| ObjectiveTo establish the spinal cord ischemia/reperfusion injury (SCII) models in rabbits by occluding the L3-L6 lumbar artery in different durations and then chose the best durations for subsequent experiments.Observing the effect of Huoxuetongdu decoction on motor function of hind limb, the expression of TNF-a、IL-β、IL-8、BDNF、Slit2、GFAP, the apoptosis of spinal cord neurons in rabbits during SCII and then explore the mechanism of the Huoxuetongdu decoction on the SCII.Methods1. Compare the different SCII models in rabbits with different occlusion durations in the L3-L6 lumbar artery24 New Zealand Rabbits were randomly divided into three groups:C20 group (n=8), C30 group (n=8), C40 group (n=8).Lumbar artery’ occlusion(L3-L6) was performed to established the SCII models in each groups(20mins in C20 group,30mins in C30 group.40mins in C40 group). The motor function of hind limb were assessed whith Jacobs scoring system at the moment 12h.24h.48h after the reperfusion and calculated paraplegia rate of the rabbits.After last scoring,the lumbar segments of spinal cord (L-L6)were removed and stained with hematoxylin and eosin (HE).then counted the number of normal neurons.2 Effect of Huoxuetongdu decoction on motor function of hind limb of rabbits during SCII16 New Zealand Rabbits were randomly divided into two groups:model group (n=8). HuoxueTongdu Decoction group (n=8).Lumbar artery occlusion(L3-L6 for 30min) was performed to established the SCII models in rabbits of each groups. Rabbits in the HuoxueTongdu Decoction group were lavaged with HuoxueTongdu Decoction for 7 days before the operation and 21 days after the operation. Rabbits in the model group were lavaged with the same dose of physiological saline for 7 days before the operation and 21 days after the operation. The motor function of hind limb were assessed whith Jacobs scoring system and BBB scoring system at the moment of 1d、3d、7d、14d、21d after reperfusion.3 Mechanism of the Huoxuetongdu decoction in SCII rabbits136 New Zealand Rabbits were randomly divided into three groups:sham-operated group (n=8). model group (n=64. divided into 8 subgroups according to the time of reperfusion at 0.5h、1h、4h、8h、1d、3d、7d、14d,each subgroups had 8 rabbits), HuoxueTongdu Decoction group (w=64, divided into 8 subgroups according to the time of reperfusion at 0.5h、1h、4h、 8h、1d、3d、7d、14d,each subgroups had 8 rabbits).Lumbar artery occlusion(L3-L6 for 30min) was performed to established the SCII models in the model group and HuoxueTongdu Decoction group, while there was no Lumbar artery occlusion in the sham-operatted group. Rabbits in the HuoxueTongdu Decoction group were lavaged with HuoxueTongdu Decoction before the operation(7 days) and after the operation. Rabbits in the model group were lavaged with the same dose of physiological saline for before the operation(7 days) and after the operation.The lumbar segments of spinal cord (L2-L6) were removed at the moment of 0.5h、1h、 4h、8h、1d、3d、7d、14d after the reperfusion.The expression of IL-1 β、IL-8、BDNF、 Slit2 in the spinal cord were measured by EL1SA. The expression of TNF-a and GFAP in the spinal cord were measured by ELISA and immunohistochemistry.The apoptosis of spinal cord neurons were observed by TUNEL detection.Results1.Compare the different SCII models in rabbits with different occlusion durations in the L3-L6 lumbar artery1.1 Jacobs scoring system:There were significant difference in Jacobs scores between C20 group.C30 group and C40 group at the same time of 12h,24h.48h after reperfusion(p<0.01). Scores of C20 group were better than C30 group and C40 group at the same time of 12h,24h,48h after reperfusion(p<0.01).There were no significant difference in Jacobs scores between C30 group and C40 group at the same time of 12h.24h.48h after reperfusion(p>0.05)1.2 Paraplegia rate:Paraplegia rate of the three groups were 0%.75% and 87.5%, respectively.Paraplegia rate of C20 group was lower than that of C30 group and C40 group(p<0.05).There were no significant difference in paraplegia rate between C30 group and C40 group (p>0.05).1.3 Pathological changes of the spinal cord:In C20 group, the appearance of the spinal cord tissue were complete.but with mild swelling.There were some bleeding point within the spinal cord. There were many neurons in anterior horn of spinal cord.The neurons were large.so that we can clearly see the nucleolus.nuclear membrane and alkaline dye particles. In C30 group and