猫运动皮层神经元和S100、GFAP阳性细胞的年龄相关性变化

比较了青、老年猫运动皮层神经元与S100、GFAP免疫阳性胶质细胞的形态学变化,并探讨其与衰老过程中运动功能衰退的关系。采用Nissl染色显示青、老年猫运动皮层分层结构和神经元。免疫组织化学方法(SABC法)显示青、老年猫运动皮层S100免疫反应阳性(S100-immunoreactive,S100-IR)细胞及胶质纤维酸性蛋白免疫反应阳性(GFAP-immunoreactive,GFAP-IR)细胞。在Olympus显微镜下,用Moitcam5000数码成像与分析系统计数运动皮层各层神经元、S100-IR细胞及GFAP-IR细胞的数量,并随机抽样测量S100-IR、GFAP-IR细胞的胞体直径。与青年猫相比,老年猫运动皮层Ⅴ、Ⅵ层神经元密度显著下降(P<0.01),老年猫运动皮层中S100-IR和GFAP-IR细胞密度与胞体直径均显著增加(P<0.01),且细胞的免疫阳性反应较强。研究结果表明,猫运动皮层的神经元密度在衰老过程中Ⅴ、Ⅵ层神经元密度显著下降,有可能会降低老年个体运动皮层对运动的调控能力;随着衰老、运动皮层的星形胶质细胞出现明显的反应性活化与增生,这对维持大脑运动皮层神经元的活性和神经元之间的通讯联系,从而延缓老年性运动功能衰退具有重要意义。     

S100在猫小脑中的分布及其表达的年龄相关性变化

用免疫组织化学ABC法标记S100免疫阳性(S100-IR)细胞,观察S100蛋白在青年猫和老年猫小脑中的分布,探讨其表达的年龄相关变化及意义。光镜下计数颗粒层和髓质中S100-IR细胞密度及浦肯野细胞(PC)层阳性细胞线密度。结果显示,颗粒层和髓质中S100-IR细胞密度较小、分布均匀,PC层阳性细胞相对密集,分子层未见阳性反应;阳性细胞胞浆深染。与青年猫相比,老年猫小脑颗粒层、髓质和PC层中S100-IR细胞密度显著增加(P<0.01),胞体较大,阳性较强。表明S100-IR细胞在小脑中的分布具区域性差异,呈明显的年龄相关性增生,推测其增生对衰老神经元的丢失起保护作用。     

猫视皮层星形胶质细胞的老年性变化

电生理研究结果显示,在衰老过程中猫的视皮层神经元对视觉刺激的反应性出现显著的功能衰退,是否这种功能性衰退伴随胶质细胞活动的改变尚无直接的实验证据。以前期电生理实验猫为材料,用免疫形态学方法比较青年猫和老年猫初级视皮层内星形胶质细胞的活动状况。利用Nissl染色显示猫初级视皮层组织结构,用免疫组织化学方法（SABC法）显示GFAP免疫阳性（GFAP-IR）星形胶质细胞。光镜下观察、拍照,对GFAP-IR细胞计数并换算成密度,测量GFAP-IR直径取平均值。老年猫初级视皮层灰质各层及白质内的GFAP-IR细胞密度比青年猫的显著升高（p〈0.001）。与青年猫相比,老年猫视皮层灰质和白质中GFAP-IR细胞的平均直径均比青年猫的显著增大（p〈0.0001）,且老年猫视皮层内GFAP阳性免疫反应较青年猫的明显增强。老年猫初级视皮层神经元功能衰退伴随着星形胶质细胞活动的增强,胶质细胞活动增强有助于神经元之间的信息交流,因而可能对衰老过程中神经元的功能衰退起补偿作用。     

