| This study aimed to investigate the difference on the maximum number of nerve fibers amplification of musculocutaneous nerve and ulnar nerve after peripheral nerve injury. Three parts were included in the study.With the results harvested by vary methods, we try to provide a convincing process for the establishment of animal model which could be taken into consideration for research on nerve amplification in upper limb of SD rats. On the other hand, we hypothesized that the ratio of nerve fibers compensation amplification on musculocutanous nerve were higher than that of ulnar nerve.Part1Establishment of animal model for comparative research on the maximum number of nerve fibers compensation amplification for ulnar nerve and musculocutanous nerve on the ratObjective To compare the efficacy and reliability of True-Blue、Fluo-Emerald and Fluo-Ruby retrograde tracing fluorescent agent, and find the details of distribution of motor neurons and sensory neurons of ulnar nerve and musculocutaneous nerve in cornu anterius medullae spinalis and dorsal root ganglions （DRG）. With the outcomes, we try to establish an animal model on the rat which could be taken into consideration for researching the difference on nerve fibers compensation amplification of ulnar nerve and that of musculocutaneous nerve.Materials and Methods60healthy adult female Sprague-Dawley rats that weighed150-200g, were seperated into six groups at random （10in each group）:for Ulnar nerve,U-TB、U-FE U-FR indicates True-Blue,Fluo-Emerald, and Fluo-Ruby retrograderacing group; for musculocutaneous nerve, M-TB、M-FE、M-FR indicates True-Blue, Fluo-Emerald and Fluo-Ruby retrograde tracing group respectively.. Values were expressed by mean±SD. Statistical analyses were performed using one-way ANOVA to analyze the distribution of the neurons and the arranging rate of spinal roots innervating ulnar nerve and musculocutaneous nerve, and the potential disparity of these three retrograding tracing agents. Differences were considered significant at p<0.05.Results （1） For U-TB group, a total number of2176±186.51blue-labelled neurons were seen from C7-T1. There were5.2±2.44（0-9）,157±29.8and150±21motor neurons located in anterior horn of C7,C8and T1respectively;31±11.32,1203.1±113.71, and651.3±78.47sensory neurons were seen in corresponding DRGs.（2） For U-FE group, green-labelled neurons were seen from C7to T1with a total number of2268.7±209.57. There were5.6±2.41（0-8）,150±19.9, and142.4±26.25motor neurons located in anterior horn of C7, C8and T1respectively;34±7.73,1305±194.8and631.2±43.92sensory neurons were seen in the corresponding DRGs.（3） For U-FR group, a total number of2216±211.56red-labelled neurons were seen from C6to T1.There were4.4±2.22（2-9）,135±23.9and153.9±18.19motor neurons located in anterior horn of C7, C8and T1, while no stained ones were found in C6.0～55,28.6±11.79,1224.5±148.74, and669.7±97.42sensory neurons were seen in corresponding DRGs from C6to T1.（4） For M-TB group, motor neurons and sensory neurons located from C4to C7segment, the total number of blue-lablled neurons was1871±120.53.There were up to11motor neurons loacted at the anterior horn of C4, and7.8±4.05sensory neurons in corresponding doral root ganglion;164±16.97motor neurons loacted at the anterior horn of C5, and253.3±36.96sensory neurons in corresponding DRG;149±25.8motor neurons loacted at the anterior horn of C6,904.6±104.92sensory neurons in corresponding DRG; up to6motor neurons loacted at the anterior horn of C7,386.5±28.77sensory neurons in corresponding DRG. No stained neurons were seen in C8、T1.（5） For M-FE group, motor neurons and sensory neurons were seen from C5to C7segments, the total number of yellow-labelled neurons was1937.9±311.22. There were171.2±49.31,143±28.6, and up to7motor neurons in anterior horn of C5,C6and C7respectively;244.4±25.49,985.1±257.57, and291.7±51.36sensory neurons were seen in the DRG of C5,C6and C7respectively. No staining neurons were found in C8and T1.（6） For M-FR group, a total number of1902±212.64red-labelled neurons were seen in the area of anterior horn and DRG from C4to C8segments. There were up to12,151.8±18.38,132.7±16.64, up to15and up to2motor neurons in anterior horn of C4, C5, C6, C7and C8respectively;0-15,240±30.4,947±203,412.6±46.94, and up to4sensory neurons located in corresponding DRG of C4to C8respectively. No neurons in T1were stained.