Root avulsion of C5-C6 of the brachial plexus is

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1 Special column of 10th anniversary Functional compensative mechanism of upper limb with root avulsion of C 5 of brachial plexus after ipsilateral transfer SONG Jie 宋捷, CHEN Liang 陈亮 * and GU Yudong 顾玉东 Objective: To investigate the compensative mechanism of no further impairment of the upper limb after ipsilateral transfer for treatment of root avulsion of C 5 of the brachial plexus. Methods: Sixty Sprague Dawley (SD) rats were randomly divided into a transection group and a control group, 30 rats each. In the transection group, the left forelimbs of the animals underwent transection of ipsilateral nerve root while C 5 nerve roots were avulsed. In the control group, the left forelimbs only underwent C 5 root avulsion. The representative muscles of (innervated mainly by ) including latissimus dorsi, triceps, extensor carpi radialis brevis and extensor digitorum communis were evaluated with neurophysiological investigation, muscular histology and motor end plate histomorphometry 3, 6 and 12 weeks after operation. The right forelimbs of all rats were taken as the control sides. Results: Three weeks after operation, the recovery rates of amplitudes of compound muscle action potential (CMAP) and CMAP latency, muscular wet weight and crosssectional area of muscle fibers, and area of postsynaptic membranes of those four representative muscles in the transection group were significantly lower than those of the control group (P <0.05 or P <0.01). Six weeks postoperatively, the recovery rates of CMAP amplitude and latency of the triceps showed no significant difference between the transection group and the control group (P>0.05). For the Department of Hand Surgery, Huashan Hospital, Fudan University, Shanghai , China (Song J, Chen L and Gu YD) *Corresponding author: Tel: , lchenhkq@online.sh.cn This study was supported by the National Natural Science Foundation of China (No ) and National Key Basic Research Development Plan ( 973 Item, No. 2003CB515305). extensor carpi radialis brevis and the extensor digitorum communis, the recovery rates of the crosssectional area of muscle fibers, the amplitude and latency of CMAP and the area of postsynaptic membranes showed no significant difference between the two groups (P >0.05), while the rest parameters were still significantly different between the two group (P <0.05 or P <0.01). As far as the ultramicrostructure was concerned in the transection group, more motor end plates of four representative muscles were observed and their ultramicrostructure also had a tendency to mature as compared with those of 3 weeks postoperatively. Twelve weeks after operation, all parameters of the transection group were not significantly different from those of the control group (P >0.05). In the transection group, the motor end plates were densely distributed and their ultramicrostructure in four representative muscles appeared to be mature as compared with those of the control group. Conclusions: After ipsilateral transfer for treatment of root avulsion of C 5 of the brachial plexus, the nerve fibers of the lower trunk can compensatively innervate fibers of representative muscles by means of motor end plate regeneration, so there is no further impairment on the injured upper limb. Key words: Nerve injuries; Brachial plexus; Spinal nerve roots; Nerve transfer; Rats Chin J Traumatol 2008; 11(4): Root avulsion of C5C6 of the brachial plexus is a common lesion in clinical practice. Presently, the conventional treatment is multiple nerve transfers. There are three generally accepted nerve transfer procedures at home and abroad: (1) the phrenic nerve transferred to the musculocutaneous nerve (or the anterior division of the upper trunk), the spinal accessory nerve to the suprascapular nerve, the motor

