The effect of 2.1 T static magnetic field on astrocyte viability and morphology

The viability and a number of morphological properties of in situ astrocytes of rat spinal cord cultures including changes in surface area and migration of both cell body and nucleus were investigated at magnetic field intensities comparable to those currently used for magnetic resonance imaging. Viability of rat spinal astrocytes was studied after up to 72 hours of 2.1T static magnetic field exposure. Surface areas and two-dimensional centroids of both soma and nucleus after 2 hours of magnetic field exposure were determined and compared with those of the same cells before magnetic field exposure. Cell membrane ruffling was quantified using fractal analysis.     

小鼠视神经再生研究动物模型的建立

目的总结制作小鼠视神经完全截断性动物模型作为视神经再生研究的经验和体会。方法将雄性Bcl-2高表达转基因小鼠(Bcl-2 transgenic mice)和受GFAP启动子控制表达疱疹病毒-胸苷激酶转基因雌性小鼠(GFAP-TK)交配产生的4只8~12周成年小鼠(20~30g),Bcl-2/GFAP-TK双转基因小鼠作为实验组,同周龄4只Bcl-2转基因小鼠作为对照组。其中Bcl-2/GFAP-TK双转基因小鼠皮下植入缓释泵,连续7d释放更昔洛韦(GCV)100mg.kg-1.d-1以选择性地去除视神经损伤后激活的星形胶质细胞。更昔洛韦缓释泵植入术后2d在两组动物中制作右侧单眼标准完全性视神经钳夹损伤模型,视神经钳夹10d后获取组织标本。采用免疫荧光染色特异性检测再生轴突纤维并进行定量分析;结合罗丹明的霍乱毒素B亚单位(CTB-R)或增强表达绿色荧光蛋白的复制缺陷型腺相关病毒(AAV-EGFP)用作顺行性标记物以显示再生轴突是否到达大脑靶器官。结果在Bcl-2/GFAP-TK双转基因小鼠中存在免疫荧光阳性的再生视神经轴突,再生轴突计数为71.99±24.04,并可见生长锥(growth cone)样结构,但是再生轴突纤维未能延伸达到大脑靶器官。在对照组Bcl-2转基因小鼠中未见明显再生迹象。结论小鼠视神经完全截断性动物模型可用于视神经病变的再生研究。     

Astrocytic energy metabolism and glutamate formation--relevance for 13C-NMR spectroscopy and importance of cytosolic/mitochondrial trafficking

Glutamate plays a double role in ¹³C-nuclear magnetic resonance (NMR) spectroscopic determination of glucose metabolism in the brain. Bidirectional exchange between initially unlabeled glutamate and labeled α-ketoglutarate, formed from pyruvate via pyruvate dehydrogenase (PDH), indicates the rate of energy metabolism in the tricarboxylic acid (V_TCA) cycle in neurons (V_{PDH, n}) and, with additional computation, also in astrocytes (V_{PDH, g}), as confirmed using the astrocyte-specific substrate [¹³C]acetate. Formation of new molecules of glutamate during increased glutamatergic activity occurs only in astrocytes by combined pyruvate carboxylase (V_PC) and astrocytic PDH activity. V_{PDH, g} accounts for ∼15% of total pyruvate metabolism in the brain cortex, and V_PC accounts for another ∼10%. Since both PDH-generated and PC-generated pyruvates are needed for glutamate synthesis, ∼20/25 (80%) of astrocytic pyruvate metabolism proceed via glutamate formation. Net transmitter glutamate [γ-aminobutyric acid (GABA)] formation requires transfer of newly synthesized α-ketoglutarate to the astrocytic cytosol, α-ketoglutarate transamination to glutamate, amidation to glutamine, glutamine transfer to neurons, its hydrolysis to glutamate and glutamate release (or GABA formation). Glutamate-glutamine cycling, measured as glutamine synthesis rate (V_cycle), also transfers previously released glutamate/GABA to neurons after an initial astrocytic accumulation and measures predominantly glutamate signaling. An empirically established ∼1/1 ratio between glucose metabolism and V_cycle may reflect glucose utilization associated with oxidation/reduction processes during glutamate production, which together with associated transamination processes are balanced by subsequent glutamate oxidation after cessation of increased signaling activity. Astrocytic glutamate formation and subsequent oxidative metabolism provide large amounts of adenosine triphosphate used for accumulation from extracellular clefts of neuronally released K⁺ and glutamate and for cytosolic Ca²⁺ homeostasis.     

