The Study Of The Role Of GM1 In The Hemisected Spinal Cord Injury Rats

ObjectiveTo establish the Wistar rats spinal cord hemisection injury model,and then applicate the neurotrophic drug of GM1 after SCI through intraperitoneal injection,after that to analyze and evaluate its effectiveness for the repairment to spinal cord injury,to explore the role of GM1 for the recovery of neurological function in rats,search for the best time window to deliver GM1 on the injuryed rats,and discuss the possibility mechanisms of GM1 for the recovery on the animals of SCI.Method1.Under the microscope,using the microblades to make the T10 spinal cord hemisection in rats,and establish the hemisection spinal cord injury model.2.12-week-old 126 male Wistar rats were randomly divided into 3 groups,named sham-operated group,control group(intraperitoneal administration of normal saline), treatment group(GM1 intraperitoneal administration),all the animals were treated with hemisection except of the sham-operated group,n=42.3.All of the three groups animals were treated and observed on seven time points:1 day pre-operated,immediately post-operated,1 day post-operated,3 days post-operated,7 days post-operated,14 days post-operated,28 days post-operated, administering GM1 in one time on the injuryed animals in accordance with the weight,then the rats were observed and recorded in general and the BBB score for 12 weeks.4.2 weeks after administering GM1,take one rat from each time point in all the three groups randomly,put them on the stereotactic frame,then use micro-syringe to inject BDA anterograde tracer into the rats' brain sensorimotor areas,and 2 weeks after the injection of BDA,execute the animals and take the spinal cord,then observe the rapid refrigerated spinal cord section under the fluorescence microscope to analysis the axons running around the injured spinal cord area.5.Excepting the BDA injected group animals,the other rats will be anaesthetized and perfused with 4%paraformaldehyde fluid,then take the injured spinal cord and carry out with HE,GFAP staining,etc..Observation the morphology of the injured spinal cord,axonal regeneration,syringomyelia,such as changes in glial scar and so on.6.Dealing the BDA sections,GFAP sections with micro-image analysis software Image pro plus,and analyzing the basic data.Using one-way ANOVA for statistical comparison.Using student's t-test for statistical comparison on differences when compare two groups of data,using analysis of variance(ANOVA) for the comparison on differences among more than two gruoups of data,p＜0.05 is considered statistically different,p＜0.01 is considered the difference among the data have significantly statistical difference.Results1.The rat spinal cord hemisection model is successful and stable,it shows as a typical BrownSequard syndrome,and the wound is clean,the modle is reproducible and the injury grade is constant,the anatomical location is accurate,the dysfunction is also confirmed.In a word,this modle is suitable for the basic research on spinal cord injury and for the neurobiological research.2.The BBB score of all the animals preoperatively was 21 points,all of the SCI animals' right lower limbs were paralyzed within the first day after injury,and from then on they began to have a spontaneous recovery,but different groups had different recovery degree.All the animals' BBB score increased fastest in the period of 3d to 2w post-operated,it increased faster in the period of 2w to 5w post-operated,and got a slow increase between the time 5w to 8w post-operated,from then on no significant change can be seen.Compared with the animals of control group:the GM1 group animals had a higher BBB scale when administrated GM1 at the time 0-14d post-operated(p＜0.05),the animals who had GM1 administration more than 14d post-operated had no significant change in BBB scale(p＞0.05).The sham-operated group animals' BBB scores nearly have no change after injury with the normal ones.3.BDA corticospinal tract anterograde tracing of nerve fibers show that,the axons in the spinal cord of the sham-operated group have stained clearly and uniformly,there is no difference between different time points,they can smoothly cross downward through the T10 spinal cord.From the injured spinal cord of control group and GM1 treated group,obvious signs of spinal cord hemisection in the right side of the spinal cord where there are fluorescent reagent concentrated can be seen.The nerve fibers in the injured spinal cord of control group can rarely cross down through the injured aera,the nerve fibers of animals whichi were administed with GM1 within 2 weeks after injury can descend through the injured aera,the animals who were treated with GM1 after injury more than 2 weeks can't be found obvious axons descending from the injuredaera.Animals injected with GM1 or control after injury more than 2 weeks, are not clearly stained with fluorescent reagent,their difference is not statistically significant at the same time.4.GFAP rarely expressed in the sham-operated group,there is also no significancet difference between each time point of this group.The GFAP expression in the injured animals are continuing to increase in the control group and GM1 treated group with time going,it lasts for two weeks after spinal cord injured,the difference between them are not obvious.Animals injected with GM1 within the first 14 days after injury, have a gradually decrease in the expression of GFAP when injured more than two weeks,while the control group nearly have no change,in this period the two group's difference is statistically significant.The animals who have been injured more than 14 days,the GM1 administrated group has no significant difference between the control group in GFAP expression.Conclusion1.The T10 spinal cord hemisection model made under the microscope on Wistar rats, is reproducible and the injury grade is constant,the anatomical location is accurate, the dysfunction is also confirmed.In a word,this modle is suitable for the research on the role of GM1 administrating to SCI rats and observing the rehabilitation of neurological function.2.The BBB motor functional score system,is objective to evaluate the spinal cord injured rats' hindlimb motor functional rehabilitation.Within the first 14 days after injure,administrated GM1 in rats can promote motor function recovery,there must be a better effect especially administrat GM1 within the first 7 days after injure.This research also found that when the rats' spinal cord injured more than 14days administrat GM1 has little effect on motor functional recover.3.Using the BDA corticospinal tract anterograde tracing technique,we can observe the nerve fibers' pathway in the normal or injured spinal cord.Via this technique we find that when the spinal cord injury accurs,the earlier use GM1 the better neural axons' regeneration will be turned up,GM1 can promote the axons growing through the injured aera and down to the distant end of the spinal cord.But it's administment effective time window is 2 weeks after injure,especially in the first week after SCI accured.Once the spine injured more than 2 weeks the effect of administrating GM1 on the animal is not evident,there will be little axons regeneration.4.GM1 administration through intra-abdominal injecting can reduces the expression of GFAP in the hemisection aera of spinal cord,and relieve the physical hindered from the glial scar which can block the regenerate of axons and influence the neurological function recover.To sum up:GM1 can promote neurological function recover after spinal cord injury, and promote axonal regeneration,reduce the glial scar's physical obstruction.The effective time window is two weeks after injury,especially in the first week after SCI accured.