| Spinal cord injury(SCI),especially upper cervical SCI,with the characteristicof high morbidity,high disability rate,and high mortality.In the past 1 century years,with the rapid development of imaging,anatomy,and biomechanics,surgical has made significant progress.About 50%of patients with early spinal cord decompression have improved spinal function scores.However,about 50%of patients still recover ASIA grade 1 or no improvement,and neuralgia complications still remain after surgery,causing a heavy burden on patients and medical care.Therefore,the search for drugs and other therapies has become the main task at present.Governor Vesel treatment is a relatively complete method of TCM treatment based on syndrome differentiation.There is a consensus that there is a close relationship between Governor Vesel and spinal cord in terms of anatomy,physiology,and pathology.Modern physicians have proposed spinal cord injury,the disease position is in the Governor Vessel,and Governor Vessel Stasis syndrome is a common clinical syndrome.For the diagnostic criteria of spinal cord injury,the doctors only proposed the macroscopic diagnostic criteria for spinal cord injury,and there is still no report on its microscopic diagnostic criteria.From the therapeutic point of view,many studies have explored the clinical efficacy and possible mechanisms of BYHWD in treating spinal cord injury.The results showed that BYHWD is effective in the treatment of spinal cord injury,but if the effective basis is related to endoplasmic reticulum apoptotic pathway has not yet been reported.In view of the important function of the spinal cord in the human body,research on the mechanism of traditional Chinese medicine and microscopic indicators can only be conducted through animal experiments,but there is still short of upper cervical spinal cord injury.Therefore,this study attempted to use a balloon catheter to construct animal models of different degrees upper cervical spinal cord injury.Based on this model,explore the mechanism of BYHWD on endoplasmic reticulum-associated apoptosis signaling pathway;From the changes of platelet activating factor in the spinal cord tissue and plasma before and after treatment,explore the mechanism of Governor Vesel treatment in spinal cord injury.Part Ⅰ Research on animal model of upper spinal cord injury of Governor VeselStasiswith different degrees using Foley’s tubeObjective:Thirty female SPF Wistar rats were randomly divided into blank group,sham operation group,mild compression group,moderate pressure group and servere pressure group.Except for the blank group,A linear midline incision was created at the median of the C1-C3 vertebrae,after incising the atlanto-occipital membrane and the C1-C2 yellow ligament,an uninflated balloon was then placed in the epidural space underneath the Cl posterior arch using micro-surgical techniques.After 24 hours,normal saline was injected into the balloon until the pressure reached 100 kPa,300 kPa and 500 kPa,resulting in mild,moderate and severe acute upper cervical compression.The cervical MRI was conducted at 1h post-compression;Neurobehavioural assessments using the BBB score and modified Tarlov scale at 1 h,3 h,12 h and 24 h post-compression,after four diagnostic methods information were collected,the balloon catheter was removed.Spinal cords were harvested after cardiac perfusion for HE staining and Tunel apoptosis.The pathological features of the upper cervical spinal cord injury were observed under a light microscope.Methods:Thirty female SPF Wistar rats were randomly divided into blank group,sham operation group,mild compression group,moderate pressure group and servere pressure group.Except for the blank group,A linear midline incision was created at the median of the C1-C3 vertebrae,after incising the atlanto-occipital membrane and the C1-C2 yellow ligament,an uninflated balloon was then placed in the epidural space underneath the Cl posterior arch using micro-surgical techniques.After 24 hours,normal saline was injected into the balloon until the pressure reached 100 kPa,300 kPa and 500 kPa,resulting in mild,moderate and severe acute upper cervical compression.The cervical MRI was conducted at 1h post-compression;Neurobehavioural assessments using the BBB score and modified Tarlov scale at 1 h,3 h,12 h and 24 h post-compression,after four diagnostic methods information were collected,the balloon catheter was removed.Spinal cords were harvested after cardiac perfusion for HE staining and Tunel apoptosis.The pathological features of the upper cervical spinal cord injury were observed under a light microscope.Results:1)T2-weighted MRI images:The spinal occupancies in blank group(N/A),sham operation group(1.000±0.000%),slight compression(53.000±8.485)%,moderate compression group(57.500±4.950)%and severe compression(67.000±11.314)%;2)The BBB scores and Tarlov scores:there was interaction between group factor(P<0.01);the scores were higher in blank group and sham-operated group compared to model group(P<0.01);From mild compression to