The Effect Of P75^NTR On Nerve Regeneration After Sciatic Nerve Injury In Mice And Its Mechanism

Research backgroundThe peripheral nerve injury caused by traffic accident and industrial injury is more and more common,but there is no effective treatment at present.Compared with the central nerve injury,the peripheral nerve injury can often be repaired because of neuronal intrinsic growth capacity.However,the prognosis of patients with peripheral nerve injury is still very poor.This is mainly due to the different effects of axonal degeneration and regeneration after peripheral nerve injury.Nerve regeneration is determined by many factors,including the patient's age and physiological state,the location of lesion injury,the time of intervention and the surgical technique selected.Most of these factors are related to the intrinsic growth capacity of neurons and the remyelination of distal nerve.Therefore,the main problem we need to solve is how to promote nerve regeneration and reduce the degree of distal denervation.As a member of the tumor necrosis factor receptor superfamily,p75 neurotrophin receptor(p75NTR)plays a key role in nerve repair.Neurotrophin binding to p75NTR regulates different signal pathways and mediates cell survival and differentiation.It can play a bidirectional biological regulatory role in the process of nerve regeneration.It has been found the expression of p75NTR in central and peripheral nerves is closed under normal conditions while upregulated under various injury.Many cell functions such as cell survival,cell apoptosis,migration,axon localization,differentiation and myelin regeneration are considered to be involved in upregulation of p75NTR.The upregulation of p75NTR expression in peripheral nerve injury is closely related to nerve regeneration and neuroprotection.However,the changes of p75NTR expression after peripheral nerve injury and the mechanism of its involvement in nerve regeneration need to be further explored due to unclear.Part I The expression pattern of p75NTR in mice after sciatic nerve crush injuryObjectiveThe time-dependent expression of p75N-TR in wild-type C57BL/6 male mice with sciatic nerve crush injury were studied by detecting the expression of p75NTR at different time points.It provides a theoretical basis for the further study of the mechanism of p75NTR on nerve regeneration after sciatic nerve injury.MethodForty-two wild-type healthy male C57BL/6 mice were randomly divided into sham-operated group and model group(n=21 per group).All mice were anesthetized with isoflurane.The sciatic nerve was clamped at 5mm distal end of sciatic tubercle by microscopically smooth vascular forceps in the model group.The stress point of nerve clamp was located at 1/3 points at the tip of vascular forceps,and the quantitative pressure was 1 buckle for 60 s to make the sciatic nerve crush injury model.The sciatic nerve was only exposed without nerve clamp in the sham-operated group.According to the time of sciatic nerve crush injury,the models were divided into pre-model group,post-model group at dayO,day3,day7,dayl4,day21 and day28 respectively.The injury of sciatic nerve was taken at each time point.The mRNA and protein expression of p75NTR was detected by RT-qPCR and Western-Blot.ResultIn the model group,the expression of p75NTR mRNA and protein increased after sciatic nerve crush injury.Its expression was related to the time after sciatic nerve crush injury.There was no significant difference in the expression of mRNA and protein of p75NTR between the sham and the model group before injury(P>0.05).The expression of p75NTR mRNA in the model group increased at day3 and reached the peak at day7 after injury.Then the expression of p75N-TR mRNA decreased gradually,and decreased to the pre-model level(P>0.05)at day 21 after injury.The expression of p75NTR mRNA at day3,day7 and dayl4 was higher than that in the sham group(P<0.01).In the model group,the expression of p75NTR protein increased at day3 and reached the peak at day14.Then the protein expression gradually decreased,it was higher than that of sham operation group at day3s day7,day 14,day 21 after injury(P<0.01),and decreased to the pre-model level at day28(P>0.05).There was no significant difference in expression of p75NTR mRNA and protein at all time points in sham group(P>0.05).ConclusionThe