小鼠股骨骨折合并坐骨神经损伤的标准化动物模型构建与评价

目的 建立并评价小鼠股骨骨折合并坐骨神经损伤的标准化动物模型。方法 将96只C57BL/6J小鼠随机分为2组,分别为A组：股骨骨折合并坐骨神经损伤组(48只)、B组：股骨骨折组(48只)。A组通过止血钳钳夹致小鼠左侧坐骨神经损伤,于同侧进行股骨开放性骨折联合髓内钢针固定,建立了小鼠股骨骨折合并坐骨神经损伤的模型;B组行左侧股骨开放性骨折联合髓内钢针固定,仅游离坐骨神经,不做钳夹处理。术后仔细观察小鼠行为学变化。于术后第3、5、7、10、14、18天6个时间点,取A组(36只)与B组(36只)左侧坐骨神经,其中每个时间点每组6只,分别通过免疫组化染色来分析坐骨神经损伤修复过程。同时,于第7、14、21和28天4个时间点取小鼠左侧股骨,分别通过股骨X线、Micro-CT、组织学染色来分析并评估在周围神经损伤条件下骨折愈合情况。结果 本实验建立的小鼠股骨骨折合并坐骨神经损伤模型,于建模后第14天小鼠步态基本恢复正常;于第14天坐骨神经纤维结构连续性基本恢复;第7天骨折断端几乎不存在骨痂;至第14天出现少量软骨痂,主要为未分化的间充质干细胞构成;第21天软骨痂开始钙化,骨痂中存在部分肥大的软骨细胞;第28天骨折断端新生骨形成,含有大量肥大的软骨细胞、成骨细胞与骨细胞。结论 通过本研究方法建立的小鼠股骨骨折合并坐骨神经损伤模型愈合过程良好,并较单纯股骨骨折的愈合过程有所迟缓,可很大程度上模拟临床骨折合并周围神经损伤后修复及愈合过程,为合并周围神经损伤的骨折愈合相关研究提供实验基础。

Construction and evaluation of a standardized animal model of femoral fracture combined with sciatic nerve injury in mice

Objective To establish and evaluate a standardized model of femoral fracture combined with sciatic nerve injury in mouse. Methods A total of 96 C57BL/6J mice were randomly divided into two groups, group A: femoral fracture combined with sciatic nerve injury group (n=48) and group B: femoral fracture group (n=48). The left sciatic nerves were crushed by hemostatic forceps, and the ipsilateral femurs were cut and fixed with intramedullary nails in the bone marrow. And then, the model of femoral fracture with sciatic nerve injury in mouse was established in group A. Mice in group B underwent fracture of the left femurs by intramedullary nails fixation, and dissociated the left sciatic nerves, but not clamped. Behavioral observation of postoperative mice was performed. The sciatic nerves were collected at 6 time points on the 3rd, 5th, 7th, 10th, 14th, and 18th day after operation. Six mice of each group at each time point were obtained for immunohistochemical staining to analyze the repairing process of sciatic nerve injury. Simultaneously, at 4 time points, the left femurs of postoperative mice were harvested on the 7th, 14th, 21st, and 28th day, including 6 mice of each group at each time point. The fracture healing under the condition of combined sciatic nerve injury was displayed via X-ray radiology and micro-CT scanning, analyzed and evaluated via histological staining. Results A mouse model of femoral fracture combined with sciatic nerve injury established. On the 14th day, the gait basically returned to normal, and the structural integrality of the sciatic nerve fiber basically recovered. On the 7th day, there were almost disappeared calluses at the fracture site. However, a small amount of cartilage calluses appeared on the 14th day, which were mainly composed of undifferentiated mesenchymal stem cells. The cartilage calluses began to calcify on the 21st day, and there were some hypertrophic chondrocytes in the calluses. On the 28th day, new bone formed, containing a large number of hypertrophic chondrocytes, osteoblasts and osteocytes. Conclusion Compared with simple fracture,the mouse model established in this research shows a good but later healing process,to a great extent,which can simulate clinical fracture with peripheral nerve injury and provide experimental base for study on this field.