Objective: To explore the mechanisms for repairing spinal cord injury (SCI) with tetramethylpyrazine-loaded electroconductive hydrogel (hereinafter referred to as "TGTP").
Mehtods: A total of 72 female Sprague-Dawley rats were randomly divided into 4 groups: sham operation group (group A), SCI group (group B), SCI+electroconductive hydrogel group (group C), and SCI+TGTP group (group D). Only the vertebral plate was removed in group A, while the remaining groups were subjected to a whole transection model of spinal cord with a 2 mm gap in the lesions. The recovery of hindlimb motor function was evaluated by Basso, Beattie, Bresnahan (BBB) score and modified Rivlin-Tator inclined plate test before operation and at 1, 3, 7, 14, and 28 days after operation, respectively. Animals were sacrificed at 7 days and 28 days after modeling. Neovascularisation was observed by immunofluorescence staining of CD31 and the expression levels of angiopoietin 1 (Ang-1) and Tie-2 were assessed by Western blot assay. At 28 days postoperatively, the expression levels of pro-angiogenic related proteins, including platelet-derived growth factor B (PDGF-B), PDGF receptor β (PDGFR-β), vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2), were also assessed by Western blot. The fibrous scar in the injured area was assessed using Masson staining, while neuronal survival was observed through Nissl staining. Furthermore, LFB staining was utilized to detect myelin distribution and regeneration. Immunofluorescence and Western blot assay were employed to evaluate the expression of neurofilament 200 (NF200).
Results: The hindlimb motor function of rats in each group gradually recovered from the 3rd day after operation. The BBB score and climbing angle in group D were significantly higher than those in group B from 3 to 28 days after operation, and significantly higher than those in group C at 14 days and 28 days after operation ( P<0.05). Masson staining showed that the collagen volume fraction in groups B-D were significantly higher than that in group A, and that in group D was significantly lower than that in groups B and C ( P<0.05); a small amount of black conductive particles were scattered at the broken end in group D, and the surrounding collagen fibers were less than those in group C. Nissl and LFB staining showed that the structure of neurons and myelin sheath in the injured area of spinal cord in group D was relatively complete and continuous, and the number of Nissl bodies and the positive area of myelin sheath in group D were significantly better than those in groups B and C ( P<0.05). NF200 immunofluorescence staining and Western blot assay results showed that the relative expression of NF200 protein in group D was significantly higher than that in groups B and C ( P<0.05). CD31 immunofluorescence staining showed that the fluorescence intensity of group D was better than that of groups B and C at 28 days after operation, and tubular or linear neovascularization could be seen. The relative expressions of Ang-1 and Tie-2 proteins in group D were significantly higher than those in groups B and C at 7 and 28 days after operation ( P<0.05). The relative expressions of PDGF-B and PDGFR-β proteins in group D were significantly higher than those in groups B and C, and group B was significantly higher than group C at 28 days after operation ( P<0.05). The relative expressions of VEGF-A and VEGFR2 proteins in group D were higher than those in groups B and C, showing significant difference when compared with group B ( P<0.05), but only the expression of VEGF-A protein was significantly higher than that in group C ( P<0.05). There was significant difference only in VEGFR-2 protein between groups B and C ( P<0.05).
Conclusion: TGTP may enhance the revascularization of the injured area and protect the neurons, thus alleviating the injury of spinal cord tissue structure and promoting the recovery of neurological function after SCI in rats.
目的: 探索携川芎嗪导电水凝胶（简称“TGTP”）修复脊髓损伤（spinal cord injury，SCI）的潜在机制。.
方法: 将72只雌性SD大鼠随机分为4组，假手术组（A组）、SCI组（B组）、SCI+导电水凝胶组（C组）和SCI+TGTP组（D组）。A组仅切除椎板，其余组构建切除2 mm长脊髓的全横断模型。通过BBB评分法和改良Rivlin-Tator斜板实验分别于术前及术后1、3、7、14、28 d评估大鼠后肢运动功能恢复情况。术后7、28 d动物取材，通过免疫荧光染色观察新生血管，采用Western blot评估血管生成素1（angiopoietin 1，Ang-1）、Tie-2蛋白表达；术后28 d采用Western blot进一步评估促血管新生相关蛋白PDGF-B、PDGF受体β（PDGF receptor β，PDGFR-β）、VEGF-A、VEGF受体2（VEGF receptor 2，VEGFR-2）的表达情况。术后28 d，采用Masson染色评估损伤区域瘢痕增生，Nissl染色观察神经元存活，LFB染色检测髓鞘分布和再生情况，免疫荧光染色和Western blot评估神经丝蛋白200（neurofilament 200，NF200）的表达。.
结果: 各组大鼠术后3 d起后肢运动功能逐渐恢复，D组术后3～28 d BBB评分和斜坡角度显著高于B组，术后14、28 d显著高于C组（ P<0.05）。Masson染色示B～D组胶原容积分数显著高于A组，D组显著低于B、C组（ P<0.05）；D组断端可见少量散在黑色导电颗粒，周围胶原纤维成分少于C组。Nissl和LFB染色示D组损伤区域脊髓组织神经元及髓鞘结构相对完整、连续，尼氏体数量和髓鞘阳性表达面积明显优于B、C组（ P<0.05）。NF200免疫荧光染色和Western blot检测示，D组损伤区域NF200蛋白相对表达量显著优于B、C组（ P<0.05）。CD31免疫荧光染色示D组术后28 d荧光强度优于B、C组，可见管状或线性新生血管影。Western blot检测示术后7 d和28 d D组Ang-1、Tie-2蛋白相对表达量高于B、C组，差异均有统计学意义（ P<0.05）。术后28 d D组PDGF-B、PDGFR-β蛋白相对表达量显著高于B、C组，B组显著高于C组，差异均有统计学意义（ P<0.05）。术后28 d D组VEGF-A和VEGFR-2蛋白相对表达量优于B、C组，与B组比较差异有统计学意义（ P<0.05），但与C组比较仅VEGF-A蛋白差异有统计学意义（ P<0.05）；B、C组间比较仅VEGFR-2蛋白差异有统计学意义（ P<0.05）。.
结论: TGTP可能增强损伤区域再血管化，同时发挥神经元保护作用，从而减轻脊髓组织结构损伤，促进大鼠SCI后神经功能恢复。.
Keywords: Spinal cord injury; angiogenesis; hydrogel; neuroprotection; tetramethylpyrazine.