低温对大鼠海马脑片缺氧无糖损伤的保护作用及其与Glu受体的关系

目的:探讨低温对离体大鼠海马脑片缺氧无糖(oxygen and glucose deprivation,OGD)损伤的保护作用及其机制.方法:①观察大鼠海马脑片在OGD条件下顺向群峰电位(orthodromic population spike,OPS)的变化及温度对它的影响.②观察谷氨酸(Glu)对海马脑片OPS的影响及低温的抗Glu毒性作用.并在人工脑脊液(ACSF)中分别加入GABA-R的特异性阻滞剂bicuculline(BMI)和NMDA-R的特异性阻滞剂D-(-)-2-Amino-5-phospho-nopentanoic Acid(AP5)或加入BMI和非NMDA-R阻滞剂6,7-Dinitroquinoxaline-2,3(1H,4H)-dione(CNQX)来观察低温对海马脑片OGD损伤保护作用的突触后受体机制.③观察OGD1h后海马CA1区锥体细胞超微结构的变化及低温对其的影响.结果:①OGD可以使海马脑片OPS迅速降低并很快消失,14 min后复氧供糖OPS极少恢复.低温(32℃、25℃)能使OPS消失时间明显延长,复氧供糖后OPS恢复良好.25℃其作用优于32℃.②2 mmol/LGlu使海马脑片OPS迅速消失,洗出后难以恢复.低温(3 2℃、25℃)能显著改善去Glu 1h后OPS的恢复.ACSF中加入BMI CNQX和BMI AP5均对25℃低温处理28min的脑保护作用没有影响.③OGD1h后CA1区锥体细胞水肿严重,胞浆内细胞器变性坏死脱失,线粒体肿胀,脊呈空泡状.低温(25℃)组细胞核膜规则,线粒体轻度肿胀.结论:低温有显著的抗脑OGD损伤作用,其作用机制可能与抗Glu的兴奋性毒性作用和维持细胞内ATP水平有关.而其抗兴奋性毒性作用可能既有NMDA-R又有非NMDA-R的参与.     

七氟醚对大鼠海马脑片缺血损伤的保护作用

目的:探讨七氟醚对脑缺血损伤的保护作用及其机制。方法:用电生理细胞外记录的方法和组织学检查的技术,观察对照组、2%七氟醚组和4%七氟醚组对缺氧无糖(OGD)及谷氨酸(Glu)损伤所致的大鼠海马脑片CA1区顺向群峰电位(OPS)的影响及各组脑片超微结构的变化。结果:对照组和2%七氟醚组在OGD和Glu损伤后海马脑片OPS很难恢复;4%七氟醚组明显改善OPS的恢复程度和恢复率,减轻海马CA1区神经元细胞损伤。电镜观察可见,对照组OGD和Glu损伤后海马CA1区锥体细胞明显水肿,核膜不完整,核染色加深,核内染色质凝聚成块,胞浆中内质网高度扩张,线粒体水肿;2%七氟醚组与对照组相似;4%七氟醚组细胞水肿不显,核膜完整,核内染色质轻度凝聚,内质网轻度扩张,线粒体无明显水肿。结论:4%七氟醚对大鼠海马脑片OGD损伤有保护作用,可能与减轻兴奋性Glu毒性有关。     