2 fibers of the cervical plexus to the posterior division of the upper trunk (or the axillary nerve); 1 (2) partial fascicles of the ulnar nerve transferred to the biceps nerve (Oberlin s procedure), the spinal accessory nerve to the suprascapular nerve, and the long head branches of the triceps to the anterior branch of the axillary nerve; 2 and (3) the phrenic nerve transferred to the musculocutaneous nerve (or the anterior divisions of the upper trunk) and the spinal accessory nerve to the suprascapular nerve 3. Gu et al. 4 (2003) applied transfer of ipsilateral nerve root in four patients with root avulsion of C 5 of the brachial plexus. Two of them underwent additional treatment of the spinal accessory nerve transferred to the suprascapular nerve. After followup for 12 years, the muscle force of the biceps recovered to M4 in all the patients. Two patients who received spinal accessory nerve transfer gained shoulder abduction >90, while the other two patients who did not receive spinal accessory nerve transfer only gained shoulder abduction of However, this procedure could decrease the power of representative muscles of root (the latissimus dorsi, the triceps brachii, and the extensor digitorum communis) by 1 degree (MRC) within 12 weeks after operation, but 46 months postoperatively, the muscle power could finally recover to normal. 45 Till now, we have found no related researches in literature to study the compensatory mechanism of the upper limb after ipsilateral transfer for treatment of C 5 root avulsion. Since the anatomical structure and functional innervation of brachial plexus in rats are similar to those in human beings, rats have been taken as ideal animal models for study of the reparation of brachial plexus injury. 67 In this study, we investigated the ipsilateral transfer for treatment of root avulsion of C 5 of the brachial plexus in rats to provide theoretical evidences for generalizaion of this surgical procedure. METHODS Animals and grouping Sixty adult Sprague Dawley (SD) rats (provided by the Experimental Animal Division of Fudan University, Shanghai, China ), regardless of gender or body weight, were randomly divided into a transection group (n=30) and a control group (n=30). The transection group underwent ipsilateral nerve root transection while the roots of C 5 were avulsed. The control group underwent C 5 root avulsion and the ipsilateral root was kept intact. Ten rats were selected respectively to determine the results by neurophysiological investigation, muscular histology and motor end plate histomorphometry for the representive muscles of 3, 6 and 12 weeks after operation. Establishment of animal model The rats were fastened supine and anesthetized by intraperitoneal injection of 1% pentobarbital sodium (5 mg/100 g body weight). A cervical incision was made on the midline. After the pectoralis major was incised along the arcuate line, the pectoralis minor was distracted laterally, and the C 5 nerve roots were exposed between the anterior and middle scalenus muscles. After the C 5 nerve roots were avulsed, the ipsilateral root was truncated for a 3mm section (blocked by 1% procaine), and the neural stumps were sutured to prevent them to regenerate. Then 0.1 g penicilin powder was administered before closing all incisions. The left forelimbs of rats in both groups were taken as the experimental sides and the right forelimbs as the control sides. Determination of parameters Compound muscle action potentials (CMAP) Neurophysiological investigation of the representative muscles of, i.e., the latissimus dorsi, the triceps, the extensor carpi radialis brevis and the extensor digitorum communis, 8 was performed by DantecNeuromatic2000 Electromyograph. Maximal current of 2.6 ma and single squarewave of 0.2 ms were used to stimulate the thoracodorsal nerve and radial nerve. The needle electrodes were inserted for 2 mm in depth for the four representative muscles. The stimulating and recording electrodes were placed in the same position at both forelimbs of the rats, and the CAMP amplitude and latency were recorded. Muscle wet weight and crosssectional area of muscle fiber The four representative muscles were harvested completely by removing the connective tissues, then weighed by an Analytic Balance (the verification scale interval for 1 mg, R200D, Germany). And 5 µmthick transverse sections of those muscle samples were stained by hematoxylin and eosin (HE), and Q WIN image analysis system (Leica) was used. A total of 5 fields were randomly selected under a zoom eyepiece (40 ), in which crosssectional areas of the muscle fibers were measured and the meanvalues were calculated.

