Experimental Study Of The Effect Of Ginkgo Biloba Extract (EGb₅₀) On Peripheral Nerve Regeneration

IntroductionPeripheral nerve injuries is a commonly encountered clinical problemand often result in long-term functional deficits. Extensiveinvestigation toward the development of methods to improve regenerationfollowing nerve injury is ongoing. Ginkgo Biloba Extract is an ancientChinese medicine. Its neuroprotective effect on functional restorationof brain has been widely confirmed. However, few works have aimed atusing Ginkgo Biloba Extract in the regeneration of peripheral nerves. Theaim of the study is to evaluate the effect of Ginkgo Biloba extract (EGb₅₀)on peripheral nerve regeneration and its dose-effect relationship, and toelucidate the possible mechanism.Part one: Effect of Ginkgo Biloba Extract on Sciatic Nerve Regenerationin Rats and its Dose-effect RelationshipObjective To provide the promoting effect of extract of leave GinkgoBiloba(EGb₅₀) on nerve regeneration and the dose-effect relationship.Methods 150 male Sprague-Dawely rats were subjected to right sciatic nervetransection and immediate repair. They were randomly divided into fivegroups: A: normal saline(NS) group, B: low dose EGb₅₀ group, C: moderatedose EGb₅₀ group, D: high dose EGb₅₀ group, E: compound Chinese herbs group.In group A, the rats were fed with 1 ml normal saline. In groups B,C andD, rats were fed with EGb₅₀ 50mg·kg^-1·d^-1, 100mg·kg^-1·d^-1, 200mg·kg^-1·d^-1dissolving in 1 ml normal saline. In group E, the rats were fed with 1 mlcompound Chinese herbs. At 7 days after injury, the regenerationdistances of sensory neurons (pinch test) were evaluated.Electrophysiological, histological examinations and functionalevaluation were used to assess nerve regeneration and the functionalrecovery in 2,4,6,8 weeks of post-operative intervals respectively.Results(1) Pinch test: Pinch test in groups B,C,D,E were better than in group A(P<0.01). The regeneration distances of group D was longer thanthat of group B,C,E(P<0.01). The difference between group B,C and E wasnot significant(P>0.05). (2)SFI: The SFI of group B,C,D,E were better thanthat of group A in all the time point(P<0.01), while the difference amonggroup B,C,D,E was not significant(P>0.05). (3)Muscular tension recovery:There was a higher muscular tension recovery of groups B,C,D,E than thatof group A in all the time point except group B two weeks after operationof muscular tetanic tension (P<0.01). Muscular tension recovery of groupD was better than that of groups B,C (P<0.05).2 weeks after operation,muscular twitch tension recovery of group E was better than that of groupB(P<0.05),but was lower than group D(P<0.01).4,6,8 weeks after operation,the difference between group E and group D was not significant of musculartwitch tension(P>0.05).In all the time point, the difference between groupE and group D was not significant of muscular tetanic tensionrecovery(P>0.05). (4) Electrophysiological evaluation: The recovery ofMNCV and AMPM in group B,C,D,E were better than those in group A(P<0.01).There was a significant difference between group B and group C, betweengroup B and group D, and between group C and group D at all the timepoint(P<0.05).2 weeks after operation, the recovery of MNCV and AMPM ingroup E were better than those in group B,C(P<0.05),while the differencebetween group E and group C was not significant 4,6,8 weeks afteroperation(P>0.05). (5) Conservation muscle mass ratio: Conservationmuscle mass ratios were the lowest in the animals of group A compared tothose in groups B,C,D and E at all the time(P<0.01). Muscle mass ratioswere higher in D group than in group B and C(P<0.05). 2,4,8 weeks afteroperation, the difference between group E and groups B,C,D are notsignificant(P>0.05), while the muscle mass ratio was higher in group Ethan in group B (P<0.01). (6) Recovery ratio of muscle fibercross-sectional area in groups B,C,D,E were better than in group A(P<0.01),compared to group B,C, groups D and E have a higher recovery ratio (P<0.05),but the difference between group D and E is notsignificant(P>0.05). (7)Histomorphometric evaluation: There was asignificant difference between the group A and group B,C,D and E ofmyelinated nerve fiber pass ritio(P<0.01).Group D has a high pass ritio than group B,C(P<0.01). 