中风膏对氧糖剥夺/再复氧损伤大鼠海马神经干细胞增殖的影响

目的：研究中风膏对体外氧糖剥夺/再复氧损伤大鼠神经干细胞增殖的影响。方法：采用悬浮培养法分离纯化新生SD大鼠大脑海马神经干细胞,免疫荧光法鉴定细胞,第3代贴壁培养神经干细胞氧糖剥夺2 h后,复氧培养24 h造模。实验分为正常对照组、模型组、中风膏5%含药血清组、中风膏10%含药血清组、中风膏20%含药血清组,每组6个复孔,分1 d、3 d、5 d、7 d 4个时间点。CCK8法检测细胞活力,流式细胞仪测定细胞凋亡。结果：悬浮培养细胞均高表达巢蛋白（nestin）。在增殖试验中,正常组与模型对照组比较,模型对照组的细胞增殖能力明显降低（P<0.01）;与模型对照组相比,第3日,中风膏20%治疗组的细胞增殖能力明显增强（P<0.01）;实验第5日、7日,中风膏5%、10%、20%组细胞增殖能力明显增强（P<0.01）。在凋亡实验中,与正常组比较,模型对照组的细胞凋亡明显升高（P<0.01）;与模型对照组相比,中风膏20%组细胞凋亡显著下降（P<0.01）。结论：中风膏含药血清对海马神经干细胞OGD/R损伤后的增殖能力有显著的修复作用,20%中风膏含药血清对干细胞损伤有治疗作用,可降低细胞凋亡。     

Effect of lipophilicity of Mn (III) ortho N-alkylpyridyl- and diortho N,N′-diethylimidazolylporphyrins in two in-vitro models of oxygen and glucose deprivation-induced neuronal death

In vivo investigations have confirmed the beneficial effects of hydrophilic, cationic Mn(III) porphyrin-based catalytic antioxidants in different models of oxidative stress. Using a cell culture model of rat mixed neuronal/glial cells, this study investigated the effect of MnTnOct-2-PyP⁵⁺ on oxygen and glucose deprivation (OGD)-induced cell death as compared to the effects of widely studied hydrophilic analogues MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ and a standard compound, dizocilpine (MK-801). It was hypothesized that the octylpyridylporphyrin, MnTnOct-2-PyP⁵⁺, a lipophilic but equally potent antioxidant as the other two porphyrins, would be more efficacious in reducing OGD-induced cell death due to its higher bioavailability. Cell death was evaluated at 24 h using lactate dehydrogenase (LDH) release and propidium iodide staining. At concentrations from 3–100 µm, all three porphyrins reduced cell death as compared to cultures exposed to OGD alone, the effects depending upon the concentrations and type of treatment. To assess the effect of lipophilicity the additional experiments were performed using submicromolar concentrations of MnTnOct-2-PyP⁵⁺ in an organotypic hippocampal slice model of OGD with propidium iodide and Sytox staining. When compared to oxygen and glucose deprivation alone, concentrations of MnTnOct-2-PyP⁵⁺ as low as 0.01 µm significantly (p<0.001; power 1.0) reduced neuronal cells similar to control. This is the first in vitro study on the mammalian cells which indicates that MnTnOct-2-PyP⁵⁺ is up to 3000-fold more efficacious than equally potent hydrophilic analogues, due entirely to its increased bioavailability. Such remarkable increase in efficacy parallels 5.7-orders of magnitude increase in lipophilicity of MnTnOct-2-PyP⁵⁺ (log P=?0.77) when compared to MnTE-2-PyP⁵⁺ (log P_ow=?6.43), P_ow being partition coefficient between n-octanol and water.     

The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P₂, but this function appears normal in Synaptojanin^RQ knock‐in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P₂, two lipids found in autophagosomal membranes. Using advanced imaging, we show that Synaptojanin^RQ mutants accumulate the PI(3)P/PI(3,5)P₂‐binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell‐derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in Synaptojanin^RQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic‐specific autophagy defects to Parkinson's disease.     

Cu2‐xS Surface Phases and Their Impact on the Electronic Structure of CuInS2 Thin Films – A Hidden Parameter in Solar Cell Optimization

The surface properties of CuInS₂ (CIS) thin‐film solar cell absorbers are investigated by a combination of electron and soft X‐ray spectroscopies. Spatially separated regions of varying colors are observed and identified to be dominated by either CuS or Cu₂S surface phases. After their removal by KCN etching, the samples cannot be distinguished by eye and the CIS surface is found to be Cu‐deficient in both regions. However, a significantly more pronounced off‐stoichiometry in the region initially covered by Cu₂S can be identified. In this region, the resulting surface band gap is also significantly larger than the E_g^Surf of the initially CuS‐terminated region. Such variations may represent a hidden parameter which, if overlooked, induces irreproducibility and thus prevents systematic optimization efforts.