3 Synapses distribution and postsynaptic membrane area of muscles A total of 8mm 3 tissues of the nerve entry point into the muscle were selected to make longitudinal frozen sections. Fixed by paraformaldehyde and rinsed in 0.01 mol/l PBS for 3 times, 1 µg/mlαbungarotoxin (αbtx) was incubated at room temperature and blocked the light ray for 1 hour, then rinsed in 0.01 mol/l PBS for 3 times, and measured by immunofluorescence staining. From each muscle, three sections were randomly selected and five fields were randomly chosen to observe the distribution features of the motor end plates under a fluorescent microscope (400 ). Two to five fields of postsynaptic membranes were selected with clear morphology and the overall areas of them were calculated by QWIN imaging analysis system. The average area was obtained by the overall areas divided by the number of the postsynaptic membranes. The values of the control sides were taken as 1, so that the recovery rates of the left sides could be obtained by the parameters of the left forelimbs divided by those of the right forelimbs. Data results were expressed as the median (m) and quartile (p25p75), and those recovery rates between the transection group and the control group were compared by Wilcoxon signed rank test. P value <0.05 was considered as the significant level. Ultramicrostructure of the muscles A total of 1mm 3 tissues of the nerve entry point into the muscle was fixed by 2.5% glutaraldehyde, dehydrated by ethanol and propanone, then infiltrated by resin618. After embedding and solidification, 5060 nm ultrathin sections were made and doubledyed by 3% uranyl acetatelead citrate. The morphology of motor end plates of the skeletal muscle, i.e., the structure of the myofilaments of the anterior synaptic membrane, the postsynaptic membrane and the sarcomere, was observed under a transmission electron microscope (CM120, Philips, Holland). RESULTS Histology of muscles Three weeks after operation, the recovery rates of wet weight and crosssectional area of muscle fibers of four representative muscles in the transection group were significantly lower than those of the control group (P<0.01). Six weeks postoperatively, the recovery rates of crosssectional area of muscle fibers of extensor carpi radialis brevis and extensor digitorum communis between the two groups had no significant difference (P>0.05), while the recovery rates of crosssectional area of the latissimus dorsi and the triceps and wet weight of four representative muscles in the transection group were still significantly lower than those of the control group (P<0.05 or P<0.01). And twelve weeks after operation, except that the recovery rate of wet weight of the latissimus dorsi in the transection group was lower than that of the control group (P<0.05), the recovery rate of crosssectional area of muscle fibers in the latissimus dorsi, the recovery rate of wet weight and crosssectional area of muscle fibers of the other three representative muscles had no significant difference between the two groups (Tables 1 and 2). Neurophysiological investigation Three weeks after operation, the recovery rates of CMAP latent period and amplitude of four representative muscles in the transection group were significantly lower than those of the control group (P <0.05 or P<0.01). Six weeks postoperatively, except that the recovery rates of CMAP latent period and amplitude of the latissimus dorsi in the transection group were significantly lower than those of the control group, the recovery rates of CMAP latent period and amplitude in other three representative muscles had no significant difference between the two groups (P>0.05). And twelve weeks after operation, there was no significant difference between the two groups in the recovery rates of CMAP latent period and amplitude (Tables 3 and 4). Distribution of motor end plates and area of postsynaptic membrane Three weeks after operation, motor end plates of four representative muscles were distributed sparsely in the transection group (Fig. 1A). The recovery rate of the postsynaptic membrane area was significantly lower in the transection group than the control group (P<0.01). Six weeks postoperatively, the number of motor end plates in four representative muscles of the transection group increased gradually. Except for the significant lower recovery rate of postsynaptic membrane area of the latissimus dorsi and the triceps between the transection group and the control group (P<0.05 or P<0.01), no significant difference of the recovery rate of postsynaptic membrane area of the exten

