Calpain 1 knockdown improves tissue sparing and functional outcomes after spinal cord injury in rats.
Calpain 1 knockdown improves tissue sparing and functional outcomes after spinal cord injury in rats.
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DOI：
10.1089/neu.2012.2561
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发表时间：
2013-04
影响因子：
4.2
通讯作者：
Chen-Guang Yu;Yanzhang Li;K. Raza;Xin Yu;S. Ghoshal;J. Geddes
中科院分区：
文献类型：
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作者：
Chen-Guang Yu;Yanzhang Li;K. Raza;Xin Yu;S. Ghoshal;J. Geddes
为了评估钙蛋白酶1敲低将减少脊髓损伤（SCI）后的病理损伤和功能缺陷的假设，我们开发了编码钙蛋白酶1 shRNA和eGFP作为报告基因的慢病毒载体（LV-CAPN 1 shRNA）。使用北方和Western印迹分析在大鼠NRK细胞中证实LV-CAPN 1 shRNA敲低钙蛋白酶1的能力。为了研究对脊髓损伤的影响，在相同胸水平的挫伤SCI、180 kdyn力或假手术前1周，通过对流增强扩散在Long-Evans雌性大鼠（200-250 g）的脊髓水平T10处施用LV-CAPN 1 shRNA或LV-错配对照shRNA（LV-对照shRNA）。脊髓内施用慢病毒颗粒导致在感染后2周在脊髓组织中的转基因表达，通过eGFP可视化。在shRNA介导的敲低后2周，钙蛋白酶1蛋白水平在T10降低了54%（p<0.05，与LV对照组相比，每组n=3），而钙蛋白酶2水平没有变化。与LV对照给药相比，在挫伤SCI前1周脊髓内给予LV-CAPN 1 shRNA导致损伤后6周运动功能的显著改善（p<0.05，每组n=10）。脊髓切片的组织学分析表明，损伤前脊髓内施用LV-CAPN 1 shRNA显著减少了脊髓损伤体积，并改善了总组织保留、白色物质保留和灰质保留（p<0.05，每组n=10）。总之，研究结果支持了这样的假设，即钙蛋白酶1激活有助于SCI后的组织损伤和受损的运动功能，并且钙蛋白酶1代表了潜在的治疗靶点。
To evaluate the hypothesis that calpain 1 knockdown would reduce pathological damage and functional deficits after spinal cord injury (SCI), we developed lentiviral vectors encoding calpain 1 shRNA and eGFP as a reporter (LV-CAPN1 shRNA). The ability of LV-CAPN1 shRNA to knockdown calpain 1 was confirmed in rat NRK cells using Northern and Western blot analysis. To investigate the effects on spinal cord injury, LV-CAPN1shRNA or LV-mismatch control shRNA (LV-control shRNA) were administered by convection enhanced diffusion at spinal cord level T10 in Long-Evans female rats (200-250 g) 1 week before contusion SCI, 180 kdyn force, or sham surgery at the same thoracic level. Intraspinal administration of the lentiviral particles resulted in transgene expression, visualized by eGFP, in spinal tissue at 2 weeks after infection. Calpain 1 protein levels were reduced by 54% at T10 2 weeks after shRNA-mediated knockdown (p<0.05, compared with the LV-control group, n=3 per group) while calpain 2 levels were unchanged. Intraspinal administration of LV-CAPN1shRNA 1 week before contusion SCI resulted in a significant improvement in locomotor function over 6 weeks postinjury, compared with LV-control administration (p<0.05, n=10 per group). Histological analysis of spinal cord sections indicated that pre-injury intraspinal administration of LV-CAPN1shRNA significantly reduced spinal lesion volume and improved total tissue sparing, white matter sparing, and gray matter sparing (p<0.05, n=10 per group). Together, results support the hypothesis that calpain 1 activation contributes to the tissue damage and impaired locomotor function after SCI, and that calpain1 represents a potential therapeutic target.