Lee Y B, Yune T Y, Baik S Y, Shin Y H, Du S, Rhim H, Lee E B, Kim Y C, Shin M L, Markelonis G J, Oh T H
Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Exp Neurol. 2000 Nov;166(1):190-5. doi: 10.1006/exnr.2000.7494.
We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha.
我们研究了肿瘤坏死因子（TNF）-α在大鼠脊髓挤压伤后神经元和神经胶质细胞凋亡起始过程中的作用。损伤后4小时，在损伤区域内及周围首次观察到少量TUNEL阳性细胞，损伤后24 - 48小时观察到的数量最多。使用细胞类型特异性标志物对细胞进行双重标记显示，TUNEL阳性细胞为神经元或少突胶质细胞。损伤后1小时，在损伤区域的神经元和神经胶质细胞中观察到对TNF-α的强烈免疫反应，在损伤部位头侧和尾侧数毫米处的细胞中也可见到。损伤后4小时脊髓中一氧化氮（NO）水平也显著升高。损伤后几分钟向损伤部位注射抗TNF-α中和抗体，显著降低了此后4小时观察到的NO水平以及脊髓损伤后24小时观察到的凋亡细胞数量。一氧化氮合酶（NOS）抑制剂N（G）-单甲基-L-精氨酸乙酸盐（L-NMMA）也减少了凋亡细胞数量。凋亡细胞数量的减少与琼脂糖凝胶电泳上DNA梯状条带的减少相关。这些结果表明：（i）TNF-α可能作为脊髓损伤后启动神经元和少突胶质细胞凋亡的外部信号；（ii）TNF-α引发的凋亡可能部分由响应TNF-α表达的NOS产生的NO介导。