（7） True-blue, Fluo-emerald and Fluo-ruby had equal efficacy in labeling neurons, with the results that no differences were found in the distribution and arranging of neurons innervating the musculocutaneous nerve or ulnar nerve. Based on the outcomes of Fluo-emerald retrograde tracing, neurons innervating musculocutaneous nerve located in segments from C5to C7,22.1percent of the total number of neurons were located in C5,62.5and15.4percent of them located in C6and C7respectively. For ulnar nerve,34.1percent of the total number of neurons located in T1,1.7and64.2percent of them located in C7and C8. Furthermore, most of the neurons in C7were sensory ones.Conclusion It is reasonable to establish a model for research on nerve fibers compensation amplification on ulnar nerve, with method of keeping the integrity of C5root and cutting the rest ones of brachial plexus, added by end-to-end repair of ulnar nerve. Likewise, keeping the continuity of T1and cutting the rest roots of brachial plexus, added by end-to-end repair of musculocutaneous nerve were adopted to establish the model for musculocutaneous nerve fibers compensation amplification. Part2A comparative study on the maximum number of nerve fibers compensation amplification of ulnar nerve and musculocutaneous nerve after nerve injuryObjective To compare the ratio of nerve fibers compensation amplification of ulnar nerve with that of musculocutaneous nerve.Materials and Methods224SD rats healthy adult female Sprague-Dawley rats that weighed150-200g, were randomly separated into sixteen groups, indicating1week,2、3、4、8、2、16and20weeks post establishment of animal model（8in each group） for ulnar nerve and that for musculocutaneous nerve respectively, the corresponding nerves in contralateral limbs were served as normal control ones.6rats underwent transection served as denervated group for each group. Myelinated nerve fibers and motor fibers of proximal and distal stump of ulnar nerve and musculocutaneous nerve were calculated; the amplification ratio was expressed by the number of nerve fibers in distal stump divide that of proximal one. For each segment, number of mean diameter of the fibers and axons, mean thickness of the myelin sheaths, and G ratio were determined. For each nerve, amplitude and latency of CMAP were determined. Results （1）The number of myelinated nerve fibers of normal musculocutanous nerve and ulnar nerve was2135±197and2535±238:the number of motor fibers was395±47and335±34respectively.（2）The number of myelinated nerve fibers of proximal stump of ulnar nerve was653.23±102.71,703.79±84.26,772.40±54.93and593.41±39.36:and that of distal stump was979.72±162.73,97235±93.30,1124.59±217.27and1980.33±216.53at the time of1week,2,3,and4weeks post surgical procedure；the compensation amplification ratio was2.20±0.53,1.41±0.24,1.57±0.39and3.34±0.51；The number of myelinated nerve fibers of proximal stump ulnar nerve was725.92±132.93,671.80±112.25,553.52±64.71and682.51±107.42,and that of distal stump was2217.72±419.84,1677.29±254.92,1844.19±202.90and1953.13±412.17at the time of8,12,16,and20weeks post surgical procedure,the compensation amplification ratio was3.01±0.44,2.76±0.37,3.15±0.46and2.31±0.98.（3）The number of myelinated nerve fibers of proximal stump of musculocutanous nerve was352.75±40.31,393.49±59.27,379.81±60.52and350.61±30.58at the time of lweek,2,3, and4weeks post surgical procedure；and that of distal stump was1079.754±242.41,477.36±83.55,453.75±76.59and1434.77±312.93.The compensation amplification ratio was4.77±1.25,1.21±0.47,1.19±0.32and4.09±0.73respectively. The number of myelinated nerve fibers of proximal stump musculocutanous nerve was387.68±52.74,362.48±33.45,372.07±43.28and363.25±37.42at the time of8week,12weeks,16weeks,and20weeks post surgical procedure,and that of distal stump was1798.60±345.37,1566.19±217.04,1549.96±198.47and1479.31±174.58,the compensation amplificationratio was4.71±1.32,4.29±0.79,4.23±0.65and4.34±0.49respectivel y.（4）For ulnar nerves,the number of motor fibers of proximal nerve stump was212.61±43.82,192.43±27.55,200.92±35.27and183.90±27.44,and that of distal stump was20.55±7.13,12.38±4.31,19.39±7.30and20.09±7.59,at the time of1week,2weeks,3weeks,and4weeks post surgical procedure,the compensation amplification ratio was0.094±0.031,0.0574±0.012,0.1104±0.031and0.102±0.037respectively.The number of motor nervers bers of proximal nerve stump was190.37±25.38,222.61±42.73,208.52±74.75and231.45±77.19,and that of distal stump was120.59±37.30,219.91±50.48,277.40±47.65and291.80±49.92at the time of8,12,16,and20weeks post surgical procedure respectively.The compen-sation amplification ratio was0.72±0.169,1.06±0.242,1.48±0.45and 1.45±0.317respectively.（5） The number of motor fibers of proximal musculocutan-eous nerve stump was235.41±32.70,229.74±61.53,202.72±55.09, and199.61±28.78, and that of distal stump was7.35±4.39,10.95±3.18,19.57±7.02and40.60±9.17at the time of1week,2,3, and4weeks post surgical procedure. The compensation amplification ratio was0.031±0.0184,0.045±0.0287,0.091±0.0221and0.195±0.049respectively. The number of motor nerve fibers of proximal musculocutanous nerve stump was254.10±47.75,218.06±27.80,231.44±30.40and230.51±0.06, at the time of8,12,16, and20weeks post surgical procedure, and that of distal stump was180.57±41.50,290.82±49.52289.53±44.47and277.49±46.59respectively, the compensation amplification ratio was0.774±0.216,1.26±0.35,1.36±0.27and1.209±0.411.（6） The diameter of nerve fibers of distal nerve stump was5.76±1.21and4.28±0.84μm, and that of axons was4.01±1.08and3.14±0.11μm; the thickness of myelin sheaths was0.72±0.24and0.53±0.19μm, the G ratio was0.73±0.12and0.62±0.09for musculocutanous nerve and ulnar nerve respectively.