Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy; Department of Biotechnology, University of Rijeka, Rijeka, Croatia.
Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy.
Brain Res. 2019 May 1;1710:8-21. doi: 10.1016/j.brainres.2018.12.029. Epub 2018 Dec 19.
The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity.
新生大鼠的孤立脊髓被广泛用于阐明网络发育的基本机制或损伤后早期的退化阶段。然而，这种制备物在 Krebs 溶液中只能存活 24 小时，因此需要探索延长其存活时间以进行纵向研究的方法。本报告显示，在富含氧气的 Eagle 基础培养基（BME）中培养脊髓可极好地保存神经元（包括运动神经元）、神经胶质和初级传入（包括背根神经节），最长可达 72 小时。使用 DMEM 培养基则不成功。脊髓网络出现了新的特征，具有强烈的自发性活动，而虚构的运动模式出现缺陷，周期刻板地缓慢。用突触蛋白标志物突触素 1 和突触小体蛋白染色显示，在体外 3 天后信号明显减弱。谷氨酸能和甘氨酸能神经元标志物的免疫组织化学染色表明后者显著减少。同样，GABA 合成酶 GAD65 的表达也较低。因此，运动网络的功能障碍似乎与抑制性突触的丧失有关。这种现象在体外保存的类似负鼠脊髓的类似制备物中并未发生。总之，尽管组织学数据表明培养的脊髓未受损（除了抑制性生物标志物），但电生理学数据显示存在重要的功能障碍。因此，抑制性突触的下调可能是大鼠脊髓网络进行性过度兴奋的原因，尽管其组织学外观似乎正常。我们的观察结果可能有助于理解脊髓损伤的某些延迟效应（如慢性疼痛和痉挛）的基础。