猫视神经中S100蛋白表达的年龄相关变化

比较老年猫和青年猫视神经S100蛋白表达及胶质细胞的年龄相关变化,探讨其可能的生理作用.取老年猫(10～13龄)和青年猫(1～3龄)各4只的颅内视神经相应部分作组织切片,用免疫组织化学ABC法标记S100免疫阳性(S100～IR)细胞,Marsland-Gless染色显示胶质细胞.光镜下采用图像分析系统计数视神经中S100-IR细胞密度、胶质细胞密度及阳性反应灰度值.视神经中棕黄色S100-IR细胞分布均匀,Marsland-Gless染色的纤维横断面及胶质细胞均呈棕红色.与青年猫相比,老年猫视神经中胶质细胞密度明显增大;S100-IR细胞密度显著增加(P<0.01),胞体较大,阳性较强(灰度值显著减小,P<0.01);S100-IR细胞在胶质细胞中所占比例亦显著增大.结果表明S100-IR细胞呈明显的年龄相关性增生,这可能对衰老的神经纤维起保护作用.     

S100蛋白与GFAP在猫外侧膝状体表达的年龄相关性变化

目的 比较青年猫与老年猫外侧膝状体（lateral geniculate nucleus,LGN）星形胶质细胞（astrocyte,AS）中S100蛋白与胶质原纤维酸性蛋白（glial fibrillary acidic protein,GFAP）表达的年龄相关性变化,探讨导致相关变化的原因及其在动物视觉功能衰老中的意义。方法 免疫组织化学方法（SABC法）示S100蛋白阳性细胞及GFAP阳性细胞。光镜下观察、拍照,计数外侧膝状体各层中S100蛋白阳性细胞及GFAP阳性细胞数量。结果 与青年猫相比,老年猫外侧膝状体各层中S100蛋白与GFAP表达均有不同程度的显著增强（P〈0.01）。结论 动物视觉衰老进程中,外侧膝状体星形胶质细胞存在着明显的反应性胶质化（reactive gliosis）,这种胶质化与老年动物视觉功能之间关系将在文中讨论。     

猫运动皮层神经元和S100、GFAP阳性细胞的年龄相关性变化

比较了青、老年猫运动皮层神经元与S100、GFAP免疫阳性胶质细胞的形态学变化,并探讨其与衰老过程中运动功能衰退的关系。采用Nissl染色显示青、老年猫运动皮层分层结构和神经元。免疫组织化学方法（SABC法）显示青、老年猫运动皮层S100免疫反应阳性(S100-immunoreactive, S100-IR)细胞及胶质纤维酸性蛋白免疫反应阳性(GFAP-immunoreactive, GFAP-IR)细胞。在Olympus 显微镜下,用Moitcam 5000数码成像与分析系统计数运动皮层各层神经元、S100-IR细胞及GFAP-IR细胞的数量,并随机抽样测量S100-IR、GFAP-IR细胞的胞体直径。与青年猫相比,老年猫运动皮层Ⅴ、Ⅵ层神经元密度显著下降(P < 0.01),老年猫运动皮层中S100-IR和GFAP-IR细胞密度与胞体直径均显著增加(P < 0.01),且细胞的免疫阳性反应较强。研究结果表明,猫运动皮层的神经元密度在衰老过程中Ⅴ、Ⅵ层神经元密度显著下降,有可能会降低老年个体运动皮层对运动的调控能力;随着衰老、运动皮层的星形胶质细胞出现明显的反应性活化与增生,这对维持大脑运动皮层神经元的活性和神经元之间的通讯联系,从而延缓老年性运动功能衰退具有重要意义。     

猫初级视皮层内Glu／GABA表达比率的衰老性变化

最近的一些研究结果显示,视皮层内抑制性递质系统作用减弱可能是导致老年性视觉功能衰退的重要因素。是否皮层内兴含性递质系统办伴随衰老而发生改变并影响皮层内神经兴奋与抑制的平衡尚不清楚。为此,利用Nissl染色和免疫组织化学染色方法以及Image—Pro Express图像分析软件对青、老年猫初级视皮层（17区）内各层神经元密度、兴奋性递质谷氦酸免疫反应阳性（Glu—immunoreactive,Glu—IR）神经元密度以及抑制性递质γ-氨基丁酸免疫反应阳性（v．aminobutyric acid-immunoreactive,GABA—IR）神绎元密度进行了统计分析。结果显示,青、老年猫初级视皮层各层神经元密度均没有明显的年龄性差异（P〉0．05）;与青年猫相比,老年猫初级视皮层Glu—IR、GABA．IR神经元密度均显著减少（P〈0．01）,而Glu—IR／GABA-IR神经元密度比率却显著增大（P〈0．01）。结果提示,老年猫初级视皮层内兴奋性递质系统作用相对增强,而抑制性递质系统的作用相对减弱,导致皮层内兴奋-抑制平衡关系失调,这可能是引起老年个体视觉功能衰退的重要原因之一。     

