitative analysis of the number and immunostaining intensity of Bcl-2 positive cells were highly significantly increased with respect to controls in the cortex in three age groups at 2 days after PIC icv injections. The semiquantitative findings were similar in the hippocampus. Bcl-2 induction in response to PICs, IFN-γ+TNF-α, varied with age, being stronger in old mice than in the younger and middle-aged at 2 days after PIC icv injections. A significant age×treatment interaction and a significant age effect were found for cell number and density of Bcl-2 positive cells at 2 days in the analyzed structures. Expression of the antiapoptotic protein Bcl-2 was found in glial cells in the cortex, septum, hippocampus and corpus callosum at 4 days after PIC icv administration (middle-aged animals, of 10-11 months of age, were used for this experiment). Double staining for Bcl-2 and GFAP showed that these glial cells were indeed astrocytes, and the processes of activated astrocytes were found to contact and surround Bcl-2 positive neurons at 4 days after icv PIC administration.Western blot analysis detecting Bcl-2 protein in the cortex and hippocampus of young, middle-aged and old mice at 4 days following treatment with PICs and PBS, respectively. Bcl-2 is a single protein band which weight is 25-26 KDa. Densitometric evaluation of 25-26 kDa band (Bcl-2) in the brain of young, middle-aged and old mice revealed that there was a significant increase in Bcl-2 protein levels in the aged mice hippocampus and cortex at 4 days after PBS and PIC (P＜0.01)icv injections as compared to levels observed in young and middle-aged mice.Conclusion By the icv injection of a mixture of interferon-γand tumor necrosis factor-α, an acute neuroinflammatory challenge was applied to the mouse brain, and an array of parameters of the response and age-dependence of this phenomenon was investigated. The exposure to PICs induced activation of astrocytes and microglia, documented by the upregulation of glial antigens (GFAP, and the antigens recognized by F4/80 and CD11b) in the mouse brain tissue. Activation of microglia preceded that of astrocytes. The brain inflammatory reaction to PICs showed a strong age-dependence, being much more intense at an advanced age than at younger ones. This indicates the old brain is more sensitive to inflammatory stimulation than that of younger subjects, and further supports the concept that the activation of glial cells participates in the central nervous system immune-regulated response.Exposure to PICs through icv injections was also found to upregulate the expression of the Bcl-2 protein, which is well known to exert an anti-apoptotic role. This may have participated in the neuroprotective response to the inflammatory challenge. Thus, the old brain is more susceptible to inflammatory stimuli than that of younger subjects. The activation of astrocytes which express Bcl-2 also indicated that this glial cell type is implicated in a self-protective response to an inflammatory insult. Finally, the finding that processes of activated astrocytes contact and surround Bcl-2 positive neurons further points out a close relationship between astrocytes and neurons.更多还原