星形胶质细胞与缺血性脑水肿

随着对神经系统疾病机制研究的不断深入,人们认识到,它在生理和病理下均发挥极其重要的作用.脑水肿及后继的颅内压升高和脑疝形成是脑缺血主要的并发症.星形胶质细胞可通过多种途径影响脑水肿的发生.发展以及消退.明确星形胶质细胞在缺血性脑水肿中的作用及其机制,可能将为脑缺血的治疗提供新的靶点.     

大鼠在空间辨别性学习记忆时海马CA1,CA3区和DG的ACh能纤维和星形胶质细胞的变化

目的通过建立大鼠空间辨别性学习记忆的动物模型,观察大鼠海马CA1,CA3区和齿状回(DG)的乙酰胆碱(ACh)能纤维密度和含量的变化以及星形胶质细胞的数量及其胶质原纤维酸性蛋白(GFAP)表达的变化,从形态学上探讨中枢ACh和星形胶质细胞与空间辨别性学习记忆的关系.方法建立空间辨别性学习记忆的动物模型,采用乙酰胆碱酯酶(AChE)的组织化学染色及GFAP标记星形胶质细胞.并用显微镜方格目测系统和图像分析仪对大鼠海马CA1,CA3区及DG的GFAP免疫阳性星形胶质细胞的数量及其GFAP表达进行检测.结果模型组与对照组相比大鼠各观察部位的AChE阳性纤维的密度和含量均增高,有显著性差异(P<0.05).而空白组和游水组相比大鼠各观察部位的AChE阳性纤维的密度和含量均无显著性差异(P>0.05),模型组内各组间两两比较大鼠各观察部位的AChE阳性纤维的密度和含量均无显著性差异(P>0.05);模型组与对照组相比大鼠各观察部位的星形胶质细胞的数量及其GFAP的表达均增加,有显著性差异(P<0.05).而空白组和游水组相比大鼠各观察部位的星形胶质细胞的数量及其GFAP的表达均无显著性差异(P>0.05),模型组内各组间两两比较大鼠各观察部位的星形胶质细胞的数量及其GFAP的表达均无显著性差异(P>0.05).结论在空间辨别性学习记忆过程中,大鼠各观察部位的AChE阳性纤维的密度和含量增高,说明中枢ACh参与空间辨别性学习记忆过程;在空间辨别性学习记忆过程中,大鼠各观察部位的星形胶质细胞的数量及其GFAP表达的增加,说明星形胶质细胞参与空间辨别性学习记忆过程.     

Symbiosis between in vivo and in vitro NMR spectroscopy: The creatine, N-acetylaspartate, glutamate, and GABA content of the epileptic human brain

High resolution ¹H NMR spectroscopy was used to analyze temporal lobe biopsies obtained from patients with epilepsy. Heat-stabilized cerebrum, dialyzed cytosolic macromolecules, and perchloric acid extracts were studied using one- and two dimensional spectroscopy. Anterior temporal lobe neocortex was enriched in GABA, glutamate, alanine, N-acetylaspartate, and creatine. Subjacent white matter was enriched in aspartate, glutamine, and inositol. The N-acetylaspartate/creatine mole ratio was lower in anterior temporal neocortex with mesial (0.66) than neocortical (0.80) temporal lobe epilepsy. Human brain biopsy samples were separated into crude and refined synaptosomes, neuronal cell bodies, and glia using density gradient centrifugation. Neuronal fractions were enriched in glutamate and N-acetylaspartate. Glial cell fractions were enriched in lactate, glutamine, and inositol. The creatine content was the same in biopsied epileptic cortex (8.8–8.9 mmol/kg) and normal in vivo occipital lobe (8.9 mmol/kg). Glutamate content was higher in epileptic cortex at biopsy (10.1–10.5 mmol/kg) than normal in vivo occipital lobe (8.8 mmol/kg). GABA content was higher in biopsies of epileptic cortex (2.3–2.2 mmol/kg) than in normal in vivo occipital lobe (1.2 mmol/kg). N-acetylaspartate content was lower in biopsied epileptic temporal cortex (5.8–6.8 mmol/kg) than normal in vivo occipital lobe (8.9 mmol/kg). Paired in vivo and ex vivo measurements are critical for a firm understanding of the changes seen in the ¹H-spectra from patients with epilepsy.