4 sor carpi radialis brevis and the extensor digitorum communis was observed between the two groups (P>0.05). And twelve weeks after operation, motor end plates of four representative muscles in the transection group were densely distributed (Fig. 1B). The recovery rate of postsynaptic membrane area in the transection group had no significant difference compared with that of the control group (P>0.05, Table 5). Ultramicrostructure of motor end plates Three weeks after operation, abnormal ultramicrostructure of the motor end plates of four representative muscles was found in the transection group, i.e., immature morphology, smaller but integral microstructure in the anterior synaptic membrane area, fewer folds and absence of deep grooves in the postsynaptic membrane, narrower synaptic cleft, irregularly ordered myocomma and myofilament. The motor end plates in part of the latissimus dorsi and the triceps were still in a primary stage of morphology in the transection group (Fig. 2A). In the control group, except that the immature morphology of few latissimus dorsi muscle, most motor end plates of other three muscles were almost mature. And twelve weeks after operation, the morphology of the motor end plates of four representative muscles had recovered maturely in the transection group, i.e., large area of anterior synaptic membrane containing chondriosomes, microtubules and abundant synaptic vesicle, deep folds in postsynaptic membrane, clear synaptic cleft, wellordered myocomma and myofilament without swelling or disruption, and wellorganized linem and linez with few muscle satellite cells (Fig. 2B). Fig. 1. Immunofluorescence staining under fluorescent microscope in the transection group (scale bar for 20 µm). A: Three weeks after operation, the motor end plates of the latissimus dorsi are distributed sparsely and the amount of them is little. B: Twelve weeks postoperatively, the motor end plates of the latissimus dorsi are distributed more densely and the amount of them is larger. Fig. 2. Uranyl acetatelead citrate doublestaining under transmission electron microscope in the transection group. Solid arrows show the anterior synaptic membrane and hollow arrows show the postsynaptic membrane (scale bar for 1 µm). A: Three weeks after operation, the motor end plates of the latissimus dorsi are morphologically immature, smaller but integral in the anterior synaptic membrane area and have less folds and are absent of deep grooves in the postsynaptic membrane. B: Twelve weeks postoperatively, the regenerated motor end plates of the latissimus dorsi tend to be morphologically mature with larger area of anterior synaptic membrane containing chondriosomes, microtubules and abundant synaptic vesicle, more deep folds in postsynaptic membrane and clear synaptic cleft. DISCUSSION Presently, three kinds of nerve transfer procedures treating root avulsion of C 5 of the brachial plexus can only provide quite limited regenerative myelinated nerve fibers: partial fascicles of the ulnar nerve, the spinal accessory nerve and branches to the long head of the triceps providing myelinated fibers totally, and the phrenic nerve, the spinal accessory nerve and motor branches of the cervical plexus providing myelinated fibers at most. 910 As the maximum number of regenerative neuraxons from all above nerves only accounts for 20% of C 5 nerve roots (approximately containing more than fibers), the effects of those three nerve transfers are not ideal. Because the nerve root contains almost myelinated nerve fibers 9 and its corresponding cortical area approximates to that of C 5 roots, the effect of ipsilateral transfer in treating C 5 root avulsion is well expectable, which has been applied widely in clinic It has been well documented by the followup materials that ipsilateral transection had no apparent impairment on the injuredextremity in patients with root avulsion of C 5 of the brachial plexus. 