| Traumatic optic nerve injury(TON)is a serious complication of brain trauma and can cause partial or complete loss of vision..There are about 0.7-6% patients of brain trauma combined optic nerve injury.There is still no consent standardized treatment procedure so far and no randomized double-blind control trials to confirm the advantages and disadvantages of various treatments.The optic nerve is part of the central nervous system and cannot regenerate or have the limited ability to regenerate after injury.Up to now,conservative treatments such as steroids,improvement of microcirculation,neurotrophic drugs as well as surgical decompression are the main measures of treatment.Recently,with the vigorous development of stem cell technology,stem cells have used in animal and human experiments and have achieved remarkable results.The advantages of this treatment in various diseases have appeared gradually.There have been scholars who used stem cells,such as neural stem cells,bone marrow mesenchymal stem cells,umbilical cord blood stem cells,the pulp mesenchymal stem cells,in the animal experiments and clinical trials and obtained encouraging results.Human Adipose derived stem cells(hADSCs)is very rich in the human body,easily get accessed,have high degree of purification in vitro and have the strong ability of amplification.More and more basic medical and clinical researchers began to express their attention of hADSCs.hADSCs are multipotent cells,which can differentiate into multiple tissues of mesodermal origin including bone or skeletal muscle endodermal and ectodermal tissues such as neurone like tissue.Lately,hADSCs is considered to be one of the most promising kind of adult stem cells which can be safely obtained from liposuction and can be used in the research of regenerative medicine.Many patients have excess fat which can be obtained from noninvasive and painless method of surgery.Neural progenitor cells originated from hADSCs bring the hope of curing the nervous system diseases.A large number of studies have confirmed that hADSCs have the ability of protecting optic nerve and repair the damage after injury.hADSCs also shows a good prospect of clinical application in clinical studies making it the ideal transplanted cells.However,stem cells fall into the heterologous cells which have many problems such as immune rejection or tumorigenicity.It is still not entirely clear for us of the biological characteristics of hADSCs and it is also unknown for us whether or not the amplification of hADSCs in vitro will affect their clinical use and will cause any security problems.The subjectregarding the TON as a research object,focus on studying the way of tail intravenous injection and injury site of injection of hADSCs in optic nerve crush injury model and observe the protection of the hADSCs for RGCs,impact on the expression GAP 43,the GFAP and the regeneration and anterograde transport of optic nerve and explore whether or not hADSCs is toxic and have influence on systemic inflammation factors in order to provide the important experimental data of evaluating the problems of amplification of hADSCs in vitro in clinical application.Part 1:Establishment and evaluation of rat optic nerve crush injury modelObjective:1.Successfully establish rat model of optic nerve crush injury.2.Identify the number of RGCs after optic nerve injury in rats3.Explore the changes of GAP43 and GFAP in the optic nerve and retinal after optic nerve injury.Method:Select SD rats randomly,24,weighing 220-250 g,and use the 3 g / 100 ml concentration of chloral hydrate to do intraperitoneal anesthesia according to the method of literature.After successful anesthesia,use the microsurgery instruments to cut the vertical incision on rat right eyelid,extend upward 1cm,cut open the eye camber of conjunctival fornix department,expose the optic nerve using blunt separation,use aneurysm clip clamping the optic nerve for 5second,observe the rat’s pupil after loosening the aneurysm clip.If the pupil dilate,direct light reflex disappear,indirect light reflex,fundus ophthalmoscope observation clearly shows that ophthalmic artery is not damaged,fundus blood supply is in good condition,then the optic nerve crush injury model of rat succeed and incorporate the rat into the experimental group.After successfully establish the optic nerve crush injury animal model,pour paraformaldehyde into rats’ blood circulation system and take optic nerve and eyeball for HE dyed,RGCs counts,Immunofluorescence staining of GAP43 and GFAP at 3 days,7 days,14 days and28 days respectively after injury and compare the differences between the 5 groups.Results:1.HE dyed staining show that the injury site of the optic nerve began to cavitate,invade small amount of inflammatory cells and optic nerve sheath membrane is completeafter 3days of injury;a large number of inflammatory cells invade and part of the blood vessels began to grow after 14 days;visible optic atrophy and glial scar hyperplasia start to appear after 28 days.2.RGCs has no obvious change after 3 days and reduce by 10.12%,10.12%,30.5%,61.87% respectively at 3 days,7 days,14 days and 28 days after injury;TUJ-1marked positive RGCs show similar changes reduced by 6.62%,31.12%,46.23%,62.25% at3 days,7 days,14 days,and 28 days after injury.3.Normal optic nerve have little or no expression of GAP 43 expression,the proximal part of injury site begin to express GAP43 at 3 days after injury,reach the peak point at 7days after injury,decline at 14 days after injury and decreased obviously at 28 days after injury.The expression of GAP43 in retinal show showed similar changes to the optic nerve which begin to express at 3 days after injury,reach the peak point at 7days after injury,decline at14 days after