脊髓薄片器官型培养及组织化学鉴定方法研究

A study on organotypic spinal cord slice culture and histochmical identification

目的探讨脊髓薄片器官型培养的方法及其腹角α运动神经元、背角中间神经元的鉴别。方法利用出生8d乳鼠的腰段脊髓组织切片建立脊髓器官型培养模型,并用神经元的特异性SMI蛳32和Calretinin单克隆抗体进行免疫组化染色,对脊髓腹角α运动神经元和背角中间神经元加以鉴定,测定培养液中乳酸脱氢酶(LDH)的含量。结果脊髓片在体外生长良好,形态完整,α运动神经元和背角中间神经元的数目及各时点培养液中LDH含量恒定,脊髓片可存活2个月以上。结论脊髓的器官培养技术为研究脊髓生理、病理改变及神经保护提供了有效的方法。

Objective To develop a method of organotypic cultrue of spinal cord slice and to establish ways to differentiate ventral α-motor neuron and dorsal interneuron. Methods The slice cultures were prepared with lumbar spinal cord from 8-day-old rat. The survival of α-motor neuron was evaluated by immunohistochemical staining with monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker. The interneurons in dorsal horn were identified by monoclonal anti-calretinin staining. Lactate dehydrogenase (LDH) levels in culture medium were also measured. Results The spinal cord explants could be maintained in the culture for more than 2 months with excellent cellular organization and stable populations of ventralαmotor neurons and dorsal interneurons. The levels of LDH at different culture times have no significant difference. Conclusions Organotypic spinal cord slice cultures may provide an effective method for physiological and pathological changes, and neuroprotection of spinal cord study.