大鼠脊髓损伤继发骨质疏松的实验观察

背景骨质疏松是脊髓损伤的常见并发症之一,其确切机制尚未完全清楚,损伤平面上下骨质是否均发生骨质疏松,不同学者观点不同.目的观察脊髓损伤后继发骨质疏松的骨组织超微结构、血液生化改变情况,分析损伤平面上下骨质受累情况和程度.设计随机对照实验.单位吉林大学第二临床医院动物试验室,吉林大学第二临床医院检验科,吉林大学白求恩医学部电镜教研室.材料实验于2002-05/2003-05在吉林大学第二临床医院动物试验室完成.Wistar大鼠110只,均为雄性,四五月龄,体质量为300～320g.方法110只Wistar雄性大鼠随机选取10只为0周空白对照组,将余下大鼠随机分为实验组和对照组,1,2,3,7,11周对照组及实验组每组各10只.用1 mL/L戊巴比妥钠腹腔注射麻醉,对照组仅行T10椎板切除不破坏硬膜,不损伤脊髓,实验组大鼠于T10椎体水平切除椎板,行Allen's法损伤脊髓.分别于0周、术后1周,2周,3周,7周,11周周末将动物下腔静脉采血4mL后麻醉状态下处死,血液离心,应用7170A HI-TACHI自动生化分析仪自动采样化验血钙、血磷的含量及碱性磷酸酶活性.另取0周空白对照组,7周实验组,11周实验组右侧肱骨、胫骨各一用25 mL/L乙二胺四乙酸脱钙1个月后制备切片,在醋酸双氧铀及柠檬酸双重染色下应用JEM-1200型透射电子显微镜观察肱骨外科颈、胫骨平台部以骨细胞为主的超微结构改变情况.结果进入结果分析的大鼠为105只.①血生化检查结果实验2周时血磷含量对照组明显低于实验组(1.54±0.21),(2.76±0.16)mmol/L,(P＜0.01)];实验3周时血钙含量实验组明显高于对照组(2.52±0.06),(2.35±0.12)mmol/L,(P＜0.01)];实验7周时碱性磷酸酶活性实验组明显高于对照组(155.86±20.42),(129.25±7.30)Nμ/mg,(P＜0.01)].②电子显微镜下观察结果术后7周实验组胫骨标本可见骨细胞与骨陷窝分离,个别细胞胞核不规则,可见絮状物质、线立体粗面内质网肿胀开空化;11周时,可见骨细胞与骨陷窝严重分离.术后7周实验组肱骨标本可见骨细胞与骨陷窝分离,有的细胞核固缩粗面内质网肿胀空化;11周时仍见上述改变,但细胞肿胀空化程度减轻.结论脊髓损伤早期破骨细胞活性增强,成骨细胞活性降低,导致骨吸收增强,骨形成减弱.鼠损伤平面上下骨质均受累,但不同部位的骨骼继发骨质疏松的程度不同,骨质疏松后骨细胞超微结构改变显著.

Experimental study of secondary osteoporosis of spinal cord injured rats

BACKGROUND: Osteoporosis is one of the complications of spinal cord injury, but its mechanism is unclear, different scholars have different points about whether all bones above and below the level of trauma are affected after spinal cord injury (SCI).OBJECTIVE: To observe the ultrastructure of bone tissue of secondary osteoporosis after spinal cord injury and the change of serum biochemical indexes, to analyze the suffered condition and injured degree of the bones above and below the level of the trauma.DESIGN: Randomized controlled experimentSETTING: Laboratory of Animals, and Department of Laboratory Medicine of the Second Clinical Hospital of Jilin University; Transmission Electron Microscope Center, Norman Bethune Division of Medical Sciences of Jilin UniversityMATERIALS: This experiment was conducted at the Laboratory of Animals, Second Clinical Hospital of Jilin University from May 2002 to May 2003. Totally 110 male Wistar rats, aged 4 to 5 month-old, with the body mass of (300 -320)g were involved.METHODS: Ten rats were randomly chosen from 110 male Wistar rats and were set as 0 week blank control group, the ther rats were randomly divided into experimental group and control group, there were 10 rats in week 1, 2, 3, 7 and 11 control group and experimental group separately.1 mL/L of pentobarbital natrium was intraperitoneal injected to perform anesthesia, in the control group, lamina with dura intact of rats were removed only, without spinal cord injury at the level of tenth thoracic vertebrae. In the experimental group, lamina of rats was also removed, the spinal cord injury model of the rats were made by the method of Allen's.4 mL venous blood was collected from the animals at the end of postoperative week 0, week 1, week 2, week 3, week 7 and week 11, then the animals were put to death, perform blood centrifuging .We observe the change of serum concentration of calcium, phosphorus and alkaline phophatase (ALP) with 7170A HITACHI auto-biochemistry analyzer. Right side humerus, tibia in week 0 blank control group, week 7 experimental group and week 11 experimental group were chosen, then 25 mL/L ethylene dinitrilotetraacetic acid was used for decalcium for 1 month for preparingthe sections. We observe the change of ultrastructure of osteocytes at the sites of tibial plateau and the surgical neck of the humerus after stained by uranyl acetate and lead citrate under transmission electron microscope.biochemistry detection: the level of phosphorus at week 2 of the experiment was significantly lower in the control group than experiment group (1.54±0.21),(2.76±0.16)mmol/L, (P ＜ 0.01)]; the level of calcium at week 3 in the experiment group was significantly higher than in the control group (2.52±0.06),(2.35±0.12)mmol/L, (P ＜ 0.01)];the level of alkaline phophatase at week 7 in the experiment group was significantly higher than in the control group (155.86±20.42), (129.25±7.30)Nμ/mg,operative week 7, the tibia sample showed that the osteocyte separated from the osseous lacuna, the nucleus showed anomaly, fluffed materials appeared. Mitochondrion swelled, rough endoplasmic hollowed; At week 11, osteocyte separated from the osseous lacuna. At postoperative 7 week,the humerus sample showed that osteocyte separated from osseous lacuna,some nucleus pycnosis and rough endoplasmic hollowed; At postoperative week 11, the above changes still were seen, but the degree of cellular swelling and hollowing were lighten.CONCLUSION: At the early stage of spinal cord injury, the activity of osteoclast is increased and the activity of osteoblast is decreased, which leads to the increase of bone absorption, the bone formation is largely weakened. The bones above and below the level of trauma are both affected after spinal cord injury, but different extents of osteoporosis can be seen in different bones .The change of ultrastructure of osteocytes is remarkable when osteoporosis has happened.