expression of p75NTR was upregulated after sciatic nerve crush injury,and correlated with the time after the injury.The expression of p75TNTR mRNA and protein was upregulated at day3 after sciatic nerve crush injury.The expression of p75NTR mRNA reached its peak at day7 after nerve injury,and its upregulation lasted until day21 after the injury,while the protein expression of p75NTR reached its peak at day 14 after injury,and the upregulation of p75NTR protein lasted until day28 after the injury.Part ??The mechanism of p75NTR promoting nerve regeneration in mice after sciatic nerve crush injuryObjectiveTo investigate the mechanism of p75NTR on nerve regeneration after sciatic nerve crush injury by observing the expression of neuronal intrinsic growth capacity related molecules and distal atrophy related protein and by observing the myelination degree of distal nerve axon at dayl4 after sciatic nerve injury in wild-type C57BL/6 mice and p75NTR knockout mice.MethodThe p75NTR gene knockout(p75NTR-/-extracellular exon III knockout)male mice with C57BL/6 genetic background were selected to mate with wild female C57BL/6 mice and their progeny genotypes were identified by PCR.30 wild-type male mice(p75NTR+/+)were randomly divided into sham-operated group and model group with 15 in each group,and 15 male mice with p75NTR gene knockout were selected as p75NTR-/-group.We used smooth forceps to make sciatic nerve crush injury models in the model group and p75NTR-/-group.In the sham group,the sciatic nerve was only exposed without nerve clamp.At day 14 after sciatic nerve crush injury,the L4-L5 segment spinal cord of three groups were taken and detected the mRNA and protein expression of neuronal intrinsic growth capacity related molecules(c-Jun,STAT-3,Gap-43,CREB)by RT-qPCR and Western-Blot respectively,and detected cAMP concentration by ELISA.The specimens of nerve tissue were collected from sciatic nerve crush injury area,and we detected the expression of p75NTR protein by Westernblot.The mRNA and protein expression of myelin regeneration related proteins(Mbp,Mpz,Mag)were detected by RT-qPCR and Westernblot.The myelination ratio of distal axons,g-ratio and the distribution of myelin thickness in myelinated axon were observed by transmission electron microscopy.Result1.At day14 after sciatic nerve crush injury,the expression of p75NTR protein in the model group was significantly higher than that in the sham group(P<0.01),and the expression of p75NTR protein in p75NTR-/-group was too low to be detected.2.At dayl4 after sciatic nerve crush injury,the expression of Mbp mRNA,Mpz mRNA and Mag mRNA in the model group was significantly higher than that in the sham group(P<0.01).The expression of Mbp mRNA,Mpz mRNA and Mag mRNA in P75NTR-/-group was significantly lower than that in the model group(P<0.01).The expression of Mbp,Mpz and Mag protein in sciatic nerve injury tissue in the model group was higher than that in the sham group(P<0.01).The expression of Mbp,Mpz and Mag protein in p75NTR-/-group was significantly lower than that in the model group(P<0.01).The expression of c-Jun mRNA,STAT-3 mRNA,STAT-3 mRNA,Gap-43 mRNA and CREB mRNA in L4-L5 spinal cord in the model group was slightly higher than that in the sham group,but the difference was not statistically significant(P>0.05)and there was no significant difference between p75NTR-/-group and the model group(P>0.05).The expression of these molecular proteins was consistent with their mRNA expression.There was no significant difference in L4-L5 spinal cord cAMP level between the model group and p75NTR-/-group(P>0.05).Compared with the sham group,the expression of BDNF mRNA and GDNF mRNA were significantly increased in the model group(P<0.05),but there was no significant difference between the model group and p75NTR-/-group(P>0.05).3.The myelination ratio of axon in the model group was(39.22±6.35)%and that in p75NTR-/-group was(24.83±4.62)%,while that in the sham group was(80.26±6.74)%.There was significant difference between the model group and the sham group(P<0.01).The myelination rate in the model group was significantly higher than that in p75NTR-/-group(P<0.01).The g-ratio in the model group was(0.74±0.053)and that in p75NTR-/-group was(0.83±0.058),while that in the sham group was(0.62±0.044).There was significant difference between