| Objective:To verify the protective effect of bromonidine on facial nerve injury by constructing a rat model of facial nerve crush,and to study the therapeutic effect and mechanism of platelet rich plasma on facial nerve injuryMethods:in the first part,to observe and verify the therapeutic effect of bromonidine on facial nerve crush injury in rats:to construct a rat model of facial nerve clamp contusion.After the rats were treated with bromonidine for 28 days,the recovery of facial nerve function(behavioral evaluation,neurophysiological evaluation)was observed on the 0,7,14,21,28 days after the administration of bromonidine.The facial nerve and nucleus were observed by HE staining histomorphology,and the nutritional growth factor S100 and MBP myelin protein were detected by immunohistochemistry The expression levels of long factor S100 and fibroblast growth factor FGF were detected by Western blot to verify the therapeutic effect of bromonidine on facial nerve crush injury in rats;In the second part,to study the therapeutic effect of platelet rich plasma on facial nerve clamp contusion in rats and explore its mechanism:firstly,the facial paralysis model of rats was established.Using bromonidine as positive drug,the rats were treated with autologous platelet rich plasma for 28 consecutive days.The functional recovery of facial nerve(behavioral evaluation,neurophysiological evaluation)was evaluated at 0,7,14,21,28 days after operation.Facial nerve and nucleus were observed by HE staining,toluidine blue and electron microscopy,and facial nerve tissue was detected by PCR Western blot was used to detect the expression levels of bndf and S-100 in order to study the repair effect of platelet rich plasma on nerve injuryResult:The first part:1.There was no death in the rat model of facial nerve crush,and the survival rate was 100%;2.Behavioral evaluation:during the whole experimental process,the normal control group rats maintained normal whisker movement and blink reflex,and could normally perceive the difference of the surrounding environment,without eye secretion;the model group and bromonidine group showed complete facial paralysis on day 0 after operation,and the facial paralysis score reached 4 points.After 21 and 28 days of drug intervention,the facial paralysis score of bromonidine group was higher than that of control group In the model group,the difference was statistically significant(P<0.05);3.Neuroelectrophysiological evaluation:the latency and amplitude of CMAPs in the normal control group were maintained at normal level throughout the experiment;compared with the normal control,the latency of CMAPs in the model group and bromonidine group was significantly prolonged and the amplitude was decreased on the 0 th day after operation;after 28 th drug intervention,the latency and amplitude of CMAPs in the bromonidine group were significantly shortened and the amplitude was significantly increased,the difference was statistically significant The clinical significance(P<0.05);4.Histomorphological results:in the normal control group,the facial nerve tissue was kept in good condition during the whole experimental process;on day 0 after operation,the boundary between the myelin sheath and its surrounding area in the model group and bromonidine group was unclear,the distribution of new axons was uneven,the cross-sectional area of axons was small and uneven,the number of mature myelin sheath was reduced,and the thickness was not inhibited;bromonidine intervention 28 The number and thickness of new myelin sheath increased significantly,and the cross-sectional area and number of nerve sheath increased significantly(P<0.05);5.PCR results showed that bromonidine could inhibit the expression of GFAP/PAF in the nucleus after facial nerve injury in rats.The mRNA expression of glial fibrillary acidic protein/platelet activating factor(GFAP/PAF)gene was changed.The mRNA expression of the injured group was significantly increased,while the mRNA expression of the bromonidine group was significantly decreased.Bromine monie one week can make gelatin fiber on acidic protein(gfap)expression returned to normal,treatment 3 weeks expression of platelet activating factor(paf)returned to normal,bromine money behind the facial nerve injury in rats will raise nucleus in the NT-4 and the expression of P75ntr,NT-4 and the expression of P75ntr levels also presents the characteristics of the change over time,increased rapidly after the injury,peaked in the first week,then gradually decline.Compared with the injury group,mRNA expression of NT-4 and p75NTR was significantly increased in the bromonidine group and lasted for at least 3 weeks.Bromonidine can reduce the expression of NF-?B,TNF-? and IL-6 in the nerve nucleus after facial nerve injury.Bromonidine can significantly reduce the expression of NF-?B,TNF-? and IL-6 in the nerve nucleus after crush injury.NF-?B was significantly higher in the 1st week after injury than in the control group(up to 4 times),and decreased after treatment with bromonidine.The expression of TNF-? increased rapidly and decreased gradually after injury,but was still higher than that in the control group at week 3,while the expression of TNF-? was gradually decreased by bromonide.IL-6 increased rapidly after injury,and reached the maximum value in the first week,then gradually decreased,and returned to normal level in the third week,while bromonidine significantly inhibited the expression of IL-6 in the first week.The results indicated that all three subtypes of ? 