猫内侧膝状体年龄相关性形态学变化

目的比较青年猫和老年猫内侧膝状体神经元及S100蛋白与波形蛋白表达的年龄相关性变化。方法Nissl染色显示内侧膝状体结构及神经元,免疫组织化学方法示S100免疫反应阳性（S100-IRS100-immunoreactive）细胞及波形蛋白免疫反应阳性（Vimentin-IR Vimentin-immunoreactive）细胞。光镜下观察,利用图像分析软件进行图像采集分析。结果青年猫和老年猫内侧膝状体神经元数量及胞体直径无明显改变（P〉0.05）;与青年猫相比,老年猫内侧膝状体各分区中S100-IR细胞与Vimentin-IR细胞密度均显著增大,且免疫阳性反应强度增强（P〈0.01）,提示老年会导致内侧膝状体S100与波形蛋白表达显著增强。结论在衰老过程中,内侧膝状体处于静息和激活状态的星形胶质细胞均出现明显的增生,这对维持老年个体内侧膝状体神经元的正常形态和功能,从而延缓老年性听觉功能衰退可能具有重要作用。     

猫初级视皮层内GIu/GABA表达比率的衰老性变化

最近的一些研究结果显示,视皮层内抑制性递质系统作用减弱可能是导致老年性视觉功能衰退的重要因素.是否皮层内兴奋性递质系统亦伴随衰老而发牛改变并影响皮层内神经兴奋与抑制的平衡尚不清楚.为此,利用Nissl染色和免疫组织化学染色方法以及Image-Pro Express图像分析软件对青、老年猫初级视皮层(17区)内各层神经元密度、兴奋性递质谷氨酸免疫反应阳性(Glu-immunoreactive,Glu-IR)神经元密度以及抑制性递质γ-氨基丁酸免疫反应阳性(γ-aminobutyric acid.immunoreactive,GABA-IR)神经元密度进行了统汁分析.结果显示,青、老年猫初级视皮层各层神经元密度均没有明显的年龄性差异(P>0.05);与青年猫相比,老年猫初级视皮层Glu-IR、GABA-IR神经元密度均显著减少(P<0.01),而Glu.IR/GABA.IR神经元密度比率去却显著增大(P<0.01).结果提示,老年猫初级视皮层内兴奋性递质系统作用相对增强,而抑制性递质系统的作用相对减弱,导致皮层内兴奋-抑制平衡关系失调,这可能是引起老年个体视觉功能衰退的重要原因之一.     

Directional tunings independent of orientation in the primary visual cortex of the cat

A family of moving 'random-line' patterns was developed and used to study the directional tuning of 91 single units in cat primary visual cortex (V1). The results suggest that, in addition to the well-known orientation-dependent mechanism, there is also some kind of orientation-independent mechanism underlying the direction selectivity. The directional tuning of the neurons varies in accordance with the increase of orientation or non-orientation element in the stimulus.     

Directional tunings independent of orientation in the primary visual cortex of the cat

A family of moving ‘random-line’ patterns was developed and used to study the directional tuning of 91 single units in cat primary visual cortex (V1). The results suggest that, in addition to the well-known orientation-dependent mechanism, there is also some kind of orientationindependent mechanism underlying the direction selectivity. The directional tuning of the neurons varies in accordance with the increase of orientation or non-orientation element in the stimulus.     

Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat

The recent introduction of fluorescent two-dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30-day-old kittens, in order to identify proteins expressed in an age-related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two-dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.     