the model group and the sham group(P<0.01),and the g-ratio in the model group was significantly lower than that in p75NTR-/-group(P<0.01).The average myelin sheath thickness in the model group was(0.54 ± 0.26)?m and that in p75NTR-/-group was(0.40±0.20)?m while that in the sham group was(0.78±0.22)?m.There was significant difference between the model group and the sham group(P<0.01),and the average myelin sheath thickness in the model group was higher than that in p75NTR-/-group(P<0.01).Conclusionp75NTR UprUgUlated the expression of myelin regeneration related gene and protein for Mbp,Mpz and Mag,and improved the myelinization degree of distal nerve after crush injury.The regulation of nerve regeneration by p75NTR had no relation with the expression of cAMP,c-Jun,STAT-3,Gap-43 and CREB which were associated with neuronal intrinsic growth capacity,and it also had no relation with the upregulaton of the expression of BDNF and GDNF in spinal nerve.Part? The effect of p75NTR on the behavior of mice after sciatic nerve crush injureObjectiveTo investigate the effect of p75NTR on the behavior of mice after the sciatic nerve crush injury by observing the behavioral function changes such as sciatic nerve function index,toe spreading test,slanting plate test and sensory score of pinprick assay in wild C57BL/6 type mice and p75NTR gene knockout mice after sciatic nerve crush injury.Method20 wild-type male mice(p75NTR+/+ were randomly divided into sham group and model group with 10 mice in each group.10 male mice with p74NTR knockout were randomly selected as p75NTR-/-group.We used microscopically smooth blood forceps to make sciatic nerve crush injury models in the model group and p75NTR-/-group.In the sham group,the sciatic nerve was only exposed without nerve clamp.Multiple behavioral functional indicators such as sciatic nerve function index(SFI),toe spreading test,slanting plate test and sensory score of pinprick assay were measured respectively before modeling and at different time points after modeling.Result1.At day7 after injury,the SFI of the model group was-(67.4±7.8)and that of p75NTR-/-groupwas-(75 9±8.1),while that of the sham group was-(14.2±5.5).There was significant difference between the model group and the sham group(P<0.01),and the SFI of the model group was higher than that of p75NTR-/-group(P<0.05).At day14 after injury,the SFI of the model group-(50.6±7.5)was higher than that of p75NTR-/-group-(65.9±8.3)(P<0.01).Comparison between the two groups at day21,day28 and day3 5 after injury,the SFI of the model group was still higher than that of p75NTR-/-group(P<0.05 or P<0.01.).2.The recovery of mouse toe-spreading in p75NTP-/-group was 1 week later than that in the model group.The initial reaction time of the model group(10.211.2)days was significantly shorter than that of p75NTR-/-group(15.3±1.6)days(P<0.01).The complete reaction time of the model group(17.1±1.9)days was shorter than that of p75NTR-/-group(24.7±2.5)days(P<0.01).3.At day7 after injury,the angle of mouse slanting plate test in the model group was(41.5±4.2)° and that in p75NTR-/-group was(36.8±3.4)°,while that in the sham group was(59.2±3.7)0.There was significant difference between the model group and the sham group(P<0.01)and the angle in p75NTR-/-group was significantly lower than that in the model group(P<0.05).The angle of the slanting plate test in the model group was increasing gradually at day 14 after injury.At day 14,day21,day28,and day35 after injury,the angle in the model group was significantly higher than that in p75NTR-/-group(P<0.01).4.The sensory score of pinprick assay in p75NTR-/-group mice completely recovered at day34 which was 14 days later than that in the model group.The initial reaction time of pinprick assay(9.8±1.6)days in the model group was significantly shorter than that in p75NTR-/-group(17.6±2.1)days(P<0.01).The full reaction time of pinprick assay(15.1 ± 1.9)days in the model group was shorter than that inp7NTR-/-group(27.3±2.5)days(P<0.01).ConclusionThe average scores of sciatic nerve function index,toe-spreading test,slanting plate test and sensory score of pinprick assay in p75NTR-/-mice were lower than those in wild type mice after sciatic nerve crush injury,which indicated that p75NTR could promote the recovery of behavioral function after sciatic nerve injury in mice.