Evans Teresa A, Barkauskas Deborah S, Myers Jay T, Huang Alex Y
Department of Neurosciences, Case Western Reserve University.
Department of Biomedical Engineering, Case Western Reserve University.
J Vis Exp. 2014 Nov 23(93):e52228. doi: 10.3791/52228.
Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.
创伤性脊髓损伤会引发一种炎症反应，涉及源自血液的巨噬细胞和中枢神经系统（CNS）驻留的小胶质细胞。活体双光子显微镜可用于研究活体动物脊髓损伤部位的巨噬细胞和小胶质细胞。这可以在成年动物的背柱遭受创伤性损伤时进行。在此，我们描述了使用辐照骨髓嵌合体来区分中枢神经系统中巨噬细胞和小胶质细胞的方法，以获得仅巨噬细胞或小胶质细胞用基因编码的绿色荧光蛋白标记的动物。我们还描述了一种脊髓背柱挤压损伤模型，由此产生一个简单、易于接近的矩形损伤，通过椎板切除术在动物体内很容易观察到。此外，我们将概述在损伤后的头几天至几周内对动物损伤解剖部位进行连续成像的程序，以研究细胞间相互作用。
