Adamchik Y, Frantseva M V, Weisspapir M, Carlen P L, Perez Velazquez J L
Playfair Neuroscience Unit, McL 12-413, Toronto Western Hospital, Department of Medicine (Neurology), University of Toronto, 399 Bathurst Street, Toronto, Canada.
Brain Res Brain Res Protoc. 2000 Apr;5(2):153-8. doi: 10.1016/s1385-299x(00)00007-6.
Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30 degrees C), which is known to be neuroprotective [F.C. Barone, G.Z. Feuerstein, R.F. White, Brain cooling during transient focal ischaemia provides complete neuroprotection, Neurosci. Biobehav. Rev. 1, 1997, pp. 31-44; V.M. Bruno, M.P. Goldberg, L.L. Dugan, R.G. Giffard, D.W. Choi, Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen glucose deprivation or excitatory aminoacids, J. Neurochem. 4, 1994, pp. 387-392; G.C. Newman, H. Qi, F.E. Hospod, K. Grundhmann, Preservation of hippocampal brain slices with in vivo or in vitro hypothermia, Brain Res. 1, 1992, pp. 159-163; J.Y. Yager, J. Asseline, Effect of mild hypothermia on cerebral energy metabolism during the evolution of hypoxic ischaemic brain damage in the immature rat, Stroke, 5, 1996, pp. 919-925.]. Low temperature incubation significantly reduced cell death, now being 9% at 24 h and 14% at 67 h. Our results show that these models of moderate mechanical trauma using organotypic slice cultures can be used to study neurodegeneration and neuroprotective strategies.
器官型脑片培养已用于多种关于神经退行性变过程的研究[K.M. 阿卜杜勒 - 哈米德、M. 蒂米安斯基，细胞内钙缓冲对暴露于缺氧/无糖血症或兴奋性毒素的器官型海马培养物中神经元存活的机制及影响，《神经科学杂志》17卷，1997年，第3538 - 3553页；D.W. 纽厄尔、A. 巴特、V. 帕皮马斯特、A.T. 马洛夫，器官型海马培养物中氧和葡萄糖剥夺引起的谷氨酸和非谷氨酸受体介导的毒性，《神经科学杂志》15卷，1995年，第7702 - 7711页；J.L. 佩雷斯·贝拉斯克斯、M.V. 弗兰采娃、P.L. 卡伦，体外缺血促进培养海马切片锥体神经元中谷氨酸介导的自由基生成和细胞内钙积累，《神经科学杂志》23卷，1997年，第9085 - 9094页；L. 斯托皮尼、L.A. 布克斯、D. 米勒，一种用于神经组织器官型培养的简单方法，《神经科学方法杂志》37卷，1991年，第173 - 182页；R.C. 塔克、J.T. 科伊尔、J.J. 沃尔诺夫，器官型海马培养物中对低血糖诱导神经毒性的区域易损性：早期河豚毒素或延迟MK - 801的保护作用，《神经科学杂志》12卷，1992年，第4298 - 4308页]。我们描述了两种在海马器官型培养物中诱导创伤性细胞损伤的方法。原发性创伤损伤是通过在器官型切片上滚动一个不锈钢圆柱体（0.9克）来实现的。继发性损伤是在从2毫米高度将一个重物（0.137克）落在器官型切片的局部区域后发生的。通过在损伤后的几个时间点测量活力指示剂碘化丙啶（PI）的荧光来确定细胞死亡的时间进程和程度。初始局部撞击损伤在损伤后24小时和67小时扩散，当切片保持在37摄氏度时，细胞死亡率分别为25%和54%。为了验证这些方法作为评估神经保护策略的模型，在相对低温（30摄氏度）下对切片施加类似的损伤，已知低温具有神经保护作用[F.C. 巴龙、G.Z. 费尔斯坦、R.F. 怀特，短暂性局灶性缺血期间脑冷却提供完全的神经保护，《神经科学与行为评论》1卷，1997年，第31 - 44页；V.M. 布鲁诺、M.P. 戈德堡、L.L. 杜根、R.G. 吉法德、D.W. 崔，低温对暴露于氧葡萄糖剥夺或兴奋性氨基酸的皮质培养物的神经保护作用，《神经化学杂志》4卷，1994年，第387 - 392页；G.C. 纽曼、H. 齐、F.E. 霍斯波德、K. 格伦德曼，体内或体外低温保存海马脑片，《脑研究》1卷，1992年，第159 - 163页；J.Y. 亚格、J. 阿塞琳，轻度低温对未成熟大鼠缺氧缺血性脑损伤演变过程中脑能量代谢的影响，《中风》5卷，1996年，第919 - 925页]。低温孵育显著降低了细胞死亡，24小时时为9%，67小时时为14%。我们的结果表明，这些使用器官型切片培养的中度机械创伤模型可用于研究神经退行性变和神经保护策略。
