Culturing adult canine sensory neurons to optimise neural repair
Culturing adult canine sensory neurons to optimise neural repair
复制标题
DOI：
10.1136/vr.100255
复制
发表时间：
2012-01-28
影响因子：
2.2
通讯作者：
Wewetzer, K.
中科院分区：
文献类型：
--
作者：
Gerhauser, I.;Hahn, K.;Wewetzer, K.
成年背根神经节（DRG）神经元作为研究神经元-神经胶质相互作用的通用工具（Päiväläinen等2008）最初从鸡胚和新生啮齿动物中分离（Barbin等1984），后来从成年大鼠中分离（Grothe和Unsicker 1987）。由于迄今为止还没有适用于分离成年犬DRG神经元的方案，因此建立了犬细胞与胎鼠神经元结合的嵌合试验（Kamishina等人，2009年）。简单地说，使用手术刀片切碎神经节，并在37 ℃下在Dulbecco改良Eagle培养基（DMEM; Gibco，Invitrogen）中的I型胰蛋白酶、IV-S型透明质酸酶和XI型胶原酶（各0.5%，Sigma-Aldrich）的混合物中孵育45分钟。在含有DNA酶I的DMEM中机械解离组织（0.05%，Roche Diagnostics曼海姆），使用连续缩窄的火焰收缩巴斯德移液器，离心沉淀（10分钟，200 xg），在Neubauer室中计数，并以每孔200个神经元的密度接种在DME培养基中的微量滴定板上（CLS 3696，Corning，Sigma-Aldrich）和聚-1-溶素/层粘连蛋白（Becton Dickinson）包被。
Adult dorsal root ganglion (DRG) neurons as a versatile tool to study neuron-glia interactions (Pä ivä lä inen and others 2008) were initially isolated from embryonic chick and neonatal rodents (Barbin and others 1984) and later on from adult rats (Grothe and Unsicker 1987). Since protocols suitable for isolation of adult canine DRG neurons have not been made available so far, chimeric assays were established combining canine cells with fetal rat neurons (Kamishina and others 2009). Briefly, ganglia were minced using scalpel blades and incubated in a mixture of type I trypsin, type IV-S hyaluronidase and type XI collagenase (each 0.5 per cent, Sigma-Aldrich) in Dulbecco's modified Eagle medium (DMEM; Gibco, Invitrogen) for 45 minutes at 37ÂC. Tissue was mechanically dissociated in DMEM with DNase I (0.05 per cent, Roche Diagnostics Mannheim) using successively narrowed flame-constricted Pasteur pipettes, pelleted by centrifugation (10 minutes, 200 xg), counted in a Neubauer chamber and seeded in DME medium at a density of 200 neurons per well on microtiterplates (CLS 3696, Corning, Sigma-Aldrich) coated with poly-l-lysin (100 μg/ml, Sigma-Aldrich) and poly-l-lysin/laminin (Becton Dickinson).