2-ARs exhibited in facial nucleus.Compared with the control group,the mRNA expression level of ? 2A-AR was obviously elevated in the injury group.The expression levels of ? 2A-and ? 2B-AR were also increased in response to brimonidine.6.Immunohistochemistry results:on day 0 after operation,S100 protein level in model group and bromonidine group was significantly lower than that in normal control group,with statistical significance(P<0.05);on the 28th day after bromonidine intervention,the expression of MBP myelin protein and S100 protein was significantly increased,which was close to the normal control group,and the difference was statistically significant(P<0.05);7.Western blot results:compared with the normal control group,the fibroblast growth factor FGF2 and S100 in the model group and bromonidine group were significantly lower than those in the normal control group(P<0.05);After 28 days of bromonidine intervention,the expression of S100 protein and growth factor FGF2 in bromonidine group were significantly higher than those in model group(P<0.05);The second part:1.The platelet concentration in platelet rich plasma was 4.23 times higher than that in whole blood platelet;2.Behavioral evaluation:during the whole experimental process,the normal control group kept normal whisker movement and blink reflex,and could normally perceive the difference of surrounding environment,without eye secretion;the model group,bromonidine positive group and platelet rich plasma administration group showed complete facial paralysis on day 0 after operation,and the facial paralysis score reached 4 points;after 7 and 14 days of group intervention,the facial paralysis score reached 4 points,There was no significant difference in the scores of facial paralysis between the two groups(P<0.05);however,after 21 and 28 days of intervention,the scores of facial paralysis in the bromonidine group were significantly lower than those in the model group(P<0.05).On the 7th and 14th days,the scores of facial paralysis in the model group decreased to varying degrees,But there was no significant difference(P>0.05);3.Neuroelectrophysiology:compared with the normal control,the latency of CMAPs in the model group,the bromonidine positive group and the platelet rich plasma group was significantly prolonged,and the amplitude of CMAPs was significantly reduced.After 28 days of treatment,compared with the model group,the latency of CMAPs in the bromonidine positive group and platelet rich plasma treatment group was significantly shortened and the amplitude of CMAPs was significantly increased(P<0.05).4.Histomorphological observation:after 28 days of treatment,there were more Schwann nuclei and regenerating myelin sheath in the facial nerve in the bromonidine positive group and platelet rich plasma group,and the diameter of nerve fibers was increased compared with that of the model group,but the diameter of each fiber was uneven.At the same time,the thickness of myelin sheath increased,but it was still thinner than that of the normal control group There was statistical significance(P<0.05);PCR detection results:the expression levels of neurotrophic factor gene bndf and NT-4 in nerve and facial nucleus of normal control group remained relatively stable at all time points,with no significant difference and no statistical significance(P>0.05);the expression of neurotrophic factor gene bndf in nerve nucleus of model group was significantly higher than that of normal control group and bromonidine positive group In group B,the difference was significant(P<0.05).On the 14th day,the expression of neurotrophic factor bndf in the nucleus reached the peak;compared with the model group,the expression level of neurotrophic factor gene NT-4 in the facial nerve and facial nucleus in the bromonidine positive group and platelet rich plasma group was significantly lower than that in the model group;and on the 14th day of grouping treatment,the NT-4 expression level of the neurotrophic nerve factor gene in the facial nerve and facial nucleus of the rats in the group treatment group was significantly lower than that in the model group The expression level reached the peak and then decreased gradually;Western blot results:compared with the normal control group,the expression levels of bndf and S100 protein in the model group were significantly lower than those in the normal control group(P<0.05).At the same time,compared with the model group,the expression of bndf and S100 protein in the bromonidine positive group and platelet rich plasma group were significantly increased in the model group,and the difference was statistically significant(P<0.05).Conclusion:bromonidine can shorten the latency of CMAPs,increase the amplitude of CMAPs,maintain the stability of the number of new axons and myelin sheath structure,up regulate the expression levels of MBP,bndf,FGF and S100,promote the myelination of nerve scar and axon,and finally protect the facial nerve;Platelet rich plasma can repair facial nerve injury to a certain extent,which may be through shortening the latency of CMAPs,increasing the amplitude of CMAPs,up regulating the expression of bndf,NT-4 and S100,instead of providing nutritional nerve factor protein in brain tissue. |
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