（7）No sign of electronic reaction was detected within2weeks after procedure in both nerves. The CMAP amplitude and latency in normal control group of ulnar nerve was50±0.22ms and6.77±1.62mV, and1.24±0.27ms and9.46±2.33mV in musculocutaneous nerve. Compared with musculocutaneous nerve,the recovery rate of CMAP amplitude and latency was lower in ulnar nerve20weeks after establishment of model, P<0.05.Conclusion At the time of20weeks post surgical procedure,（1） The compensation amplification ratio of motor fibers had no difference between musculocutanous nerve and that of ulnar nerve （P>0.05）;（2）The compensation fiber amplification ratio of sensory fibers in ulnar nerve was lower than that of musculocutanous nerve （P<0.05）;（3） The average diameters of nerve fibers and axons, and the thickness of myelin sheaths were lower in ulnar nerve （P<0.05）.（4） The recovery rate of CMAP amplitude and latency were lower in ulnar than that of musculocutanous nerve （P<0.05） Part3A comparative study of the changes in neurotrophic factor expression in distal stump of ulnar nerve and musulocutaneous nerve on the model of nerve fibers compensation amplification Objective To investigate the changes in neurotrophic factor expression in distal stump of ulnar nerve and musulocutaneous nerve and its relation with the amplification ratio of sensory fibers.Materials and Methods224healthy adult female Sprague-Dawley rats that weighed150-200g, were randomly seperated into sixteen groups. U-1, U-2、U-3、U-4、 U-8、U-12、U-16and U-20indicating1week,2、3、4、8、12、16and20weeks post estalblishment of animal model （8in each group） for ulnar nerve. M-1、M-2、M-3、 M-4、M-8、M-12、M-16and M-20for that of musculocutaneous nerve. The corresponding nerve in contralateral limbs were served as control ones.6rats underwent transection served as denervated group for each group respectively. Real-time PCR of BDNF mRNA, GDNF mRNA, CNTF mRNA, NGF mRNA in the distal stump of ulnar nerve and musculocutaneous nerve were performed at each time point.Results An one-way ANOVA （P<0.001） followed by post hoc Turky test revealed increased mRNA more than0.1μg/mg were detected from distal nerve stumps for all operated groups, among which0.38～0.46μg/mg were detected in groups post procedure within4weeks. The amount of RNA per mg tissue returned to normal by20weeks after operation.For ulnar nerve:（1） There were significant higher NGF mRNA levels in model groups and denervated ones within4weeks （P<0.05）, declining thereafter in all groups.（2） BDNF mRNA were rarely detected in control groups. In model groups, copy numbers of BDNF mRNA elevated, reaching a peak at3weeks after operation, and declining sharply afterward. In denervated groups, BDNF levels peaked higher than in model groups （P<0.05）.（3） CNTF mRNA levels were highly expressed in control groups, and declined significantly1week after operation, reaching a peak at20weeks below control levels in model groups （P<0.05）. In denervated group, CNTF mRNA levels were significantly lower than that of model groups4weeks after operation.（P<0.05）.（4） Also like BDNF, GDNF mRNA were rarely detected in control groups. BDNF copies elevated higher in denervated groups than model groups after transaction at stable levels from1week to4weeks （P<0.05）, declining gradually afterward with no significant difference from each other.For musculocutaneous nerve:（1） There were significant differences in levels of NGF mRNA between groups within3weeks, declining thereafter in all groups.（2） BDNF mRNA were rarely detected in control groups, whereas BDNF mRNA levels elevated in model groups, reaching a peak at3weeks, and declining after8weeks after operation. Meanwhile, there were significantly higher levels were found in denervated groups （P<0.05）.（3） CNTF mRNA levels were highly expressed in control groups, and declined significantly1week after operation, reaching a peak at20weeks below control in model groups. In denervated group, CNTF mRNA levels were significantly lower than that of model groups4weeks after operation （P<0.05）.（4） GDNF mRNA were rarely detected in control groups. GDNF mRNA copy numbers were detected in denervated groups as well as model groups with a declining trend （P<0.05）Compared with the musculocutaeous nerve, BDNF mRNA levels were lower in ulnar in each time point （P<0.05）; GDNF mRNA levels had no differences between the two nerves in each time point, P>0.05; NGF mRNA levels were the same within3weeks, however, lower levels were detected at the time of4,8and12weeks, P<0.05; the expression of CNTF mRNA were lower in ulnar nerve since3weeks after procedure, P<0.05.Conclusion The lower levels of BDNF mRNA and NGF mRNA in distal stump of ulnar nerve, was the cause of lower amplification ratio of sensory fibers compared with that of musculocutaneous nerve. Athough a similar change was found in the expression of CNTF mRNA, further studies were needed to explain its relation with the discrepancy of amplification ratio of sensory fibers in these two nerves. The change of GDNF mRNA levels had no association with the potential of amplification ratio of sensory fibers. |
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