4 Lu et al. 13 found that two weeks after transection of upper and middle trunks of brachial plexus of rats, CMAP amplitude of the ipsilateral latissimus dorsi muscle and triceps decreased dramatically, and CMAP latency of those two tested muscles were obviously prolonged. However, 2 months postoperatively, the above two parameters had recovered to normal. In this study, the histological and electrophysiological

5 changes of four representative muscles of (the latissimus dorsi, the triceps brachii, the extensor carpi radialis brevis and the extensor digitorum communis) after ipsilateral root transection on the condition of C 5 C 6 root avulsion showed that in the early stage after operation, the transection group had reduced wet weights and crosssectional areas of muscle fibers as well as declined CAMP amplitudes and prolonged latencies of the four representative muscles of root as compared with the control group. However, 12 weeks postoperatively, the recovery rates of all parameters determined were similar to those of the control group. The experimental result was consistent with the clinical appearance that we had observed, indicating that this animal experiment has a well clinical simulation. The immunofluorescent staining and electron microscopic morphological results of motor end plates of four representative muscles in transection group demonstrated that, 3 weeks after operation, motor end plates were distributed sparsely and the area of postsynaptic membrane was reduced. The regenerated motor end plates had a smaller size but an integral structure. The anterior synaptic membrane was atrophic and the postsynaptic membrane had less and superficial folds. The synaptic cleft was narrower and the myocomma and myofilament were not clear (Fig. 2A). However, 12 weeks postoperatively, more synapses in transection group were observed and the recovery rate of postsynaptic membrane area in transection group was near to the level in the control group. In the transection group, the ultramicrostructure of the motor end plates regenerated by axon sprouting tended to mature, for example, abundant synaptic vesicles were found in the anterior synaptic membrane, and affluent folds were observed in the postsynaptic membrane, so did clear synaptic cleft, ordered myocomma and myofilament (Fig. 2B). The results suggested that after transection of ipsilateral root with root avulsion of C 5 of the brachial plexus, the nerve fibers of the lower trunk could reconstruct functional neuromuscular junction of the denervated muscle fibers in an endingregeneration way and finally played a compensative role in innervating the representative muscles of. Therefore, there would be no further functional impairment on the injured upper limb. Table 1. Comparison of recovery rate of wet weight of representative muscles in the two groups [%, M(p25p75)] Latissimus dorsi Triceps Extensor carpi radialis brevis Extensor digitorum communis 3* 6* 12* 3* 6* 12* 3* 6* 12* 3* 6* 12* A 50.28( ( ( ( ( ( ( ( ( ( ( ( ) ) 79.10) 91.92) ) 75.05) 89.46) ) 84.53) ) B 80.36( ( ( ( ( ( ( ( ( ( ( ( ) ) 89.01) 98.76) ) 92.74) 99.40) ) 92.74) 99.40) ) Z value P value <0.01 <0.01 <0.05 <0.01 <0.05 >0.05 <0.01 <0.05 >0.05 <0.01 <0.01 >0.05 Notes: *: weeks after operation. Group A: transection group, ipsilateral root transection + root avulsion of C 5 of the brachial plexus; Group B: control group, simple root avulsion of C 5 of the brachial plexus. Wilcoxon signed rank test is used to test the variance of the recovery rate of the two groups. Statistical significance is defined as P<0.05. Number of rats in Group A 12 weeks postoperatively was 9 and number of those in Group B 6 weeks postoperatively was 10, who were used in other time periods in both groups.