injury and decrease obviously at 28 days after injury.4.The immune fluorescence intensity of GFAP in the damage area of optic nerve decrease obviously,the GFAP in the proximal part of injury site begin to increase at 3days after injury,reach the peak point at 7days after injury,decline at 14 days after injury and decreased obviously at 28 days after injury.The immune fluorescence intensity of GFAP in retinal begin to increase after 3 days of injury,reach the peak point after 7 days of injury,decreased obviously after 28 days of injury.Conclusion:The optic nerve crush injury model of rat has high stability,repeatability and good maneuverability and the process of operation is very simple and can be done in a general aseptic animal laboratory.This animal model can effectively simulate the pathological and physiological change,principle of injury and related clinical symptoms of TBI patients.Part 2:Protection of hADSCs to RGCs after optic nerve crush injuryObjective:Explore the protection of hADSCs to RGCs after the tail intravenous injection and injury site injection and its dynamic changes.Method:Select SD rats randomly,120,weighing 220-250 g,randomly divided into 5 groups:simple injury group,injury site of hADSCs group,injury site of freeze-stored liquid group,tail intravenous injection of hADSCs group,tail intravenous injection of freeze-storedliquid group respectively,each group have 24 SD rats.After successfully establish animal model,simple injury group do not do any processing,hADSCs were injected to injury site by the way of intrathecal injection to the injury site of hADSCs group,The freeze-stored liquid were done the same to the freeze-stored liquid group,hADSCs were injected through tail vein to the tail intravenous injection of hADSCs group,freeze-stored liquid were done the same with tail intravenous injection of hADSCs group to tail intravenous injection of freeze-stored liquid group immediately after injury.After completion of treatment,HE dyed staining and immunofluorescence staining of TUJ-1,RGCs counts,were carried out at 3 days,7 days,14 days,28 days respectively after injury and compare the differences between the 5 groups.Results:1.There was no obvious difference and no significant statistical difference of RGCs counts between tail intravenous injection of hADSCs group,simple injury group and injury site of hADSCs group after 3 days of injury(P>0.05).RGCs in tail intravenous injection of hADSCs group decline at a slower pace than those in simple injury group at 7days and 14 days respectively after injury.There was a significant statistical difference of RGCs counts between these groups(P<0.05).There was no significant statistical difference between simple injury group and injury site of hADSCs group(P>0.05).There was also no obvious difference and no significant statistical difference of RGCs counts between tail intravenous injection of hADSCs group,simple injury group and injury site of hADSCs group after 28 days of injury(P>0.05).2.There was no obvious difference and no significant statistical difference of RGCs counts between tail intravenous injection of freeze-stored liquid group,injury site of freeze-stored liquid group and simple injury group at all time node.Conclusion:Tail intravenous injection of hADSCs can slow down the speed of retinal ganglion cells and have a certain protective effect,but the duration of this kind of protection is relatively short.Part 3:Effect of hADSCs for the regeneration of optic fiber after optic nerve crush injuryObjective:Explore the effect of hADSCs for the regeneration of optic nerve after optic nervecrush injuryMethod:Select SD rats randomly,120,weighing 220-250 g,randomly divided into 5 groups:simple injury group,injury site of hADSCs group,injury site of freeze-stored liquid group,tail intravenous injection of hADSCs group,tail intravenous injection of freeze-stored liquid group respectively.Each group have 24 SD rats.The method is the same with the second part.After completion of treatment,carry out the experiment of immunofluorescence staining(GAP 43,GFAP)of optic nerve,eyeball at 3 days,7 days,14 days and 28 days respectively after injury,detect the IOD of optic nerve and eyeball section and measure the number of regenerative optic fiber which pass the crush site.Select SD rats randomly,30,use the Western Blot method to detect content of protein of GAP – 43 in retinal after 7 days and 14 days of injury.Select SD rats randomly,30,use the rhodamine B as the anterograde tracer to detect the length of regenerative optic fiber.Results:1.The tail intravenous injection of hADSCs group: The expression of GAP-43 in optic nerve begin to increase after 3 days of injury,reach the peak point after 7days of injury,decline after 14 days of injury and decreased obviously after 28 days of injury;there was no significant statistical difference of IOD at 3 days and 28 days after injury(P>0.05)and there was significant statistical difference of IOD at 7days and 14 day after injury s(P<0.05)compared to simple injury group.The IOD of GAP-43 in retinal have the similar change and there was no significant statistical difference of IOD at3 days and 28 days after injury(P>0.05)and there was significant statistical difference of IOD at 7days and 14 days after injury(P<0.05)compared to simple injury group.The expression of GFAP in optic nerve begin to increase after 3 days of injury,reach the peak point after 7days of injury and decreased obviously after 28 days of injury compared to simple injury group and there was no significant statistical difference at all time node.The expression of GFAP in retinal begin to increase after 3 days of injury,reach the peak point after 14 days,and decreased a certain degree after 28 days of injury and there