| Part1Eestablishment of Delayed Nerve Repair Rat ModelObjective To establish a standardized delayed nerve repair model in rats and validate the feasibility of direct secondary neurrorraphy after various delay intervals.Methods58female Lewis rats were randomly divided into the following6groups:immediate repair (BO group, n=8),1week (B1group, n=11),4weeks (B2group, n=10),6weeks (B3group, n=11),8weeks (B4group, n=11) and12weeks (B5group, n=7) delayed repair group. Left sciatic nerve was transected, the proximal nerve stump was turned around, embedded into the neighboring muscles and the distal nerve stump was tagged onto obturator tendon by two9-0monofilament nylon sutures to prevent retraction. The sciatic nerve stumps were sutured by10-0monofilament nylon sutures directly during the secondary nerve repair surgery after various delay intervals. A successful direct nerve repair without tension was defined when the gap was no longer than4.0mm and the nerve repair with10-0stitches did not rip apart upon repeated maximal passive movements of the limb. Intra-operative parameters such as the size、gap、scarring and neuroma size of the nerve stumps were measured to insure the reproducibility of the standardized animal model. Compound muscle action potential (CMAP) was recoreded to demonstrate successful nerve regenenration.Results Gaps between the two nerve stumps ranged from0to9mm, the average being1.36,2.85,3.43,3.83and6.40mm in rats with1,4,6,8and12week delay, respectively. The overall rate of successful repair without tension was78%among50delayed nerve repair rats. CMAP values of1and4week delay groups were not different from the immediate repair group, whereas CMAP amplitudes of6,8and12week delay groups were significantly lower (P<0.05).Conclusion A novel, standardized delayed nerve repair model is established. For this model to be sensitive, the interval between nerve injury and secondary repair should be at least over4weeks. Thereafter the longer the delay, the more challenging the model is for nerve regeneration. The choice of delay intervals can be tailored to meet specific requirements in future studies. Part2Characteristics and Time Courses of the Microenvironment Changes in the Denervated Muscles with Various Denervation Intervals:An Experimental Study in RatsObjective To detect the characteristics and time courses of microenvironmental changes in muscles after denervation with various intervals.Methods40female Lewis rats with body weight of200±5gram were subjected to sciatic nerve transection and randomized into the following5groups:sciatic nerve transection and denervation for1week (Al group, n=8),4weeks (A2group, n=8),6weeks (A3group, n=8),8weeks (A4group, n=8) and12weeks (A5group, n=8). Changes in the denervated muscles including electrophysiologic (fibrillation potential), histologic (muscle wet weight, muslce fiber types, muscle cross-sectional area, extent of fibrosis) and molecular biologic examinations (MRF4, Myogenin, nAChR, MUSK, P21mRNA) were performed for all rats postoperatively.Results Fibrillation potential (Fib) appeared in the medial gastrocnemius (MGS) muscle on the third day, reached peak at1to4weeks, decreased dramatically at6weeks, postoperatively; The manintanence rate of muscle wet weight of MGS, soleus and tibialis anterior (TA) was approximately80%,40%,20%and20%, respectively, at1,4,8and12weeks postoperatively; The muscle cross sectional area (CSA) of TA and MGS gradually decreased, while the amount of connective tissues in TA and MGS gradually increased over the time course of denervation (P<0.05); In denervated MGS (mixed fiber muscle) and TA (fast fiber dominant muscle), the proportion of type IIB obviously decearsed, while type Ⅰ and ⅡA fiber proportions obviously increased over the time course of denervation (P<0.05), the fast fibers converted to slower ones. Changes in soleus (slow fiber dominant muscle) were totally different: the proportion of type Ⅰ fibers decreased, while type ⅡA fiber proportion obviously increased over the time course of denervation (P<0.05), the slow fibers converted to fast ones. These changes were especially obvious at4-6weeks postoperatively. The mRNA relative expression level of Myogenin, MUSK, nAChR, P21and MRF4in MGS increased initially, peaked at4-6weeks postdenervation, and then decreased to a low level.Conclusion The maximal fibrillation potential amplitude, muscle weight, muscle CAS, amount of connective tissues, muscle fiber phenotyping, mRNA relative expression levels of Myogenin, MUSK, nAChR, P21and MRF4had specific and obvious changes after denervation. These changes were closely related to the time course of denervation, being most significant at4-6weeks postdenervation. Part3Functional Recovery of Target Muslces after Nerve Repair with Various Delay Intervals and Identification of Sensitive Prognostic MarkersObjective To compare the functional recovery of target muscles after nerve repair with various delay intervals and identify markers that are sensitive for predicting prognosis of nerve reconstruction.Methods43female Lewis rats were subjected to sciatic nerve transection. Nerve repair was done at different time intervals according to group assignment:1week delay (B1, n=8),4week delay (B2, n=8),6week delay (B3, n=8),8week delay (B4, n=8) and12week delay (B5, n=3). Sciatic nerve transection and immediate repair group (B0, n=8) served as control. Compound muscle action potential (CMAP) was tested post-operatively every four weeks. Maximum isometric force measurement, histologic examinations of nerves and muscles, gene expression levels were acquired at16weeks post-operatively. By analyzing the relationships between functional recoveries and changes in denervated muscles, we set out to identify sensitive prognostic markers that could predict the functional recovery of delayed nerve repair.Results Functional evaluations at16weeks post-nerve reconstruction including CMAP, muscle wet weight, maximum isometric muscle force, myelinated nerve fibers count and diameter, axon denstity and axon diameter in group B1and B2were as good as those in the immediate nerve repair group, but were significantly better than those in groups B3, B4and B5(P＜0.05). The cross sectional areas (CSA) of tibialis anterior (TA) and medial gastrocnemius (MGS) in immediate and1week delay repair groups were larger than those of6,8and12week delay repair groups (P＜0.05). The proportion of muscle phenotyping, the relative expression levels of MRF4, Myogenin, nAChR, P21and MUSK mRNA were not different between various groups (P>0.05). The maximum fibrillation potential amplitude, CSA of TA and MGS, connective tissue amount of TA and MGS, the proportion of type ⅡB and ⅡA muscle fibers, and nAChR mRNA relative expression level had a linear correlation with the functional recovery after nerve reconstruction. Conclusion Delayed nerve repair with various intervals resulted in various extents of functional recovery. The longer the interval, the worse the recovery. Functional recovery in1and4week delayed repair groups did not differ from that in the immediate nerve repair group, but was significantly better than that in groups of longer delays. The maximum fibrillation potential amplitude, CSA, connective tissue content, proportion of muscle fiber type ⅡB and ⅡA and nAChR mRNA relative expression level were sensitive markers which could predict the functional recovery after delayed nerve repair. |
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