C40 group, there were obvious pathological damage. There were significant swelling and bleeding point in the spinal cord.and the number of neurons in anterior horn of spinal cord was less than normal.There were vacuolization around neurons.and the neurons were small than normal. The Nuclear were deeply stained.and the nucleolus.nuclear membrane and alkaline dye particles were blurry.1.4 Number of normal neurons:Number of normal neurons of the C20 group was the most of the three group, C30 group the second, C40 group the third. There were significant difference in Number of normal neurons between C20 group,C30 group and C40 group (p<0.05).2.Effect of Huoxuetongdu decoction on motor function of hind limb of rabbits during SCII2.1 Jacobs scoring system:The Jacobs scores of model group and HuoxueTongdu Decoction group were increased from Id to 21 d after reperfusion. The Jacobs scores of HuoxueTongdu Decoction group was significant higher than model group at 7d、14d、21d after reperfusion(p<0.05).2.2 BBB scoring system:The BBB scores of model group and HuoxueTongduDecoction group were increased from 1d to 21d after reperfusion.The BBB scores of HuoxueTongdu Decoction group was significant higher than model group at 3d、7d、14d、21d after reperfusion(p<0.05 at 3d、7d、14d, p<0.01 at 21d).3.Mechanism of the Huoxuetongdu decoction in SCII rabbits3.1 ELISA:3.1.1 Expression of TNF-a. IL-1β、IL-8Expression of TNF-a、IL-1β、IL-8 of model group were significantly higher than sham group at 0.5h、1h、4h、8h after reperfusion(p<0.01).Expression of TNF-a、IL-1β、L-8 of HuoxueTongduDecoction group were significantly lower than model group at 0.5h、1h、4h、8h after reperfusion (p<0.05 or p<0.01).3.1.2 Expression of BDNF、Slit2、GFAP:Expression of BDNF、Slit2 of model group were significantly higher than sham group at 1d、3d、7d、4d after reperfusion(p<0.01).Expression of BDNF、Slit2 of HuoxueTongduDecoction group were significantly higher than model group at 1d、3d、7d、14d after reperfusion (p<0.05 or p<0.01). Expression of GFAP of model group were significantly higher than sham group at 1d、3d、7dx 14d after reperfusion(p<0.05 or p<0.01).Expression of GFAP of HuoxueTongduDecoction group were significantly lower than model group at 3d、7d、14d after reperfusion (p<0.05 or p<0.01).3.2 Immunohistochemistry:3.2.1 Expression of TNF-a:There were no yellow or brown stained neurons in sham group.while there were some of yellow or brown stained neurons and glial cells in model group and HuoxueTongduDecoction group.Optical delnsity of model group were significantly higher than sham group at 0.5h、1h、4h、8h after reperfusion(p<0.01).Optical delnsity of HuoxueTongduDecoction group were significantly higher than sham group at 0.5h、1h、4h、 8h after reperfusion(p<0.01).while lower than model group at 0.5h、1h、4h、8h after reperfusion(p<0.05 at 0.5h. p<0.01 at 1h、4h、8h).3.1.2 Expression of BDNF:There were no yellow or brown stained neurons in sham group.while there were some of yellow or brown stained neurons and glial cells in model group and HuoxueTongduDecoction group.Optical delnsity of model group were significantly higher than sham group at 1d、3d、7d、4d after reperfusion(p<0.01).Optical delnsity of HuoxueTongduDecoction group were significantly higher than sham group at 1d、3d、7d、 14d after reperfusion(p<0.01 at 1d、3d、7d、14d).Optical delnsity of HuoxueTongduDecoction group were significantly higher than model group at 1d、3d、7d、14d after reperfusion(p<0.05 at 3d,p<0.01 at 1d、7d、14d).3.3 TUNEL:Apoptotic neurons was brown or yellow dyeing.There were less apoptotic cells in sham group while there were some apoptotic cells in model group and HuoxueTongduDecoction group.Apoptosis index of model group were significantly higher than sham group at 4h、8h、1d、3d after reperfusion(p<0.01 at4h、8h、1d、d). Apoptosis index of HuoxueTongduDecoction group were significantly lower than model group at4h.8h、1d、 3d after reperfusion(p<0.05 at 4h, p<0.01 at 8h、1d、3d).Conclusions1.Blocking lumbar artery(L3-L6) for 30min will cause irreversible damage in spinal cord.which means it is suit for establish the SCII model.2.HuoxueTongdu Decoction can improve motor function of hind limb of rabbits during SCII.which means it can treat SCII to some extent.The mechanism of HuoxueTongdu Decoction on SCII are reduce inflammatory admage.inhibit apoptosis of neurons and improve nerve regeneration environment after spinal cord injury. |
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