severe compression,BBB scores and Tarlov scores gradually decrease;There was no statistical difference between different timepoints and time*group points(P>0.05);3)Four diagnostic methods information:The normal group and the sham-operated group in good mental states and liked activities and presented with flexible movement,reddish lips,light-colored ears and reddish paws.The rats in model group were in bad mental states and dislike activities and presented with instability of gait,dull-red lips and ears and livid paws;4)Hematoxylin&eosin staining(HE)showed broad neuronal atrophy,necrosis and hemorrhage in gray matter,diffuse demyelination changes in white matter and obvious vacuolar degeneration in axons from mild compression to severe compression;5)Apoptotic staining by TUNEL method showed that there were no obvious apoptotic cells in blank group,sham operation group,and light pressure group;the apoptosis index of moderate and severe pressure group increased slightly,but there was no statistical difference in apoptosis index between the two groups(P>0.05).Conclusions:In this experiment,we successfully created upper spinal cord injury animal model of Governor Vesel Stasis using Foley’s tube.Which conforms to the clinical pathological features of the upper cervical spinal cord injury of Governor Vessel stasis,pressure time and degree is controllable.Part II Effect of Buyanghuanwu decoction on the expression of Endoplasmicreticulum related factors after acute upper cervical spinal cord injury in ratsObjective:In this experiment,based on the acute upper cervical spinal cord injury model,treatment of Buyang Huanwu Decoction,methylprednisolone as a positive control group.After 14 days,investigate the role of Buyang Huanwu Decoction in acute upper cervical spine cord injury(SCI)in its regulation of Caspase-12,Caspase-9 and Caspase-3.Methods:Wistar Female rats were randomly divided into the sham operation,model,methylprednisolone(MP)and Buyanghuanwu decoction(BYHWD)groups,10 rats in each group.The expression of Caspase-12,Caspase-9 and Caspase-3 proteins were detected through Western blotting at 14 d after injury.Motor functions were evaluated using the 21-point locomotor rating scale developed by Basso,Beattie and Bresnahan(BBB)at 1 d,3d,7 d,and 14 d after surjury.Results:1)At 14 days after treatment,levels of Caspase-12,Caspase-9 and Caspase-3 in MP and BYHWD groups were lower than model group(P<0.01);there was no significant difference between MP and BYHWD groups(P>0.05);2)First day after surgery,BBB score in model,BYHWD and MP groups were lower than that of SO group,the difference was statistically significant(P<0.01);At 3d,7 d and 14 d after surgery,BBB score in BYHWD and MP groups were higher than that of model group,the difference was statistically significant(P<0.01);There are no significant difference between BYHWD and MP group(P>0.05).Conclusion:BYHWD can significantly improve locomotor recovery by inhibiting the expression of ER apoptosis-related proteins Caspase-12,Caspase-9 and Caspase-3 after acute spine cord injury.Part Ⅲ Effect of Buyanghuanwu decoction on the expression of Platelet ActivatingFactor after upper cervical spinal cord injury of Governor Vesel Stasis in ratsObjective:This part of the experiment is to investigate the effect and mechanism of Buyanghuanwu decoction on upper cervical spinal cord injury of Governor Vesel Stasis in rats,from the changes of platelet activating factor content in spinal cord tissue and plasma after treatment.Methods:Wistar Female rat s were randomly divided into the sham operation,model,methylprednisolone(MP)and Buyanghuanwu decoction(BYHWD)groups,6 rats in each group.The expression of platelet activating factor in spinal cord tissue and plasma were detected through Elisa at 14 d after injury.Motor functions were evaluated using the 21-point locomotor rating scale developed by Basso,Beattie and Bresnahan(BBB)at 1 d,3d,7 d,and 14 d after surjury.Results:1)At 14 days after treatment,levels of platelet activating factor in plasma in MP and BYHWD groups was lower than model group(P<0.01);PAF in BYHWD groupswas lower than MP group(P<0.01);there was no significant difference during MP、BYHVDand SO groups(P>0.05);2)At 14 days after treatment,levels of PAF in spinal cord tissue in BYHWD groupwas lower than model group(P<0.01);PAF in MPgroupwas lower than model group(P>0.05);PAF in BYHWD and MP groupswerehigher than SO group(P<0.05);3)First day after surgery,BBB score in model,BYHWD and MP groups were lower than that of SO group,the difiference was statistically significant(P<0.01);At 3d,7 d and 14 d after surgery,BBB score in BYHWD and MP groups were higher than that of model group,the difference was statistically significant(P<0.01);There are no significant difference between BYHWD and MP group(P>0.05).Conclusion:BYHWD can significantly improve locomotor recovery by inhibiting the expression of platelet activating factor content in spinal cord tissue and plasma after acute spine cord injury. |
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