was no significant statistical difference after 3 days of injury(P>0.05)and there was significant statistical difference at 7days,14 days and 28 days after injury compared to simple injury group(P<0.05).2.The injury site of hADSCs group: The expression of GAP-43 in optic nerve begin to increase after 3 days of injury,reach the peak point after 7days of injury,decline after14 days of injury and decreased obviously after 28 days of injury;there was no significant statistical difference of IOD at 3 days and 28 days after injury(P>0.05)and there was significant statistical difference of IOD at 7days and 14 days after injury(P<0.05)compared to simple injury group.The IOD of GAP-43 in retinal have no significant statistical difference of IOD at 3 days and 28 days after injury(P>0.05)and have a significant statistical difference at 7days and 14 days after injury(P<0.05)compared to simple injury group.The IOD of GFAP in optic nerve have no significant statistical difference at all time node compared to simple injury group(P>0.05).The expression of GFAP in retinal begin to increase after 3 days of injury,have a significant increase at 7days after injury,reach the peak point at 28 days after injury and there was no significant statistical difference after 3 days of injury(P>0.05)and there was significant statistical difference at 7days,14 days and 28 days after injury compared to simple injury group(P<0.05).3.The expression of GAP-43 and GFAP of optic nerve and retinal in the injury site of freeze-stored liquid group and tail intravenous injection of freeze-stored liquid group consistent with simple injury group and there was no significant statistical difference between injury site of freeze-stored liquid group and tail intravenous injection of freeze-stored liquid group at all time node compared to simple injury group(P>0.05).4.The results of Western Blot show that the expression of GAP43 is higher in the tail intravenous injection of hADSCs group and the injury site of hADSCs group and the relative grayscale values in the tail intravenous injection of hADSCs group and the injury site of hADSCs group were higher than simple injury group at each time node which consistent with the results of immunofluorescence staining(P<0.05).However,the expression of GAP43 in the tail intravenous injection of hADSCs group is highest.There was no significant statistical difference of the relative grayscale values between injury site of hADSCs group,injury site of freeze-stored liquid group and tail intravenous injection of freeze-stored liquid group(P<0.05).5.The number of regenerative GAP43 positive optic nerve fibers through crush site:The number of regenerative GAP43 positive optic nerve fibers through crush site in the injury site of hADSCs group and the tail intravenous injection of hADSCs group is much higher than the simple injury group and there was significant statistical difference(P<0.05)and the number in the tail intravenous injection of hADSCs group is more than the the injury site of hADSCs group.There was no significant statistical difference of the numberof regenerative GAP43 positive optic nerve fibers through crush site among njury site of freeze-stored liquid group,tail intravenous injection of freeze-stored liquid group and the simple injury group(P<0.05).6.Rhodamine B anterograde tracer display that the largest distance of regenerative optic nerve is much longer after crossing the injury site in the tail intravenous injection of hADSCs group and injury site of hADSCs group compared to simple injury group and the statistical difference is significant between these groups(P<0.05).But the effect of the tail intravenous injection of hADSCs group is much better than the injury site of hADSCs group.There was no significant statistical difference between injury site of freeze-stored liquid group and tail intravenous injection of freeze-stored liquid group.Conclusion:hADSCs can promote the regeneration of optic nerve fiber,have the advantage of repairing optic nerve fiber and improve the function of axonal transport.Part 4:Toxicity and immunology test of hADSCs on optic nerve crush injury model ratObjective:Investigate the influence of hADSCs on systemic toxicity and immune related factors in rat.Method:The process of making animal model and treatments was the same to the second part.After completion of treatment,rats were executed at 3 days,7days,14 days and 28 days respectively after injury.Blood samples were collected from heart and put in the blood into EDTA anticoagulant tube,biological specimen tube and sodium citrate anticoagulation tube respectively for preservation.The biological specimen tube do not have anticoagulation.The blood sample in the biological specimen tube were taken for centrifuge after collecting and separating the serum.Then the serum was preserved in-80 ℃refrigerator.After the completion of collection of specimen,all samples of serum were tested at the same time.Blood routine and blood coagulation function tests were carried out shortly after collecting blood sample.Blood routine,electrolyte,liver and kidney function,blood coagulation function were detected respectly and the IFN-γ,IL-4elisa kit were used to detect levels of IFN-γ,IL-4 in the serum and all results were compared the differences between the 5 groups.Results:There was no significant statistical difference of the results of blood routine,electrolyte,liver and kidney function,blood coagulation function and the levels of IFN-γand IL-4 in serum among the simple injury group,injury site of hADSCs group,injury site of freeze-stored liquid group,tail intravenous injection of hADSCs group and tail intravenous injection of freeze-stored liquid group(P>0.05). |
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