2,4 weeks after operation the difference betweengroup E and group B,C,D were not significant(P>0.05),but the myelinatednerve fiber pass ritio of group E was better than group B,C 6 weeks afteroperation (P<0.01). 8 weeks after operation, the myelinated nerve fiberpass ritio of group E were better than group B,C,D(P<0.05).Conclusions EGb₅₀ has the effect of promoting regeneration of injuredperipheral nerve. The higher the dose, the better the result.Part two: In vitro study of proliferation of Schwann cells cultured withGinkgo Biloba leaves(EGb₅₀)Objective To investigate the effects of EGb₅₀ on the proliferation ofSchwann cells cultured in vitro.Methods Purified Schwann cells were cultured under different conditionsaccording to group assignment. In the experiment group, Schwann cells werecultured in FBS-DMEM media supplemented with 50ug/ml EGb₅₀, while in thecontrol group the cells were cultured in the same culture media withoutEGb₅₀. The OD of different intervals was determined by XTT assay to obtainthe growth curves 1,3,5,7,9 days of culture respectively. Thymidineincorporation assay was applied after 48 hours and 72 hours of culturerespectively to determine the DPM value.Results The optical density (OD) of Schwann cells were higher in theexperiment group than that in the control group in all the time point.The DPM of Schwann cells cultured with EGb₅₀ for 48 hours and 72 hours werehigher than that of those cultured without EGb₅₀. All those differenceswere statistically significant (P<0.01).Conclusion EGb₅₀ can enhance the proliferation of Schwann cells culturedin vitro. This chould be one of the underlying mechanisms of its effectfor promoting nerve regeneration.Part three: Effect of Extract of Ginkgo Biloba leaves(EGb₅₀) onexpression of NGF, GAP-43 and iNOS after sciatic nerve injuries at rats Objective To investigate the effect of Extract of Ginkgo Bilobaleaves(EGb₅₀) on the expression of NGF, GAP-43 and iNOS in nerve tissue,dorsal root ganglia and spinal cord after sciatic nerve injuries at rats.Methods Sciatic nerve injury model was established by transaction of rightsciatic nerve in 144 male SD rats, which were randomly divided into twogroups. The rats were fed with EGb₅₀ (200mg·kg^-1·d^-1)at the experimentalgroup and normal saline in the control group. At the same time, 12untreated rats served as the normal group. The distal part of injurednerve, dorsal root ganglia(L5, L6) and spinal cord(L4～L6) were harvested1,3,7,14,21 and 28 days after the operation. The expression ofNGF, GAP-43, iNOS protein and mRNA were determined by immunohistochemicaland RT-PCR analysis.Results The mRNA expression of NGF in sciatic nerve was much higher ofexperimental group than that of control group in 7,14,21 days(P<0.05).7and 14 days after operation, the experimental group has a higher mRNAexpression of NGF in dorsal root ganglia and spinal cord (P<0.05). Theoptical density and positive area of NGF in sciatic nerve, dorsal rootganglia and spinal cord were higher of experimental group than that ofcontrol group in 7,14,21, and 28 days(P<0.05). The mRNA expression ofGAP-43 was much higher of experimental group than that of control groupin 7,14 days after operation (P<0.05). The optical density and positivearea of GAP-43 in sciatic nerve, dorsal root ganglia and spinal cord werehigher of experimental group than that of control group in 7,14 and 21days(P<0.05). The experimental group has a lower mRNA expression of iNOSthan that of control group in 1,3,7 days (P<0.01). The mRNA expressionof iNOS in dorsal root ganglia and spinal cord were higher of experimentalgroup than that of control group in 7 days(P<0.05).The optical densityand positive area value of iNOS of sciatic nerve was much lower ofexperimental group than that of control group in 3,7,14,21 days(P<0.05).The optical density of iNOS in dorsal root ganglia was lower ofexperimental group than that of control group in 7,14 days(P<0.05). Theoptical density and positive area value of iNOS in spinal cord were lowerof experimental group than that of control group in 7 days(P<0.05).Conclusion EGb₅₀ can increase the expression of NGF, GAP-43 and inhibit the expression of iNOS after sciatic nerve injuries in rats.