Department of Pharmacy, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, PR China; Department of Pharmacy, Huashan Hospital, Fudan University, 12 Middle Urumqi Road, Shanghai, 200040, PR China.
Center of Clinical Research, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, PR China.
Brain Res Bull. 2018 Oct;143:171-180. doi: 10.1016/j.brainresbull.2018.08.005. Epub 2018 Aug 4.
To characterize the microenvironment following blood-spinal cord barrier (BSCB) damage and to evaluate the role of BSCB disruption in secondary damage of spinal cord injury (SCI).
A model of BSCB damage was established by co-culture of primary microvascular endothelial cells and glial cells obtained from rat spinal cord tissue followed by oxygen glucose deprivation/re-oxygenation (OGD/R). Permeability was evaluated by measuring the transendothelial electrical resistance (TEER) and the leakage test of Fluorescein isothiocyanate-dextran (FITC-dextran). The expression of tight junction (TJ) proteins (occludin and zonula occludens-1 (ZO-1) were evaluated by Western blot and immunofluorescence microscopy. Proinflammatory factors (TNF-α, iNOS, COX-2 and IL-1β), leukocyte chemotactic factors (MIP-1α, MIP-1β) and leukocyte adhesion factors (ICAM-1, VCAM-1) were detected in the culture medium under different conditions by enzyme-linked immuno sorbent assay (ELISA).
The model of BSCB damage induced by OGD/R was successfully constructed. The maximum BSCB permeability occurred 6-12 hours but not within the first 3 h after OGD/R-induced damage. Likewise, the most significant period of TJ protein loss was also detected 6-12 hours after induction. During the hyper-acute period (3 h) following OGD/R-induced damage of BSCB, leukocyte chemotactic factors and leukocyte adhesion factors were significantly increased in the BSCB model. Pro-inflammation factors (TNF-α, IL-1β, iNOS, COX-2), leukocyte chemotactic factors (MIP-1α, MIP-1β) and leukocyte adhesion factors (ICAM-1, VCAM-1) were also sharply produced during the acute period (3-6 hours) and maintained plateau levels 6-12 hours following OGD/R-induced damage, which overlapped with the period of BSCB permeability maximum. A negative linear correlation was observed between the abundance of proinflammatory factors and the expression of TJ proteins (ZO-1 and occludin) and transepithelial electrical resistance (TEER), and a positive linear correlation was found with transendothelial FITC-dextran.
Secondary damage continues after primary BSCB damage induced by OGD/R, exhibiting close ties with inflammation injury.
描述血脊髓屏障（BSCB）损伤后的微环境，并评估 BSCB 破坏在脊髓损伤（SCI）继发损伤中的作用。
通过共培养大鼠脊髓组织来源的原代微血管内皮细胞和神经胶质细胞，建立 BSCB 损伤模型，然后进行氧葡萄糖剥夺/再氧合（OGD/R）。通过测量跨内皮电阻（TEER）和荧光素异硫氰酸酯-葡聚糖（FITC-dextran）渗漏试验来评估通透性。通过 Western blot 和免疫荧光显微镜评估紧密连接（TJ）蛋白（occludin 和 zonula occludens-1（ZO-1）的表达。在不同条件下，通过酶联免疫吸附试验（ELISA）检测培养基中的促炎因子（TNF-α、iNOS、COX-2 和 IL-1β）、白细胞趋化因子（MIP-1α、MIP-1β）和白细胞黏附因子（ICAM-1、VCAM-1）。
成功构建了 OGD/R 诱导的 BSCB 损伤模型。BSCB 通透性的最大值发生在 OGD/R 损伤后 6-12 小时，但不在 3 小时内。同样，在诱导后 6-12 小时也检测到 TJ 蛋白丢失的最显著时期。在 OGD/R 诱导的 BSCB 损伤后的超急性期（3 小时），BSCB 模型中的白细胞趋化因子和白细胞黏附因子显著增加。在急性期（3-6 小时）也大量产生促炎因子（TNF-α、IL-1β、iNOS、COX-2）、白细胞趋化因子（MIP-1α、MIP-1β）和白细胞黏附因子（ICAM-1、VCAM-1），并在 OGD/R 损伤后 6-12 小时保持平台水平，与 BSCB 通透性最大值重叠。促炎因子的丰度与 TJ 蛋白（ZO-1 和 occludin）和跨上皮电阻（TEER）的表达呈负线性相关，与 FITC-dextran 经内皮呈正线性相关。
OGD/R 诱导的原发性 BSCB 损伤后，继发性损伤仍在继续，与炎症损伤密切相关。
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