Expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax after delayed paraplegia induced by ischemia/reperfusion injury

Bibo Liu; Miao Liu; Duoning Wang; Wei Ma; Shengli Dang

摘要：BACKGROUND:Operation of spine does not involve in spinal cord;however,spinal cord injury occurs at post-operation induced by unclear factors.Meanwhile,after decompression of lumbar spinal canal,symptoms are severer and severer.In addition,during extirpation of oervical and lumbar intervertabral disc,spinal cord and its vessels are not damaged,but spinal cord injury is also suffered from patients with partial improvement.All statuses mentioned above are related to expressed changes of bc/-2 gene inhibiting apoptosis and bax gene accelerating apoptosis.OBJECTIVE:To observe motor function of hindlimbs of ischemia/reperfusion model in rabbits at various time points after reperfusion and expressions of apoptosis correlated protein Bcl-2 and Bax.DESIGN:Completely randomized grouping design and contrast study.SEITING:Department of Orthopedics,the First Affiliated Hospital of Medical School,Xi'an Jiao Tong University.MATERIALS:Forty-eight New Zealand white rabbits of both genders were randomly divided into sham operation group(n=24) and model group(n=24),and then,rabbits in each group were observed at four time points:8,24,72 and 168 hours after reperfusion,with 6 in each time point.Rabbit-anti-rabbit Bcl-2 antibody and rabbit-anti-rabbit Bax antibody were provided by Boster Company.The procedures were accordant to the METHODS:The experiment was carried out at Laboratory of Orthopaedics of First Affiliated Hospital of Medical School,Xi'an Jiao Tong University from April to August 2005.①Delayed paralysis models of spinal cord after ischemia/reperfusion were established based on method of Zivin et al.Animals in sham operation group underwent an exposure of abdominal aorta but the aorta was not occluded.②Motor function of hindlimb was observed 8,24,72 and 168 hours after reperfusion.A grade of 0-5 was assigned to each animal (grade 0:no voluntary hind limb function;grade 5:normal hop;grades 0-3:paraplegia).③The lumbar segment of the spinal cord(L3 to L5)was used for morphological studies at 168 hours after reperfusion and cut into sections with the thickness of 4μm:and then,the sections were stained with hematoxylin and eosin and observed under optic microscope.④After dewaxing,paraffin sections were used to detect expression of Bcl-2 and Bax proteins With Strept-Avidin-Biotin Complex,(SABC)kit(Wuhan Boster Company).The detailed operation was accordant to kit instruction.⑤Sections under the same staining condition but at various time points were measured with Image-Pro Plus Version 5.1 for Window TM (Median Cybemetics,Inc.USA.)to detect average integrated absorbency of positive cells of Bcl-2 and Bax protein.⑥Measurament data were compared with one-way analysis of variance and ttest. MAIN OUTCOME MEASURES:①Hind limb motor function deficit;②Histopathological examination;③Expression of Bcl-2 and Bax proteins at different reperfusion time points in spinal cord.RESULTS:All the 48 rabbits were involved in the analysis of results.①Motor function of hindlimb:Rabbits did not have paralysis in sham operation group.Rabbits in model group did not have paralysis at 8 hours after repertusion,but had delayed paralysis at 24,72 and 168 hours after reperfusion.Grade of motor function was lower than that in sham operation group(t=15.65,20.55.29.00,P.＜0.01),similar to that in sham operation group at 8 hours after reperfusion and decreased obviously at 24 hours after reperfusion.②Pathological changes of spinal cord tissue:Spinal cord tissue was normal in sham operation group.With the reperfusion time passing by.necrosis of neurons in spinal cord tissue in model group was increased,inflammatory reaction was strengthened gradually and pathological changes were obvious at 72 hours after reperfusion.③Exprasslons of Bcl-2 and Bax protein:At 8 hours after reperfusion,expressions were similar in both sham operation group and model group(Bcl-2:20.42±4.22,19.63±2.72;Bax:5.22±1.24,4.82±1.52,P＞0.05);expression of Bcl-2 was decreased obviously at 24 hours after reperfusion(8.09±0.96,t=9.78,P＜0.05)and reached the lowest value at 72 hours after reperfusion(6.08±1.51,t=11.06,P＜0.05).However,expression of Bax was increased obviously at 24 hours after reperfusion(12.84±1.77,f=7.88,P＜0.05)and reached peak at 72 hours after reperfusion(15.84±5.19,t=4.42,P＜0.05).At 168 hours after reperfusion,structure of gray matter was damaged severely and only a few of neurons survived.However,numbers of positive glial cells in white matter were increased,and expression of Bax was higher than that in sham operation group(9.41±1.53,t=6.44,P＜0.05).CONCLUSION:Ischemia/reperfusion injury of spinal cord can induce decrease of motor function of experimental animals and cause apoptosis.Changes of both of them are general coincidence with changes of Bcl-2 and Bax expressions with the reperfusion time passing by.