Study On Draxin’s Expression Changes And Its Locations In The Process Of Spinal Cold Injury And Regeneration In Mice

Objective:In this paper, the authors try to build a model of spinal cord dorsalhemisection by crosscutting the dorsal spinal cord of adult mouse with a sharpblade. By observing the expression changes and the location of draxin indifferent periods after the spinal cold injury, the authors hope to make acontribution to the application of axon guidance factors in clinical disease ofspinal cold injury.Methods:1Animals and grouping: The experiment needs100two-month-old micewhich weigh about35g. After models of spinal cord dorsal hemisection are setup, the authors use Reuter sensory evaluation methods to make sure if modelsare set up sucessfully.Models of spinal cord dorsal hemisection are randomlydivided into several groups: uninjured group, one day-injured group, twoday-injured group, three day-injured group, four day-injured group, fiveday-injured group, six day-injured group and seven day-injured group. Thereare10mice in each group which are used to test RT-PCR and Western Blot. Asfor other models, they are divided into one day-injured group, two day-injuredgroup, three day-injured group, four day-injured group and five day-injuredgroup. Each group consists of5mice. The authors intend to observeimmunohistochemical draxin antibody and DCC antibody through thesemodels.2Extracting mRNA and RT-PCR: pick up5mice from each group of theuninjured, one day injured, two day-injured, three day-injured, fourday-injured, five day-injured, six day-injured and seven day-injured. First,anaesthetize the mice with10%chloral hydrate. Then, take out the spinal cordfrom the injured spine, removes their vertebras immediately and exposes spinal cord tissue. After washed with PBS that is compounded by DEPCtreated water, spinal cord tissue is shifted into Trizol in order to extract mRNAconventionally.Next, quantitate the spinal cord tissue with NanoDrop2000C.Finally, transcribe it into cDNA reversely and preserve it at minus twentydegrees centigrade.3Protein extraction and Western Blot: pick up5mice from each group ofthe uninjured, one day injured, two day-injured, three day-injured, fourday-injured, five day-injured, six day-injured and seven day-injured. First,anaesthetize the mice with10%chloral hydrate. Then, take out the spinal cordfrom the injured spine, removes their vertebras immediately and exposesspinal cord tissue. After washed with normal PBS, spinal cord tissue is shiftedinto lysate. Homogenate it fully and then put it on the ice for30minutes.Afterwards, the precipitate is removed by centrifugation while supernate is left.At last, draw a fraction of the supernate and test its protein level by Coomassiebrilliant blue. Other supernate is preserved in a refrigerator at minus eightydegrees centigrade for the test of Western Blot.4Equipment and the observation of immunohistochemistrial staining:first of all, pick up5mice from the group of one day injured, two day-injured,three day-injured, four day-injured, five day-injured. Anaesthetize the miceand expose their hearts. Then, pierce a transfusion needle into the left ventricle.Meanwhile, open right auricles of the mice with scissors. Next, irrigate themwith physiological saline quickly. When no blood is left, irrigate them with4%paraformaldehyde. At the end, take out the spinal cord from the injured spine,wash it with normal PBS, put it into4%paraformaldehyde to fix for two hoursand store it in OCT at minus eighty degrees centigrade forimmunohistochemistrial staining.Results:1Expression changes of draxin mRNA in different periods after adultmouse spinal cord is injured.Expression changes of draxin mRNA exist in uninjured group, oneday-injured group, two day-injured group, three day-injured group, four day-injured group, five day-injured group, six day-injured group and sevenday-injured group. As time goes on, the expression increases gradually. On thethird day, it reaches a climax. After that, expression decreases little by little.There are a lot of significant differences in the expression in different periods(P<0.05).2Expression changes of draxin protein in diffetrent periods after adultmouse spinal cord is injured.Expression changes of draxin protein exist in uninjured group, oneday-injured group, two day-injured group, three day-injured group, fourday-injured group, five day-injured group, six day-injured group and sevenday-injured group. As time goes on, the expression changes increase gradually.On the third to fifth day, expression reaches a climax, after that it decreases alittle. There are a lot of significant differences in the expression in differentperiods(P<0.05).3Draxin protein expression changes and its locations in adult mouseinjured spinal cord.In draxin positive area, fluorescence staining tends to be red whichspreads around the injured part. Besides, expression increases as time goes onand reaches a climax on the third to fifth day.4Expression of DCC, the receptor draxin, in injured adult mouse spinalcord.DCC is one of the draxin’s receptors. In draxin positive area,fluorescence staining tends to be red, but in DCC positive area, fluorescencestaining is green. All draxin positive red signals and DCC positive greensignals are concentrated on the incision. Furthermore, the location of draxinand DCC protein are the same. All of their locations are reflected in the darkyellow area.Conclusion:1Draxin mRNA and draxin protein are expressed in normal adult mousespinal cord tissue. The expression amount is little, but it is increased when thespinal cord tissue is injured. 2Draxin’s expression is different in different periods after adult mousespinal cord is injured. Within seven days after the injury, draxin mRNA anddraxin protein’s expression peaks on the third to fifth day.3Draxin protein increases when adult mouse spinal cord is injued and theexpression is concentrated on the incision. Moreover, expression increasesgradually on the first injured day, the second injured day, the third injured dayand the fifth injured day.4As one of draxin’s receptor, DCC’s expression increases in adult injuredmouse spinal cord. The expression spreads around the incision and its locationare the same with drxin pretein.