Age-related changes of structures in cerebellar cortex of cat

We studied the structures of the cerebellar cortex of young adult and old cats for age-related changes, which were statistically analysed. Nissl staining was used to visualize the cortical neurons. The immunohistochemical method was used to display glial fibrillary acidic protein (GFAP)-immunoreactive (IR) astrocytes and neurofilament-immunoreactive (NF-IR) neurons. Under the microscope, the thickness of the cerebellar cortex was measured; and the density of neurons in all the layers as well as that of GFAP-IR cells in the granular layer was analysed. Compared with young adult cats, the thickness of the molecular layer and total cerebellar cortex was significantly decreased in old cats, and that of the granular layer increased. The density of neurons in each layer was significantly lower in old cats than in young adult ones. Astrocytes in old cats were significantly denser than in young adult ones, and accompanied by evident hypertrophy of the cell bodies and enhanced immunoreaction of GFAP substance. Purkinje cells (PCs) in old cats showed much fewer NF-IR dendrites than those in young adults. The above findings indicate a loss of neurons and decrease in the number of dendrites of the PCs in the aged cerebellar cortex, which might underlie the functional decline of afferent efficacy and information integration in the senescent cerebellum. An age-dependent enhancement of activity of the astrocytes may exert a protective effect on neurons in the aged cerebellum     

A neuronal basis of iconic memory in macaque primary visual cortex

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Retinoic acid increases expression of the calcium-binding protein S100P in human gastric cancer cells

Retinoids mediate a wide spectrum of antitumor activities through induction of growth arrest, differentiation or apoptosis. To determine whether the effects of retinoids are mediated by specific gene activation or repression, one-day treatments of SC-M1 CL23 gastric cancer cells with vehicle alone or all-TRANS retinoic acid (tRA) (10 microM) were compared using differential display analysis. A 432-bp cDNA fragment from the tRA-treated cells was differentially amplified and its sequence analysis indicated homology with the calcium-binding protein S100P. Levels of S100P mRNA were increased 3.5-fold in SC-M1 CL23 gastric cancer cells treated with 10 microM tRA for 1 day, and the regulation was time- and concentration-dependent. Treatment with tRA (10 microM) also increased S100P mRNA levels in tRA-sensitive HtTA cells but not in inherent RA-resistant TMC-1 cells. However, the tRA-mediated increase in S100P expression was maintained in SC-M1/R cells that were established long-term in tRA-containing medium and had acquired partial RA resistance to tRA-induced growth suppression. In conclusion, tRA increases S100P expression, and the regulation remains intact in cells which develop acquired RA resistance.     

Bovine Brain S100 Proteins: Separation and Characterization of a New S100 Protein Species

Three S100 protein species (S100a, S100b, S100a') have been purified from bovine brain using a modification of standard preparative methods. A higher yield for each protein was obtained at the last separation step. Characterization by urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis, UV absorption spectra, and fluorescence parameters provided evidence of a new tryptophan-containing S100 protein called S100a', which exhibits, as S100a and S100b, the properties of a Ca2+ binding protein.     

Oncogenic Kras expression in postmitotic neurons leads to S100A8-S100A9 protein overexpression and gliosis

Previous studies suggest that up-regulation of Ras signaling in neurons promotes gliosis and astrocytoma formation in a cell nonautonomous manner. However, the underlying mechanisms remain unknown. To address this question, we generated compound mice (LSL Kras G12D/+;CamKII-Cre) that express oncogenic Kras from its endogenous locus in postmitotic neurons after birth. These mice developed progressive gliosis, which is associated with hyperactivation of Ras signaling pathways. Microarray analysis identified S100A8 and S100A9 as two secreted molecules that are significantly overexpressed in mutant cortices. In contrast to their usual predominant expression in myeloid cells, we found that overexpression of S100A8 and S100A9 in the mutant cortex is primarily in neurons. This neuronal expression pattern is associated with increased infiltration of microglia in mutant cortex. Moreover, purified S100A8-S100A9 but not S100A8 or S100A9 alone promotes growth of primary astrocytes in vitro through both TLR4 and receptor of advanced glycation end product receptors. In summary, our results identify overexpression of S100A8-S100A9 in neurons as an early step in oncogenic Kras-induced gliosis. These molecules expressed in nonhematopoietic cells may be involved in tumorigenesis at a stage much earlier than what has been reported previously.