6 Table 2. Comparison of recovery rate of area of crosssectional muscle fibers of representive muscles in the two groups [%, M(p25p75)] Latissimus dorsi Triceps Extensor carpi radialis brevis Extensor digitorum communis 3* 6* 12* 3* 6* 12* 3* 6* 12* 3* 6* 12* A 45.59( ( ( ( ( ( ( ( ( ( ( ( ) 76.11) 97.43) 72.98) 78.78) 97.53) 86.80) 95.00) 99.50) 88.30) 95.40) 99.50) B 75.46( ( ( ( ( ( ( ( ( ( ( ( ) 90.45) 99.23) 85.60) 94.55) 99.78) 94.00) ) ) 95.80) ) ) Zvalue Pvalue <0.01 <0.01 >0.05 <0.01 <0.01 >0.05 <0.01 >0.05 >0.05 <0.01 >0.05 >0.05 Table 3. Comparison of recovery rate of CAMP latent period of representative muscles in the two groups [%, M(p25p75)] Latissimus dorsi Triceps Extensor carpi radialis brevis Extensor digitorum communis 3* 6* 12* 3* 6* 12* 3* 6* 12* 3* 6* 12* A ( ( ( ( ( ( ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) ) ) ) B ( ( ( ( ( ( ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) ) ) ) Z value Pvalue <0.05 <0.05 >0.05 <0.05 >0.05 >0.05 <0.05 >0.05 >0.05 <0.05 >0.05 >0.05 Table 4. Comparison of the recovery rate of CMAP amplitude of representive muscles in the two groups [%, M(p25p75)] Latissimus dorsi Triceps Extensor carpi radialis brevis Extensor digitorum communis 3* 6* 12* 3* 6* 12* 3* 6* 12* 3* 6* 12* A 50.39( ( ( ( ( ( ( ( ( ( ( ( ) 73.43) 97.44) 68.20) 88.80) ) 79.87) 94.07) ) 89.87) 93.78) ) B 70.56( ( ( ( ( ( ( ( ( ( ( ( ) 93.43) ) 91.20) 99.30) ) 92.90) 96.29) ) 92.90) 96.29) ) Zvalue Pvalue <0.01 <0.01 >0.05 <0.01 >0.05 >0.05 <0.01 >0.05 >0.05 <0.05 >0.05 >0.05

7 REFERENCES Table 5. Comparison of recovery rate of postsynaptic membrane area of representive muscles in the two groups [%, M (p25p75)] Latissimus dorsi Triceps Extensor carpi radialis brevis Extensor digitorum communis 3* 6* 12* 3* 6* 12* 3* 6* 12* 3* 6* 12* A 47.17( ( ( ( ( ( ( ( ( ( ( ( ) 64.61) 96.26) 62.77) 85.57) 97.35) 81.67) 91.92) 99.89) 85.05) 95.92) 99.89) B 74.22( ( ( ( ( ( ( ( ( ( ( ( ) 89.55) 98.89) 86.90) 91.01) 99.27) 92.64) 97.95) ) 93.16) 97.53) ) Zvalue Pvalue <0.01 <0.01 >0.05 <0.01 <0.05 >0.05 <0.01 >0.05 >0.05 <0.01 >0.05 > Gu YD. Diagnosis and treatment of brachial plexus injury. 2nd ed. Shanghai: Publishing House of Shanghai Medical University, 2001: Bertelli JA, Ghizoni MF. Reconstruction of C 5 brachial plexus avulsion injury by multiple nerve transfers: spinal accessory to suprascapular, ulnar fascicles to biceps branch, and triceps long or lateral head branch to axillary nerve. J Hand Surg Am 2004;29(1): Birch R, Bonney G, Wynn Parry CB. Surgical Disorders of the Peripheral Nerves. Edinburgh: Churchill Livingstone, 1998: Gu YD, Cai PQ, Xu F, et al. Clinical application of ipsilateral nerve root transfer for treatment of C5 and C6 avulsion of brachial plexus. Microsurgery 2003; 23(2): Xue F, Cai PQ, Gu YD, et al. Initial observations of effects of ipsilateral root transferc on its innervated muscles. Chin J Hand Surg 2001;17(3): Bertelli JA, Mira JC, Gilbert A, et al. Anatomical basis of rat brachial plexus reconstruction. Surg Radiol Anat 1992;14(1): Chen L, Gu YD. An experimental study of contralateral root transfer with vascularized nerve grafting to treat brachial plexus root avulsion. J Hand Surg Br 1994;19(1): Gu YD, Shen LY. Electrophysiological changes after severance of the nerve root. J Hand Surg Br1994;19(1): Gilbert A, Pivato G, Kheiralla T. Longterm results of primary repair of brachial plexus lesions in children. Microsurgery 2006; 26(4): Witoonchart K, Leechavengvongs S, Uerpairokjkit C, et al. Nerve transfer to deltoid muscle using the nerve to the long head of the triceps, part I: an anatomic feasibility study. J Hand Surg Am 2003;28(4): Yu C, Xu JG, Lao J, et al. Relationship between effect of ipsilateral transfer in patients with patial injuries of truncus superior plexus brachialis and function of latissimus dorsi muscle. Chin J Hand Surg 2002;18(1): Cai PQ, Gu YD. Clinical applications of ipsilateral nerve root transfer. Chin J Hand Surg 2002;18(1): Lu W, Xu JG, Xiao JD, et al. An experimental study of triceps brachii and latissimus dorsi muscle function alterations after superior and middle trunk injuries of brachial plexus in rats. Chin J Orthopedic 2004;12(18): (Received April 12, 2008) Edited by LIU Yange