Animals and preparation before surgery
Animal handling procedures in this study were in agreement with the guidelines of the Animal Care and Use Committee of Fudan University. Male Sprague‑Dawley（S-D）rats (n=80; weight, 200‑250 g; age, 8 weeks) were kept in an environment with the temperature of 20˚C and humidity of 50%. All the rats were supplied by the company (Shanghai Slake Laboratory Animals Company, Shanghai, China). The rats were maintained on a 12/12‑h light/dark cycle and allowed free access to food and water. The animal use protocol was reviewed and approved by the Animal Ethics Committee Review Board of Fudan University.
Eighty rats were divided into 4 groups randomly on average as follows:
Group A: Rats with TBPA and cC7 transfer to median nerve (cC7 --- median nerve)
Group B: Rats with TBPA and cC7 transfer to axillary nerve (cC7 --- axillary nerve)
Group C: Rats with TBPA and cC7 transfer to both median and axillary nerves (cC7 --- median and axillary nerves)
Group D: Rats with TBPA without repair
The right side of each rat was selected as the injure side in all groups. Intraperitoneal injection of 1% pentobarbital sodium (1 ml/100 g body weight; Shanghai Reagent Company, Shanghai, China) was used before operation. After anesthesia, the rats were fixed and disinfected in the supine position.
Surgical techniques
TBPA Rat model
A right supraclavicular incision was made. Part of sternocleidomastoid and anterior scalene muscles were cut off. C5 and C6 nerve roots were identified. Then the clavicle was pulled down to explore C7, C8 and T1. After the right brachial plexus was completely exposed, the C5-T1 nerve roots were pulled out until avulsion at the foramen levels in Group A, B, C and D.
The first stage of cC7 transfer
CC7 nerve transfers were performed after TBPA in Group A, B, and C. A longitudinal incision was made along the ulnar side of the right upper limb. The ulnar nerve was exposed from the wrist to the axilla. Then the ulnar nerve was cut off at the wrist and separated from the wrist to the axilla. The superior ulnar collateral artery was reserved. A transverse incision was made on the left supraclavicular fossa. The cervical 7 nerve root was explored and separated from the intervertebral foramen. 2% lidocaine (Shanghai Reagent Company, Shanghai, China) was used for nerve block before the whole cC7 root was cut. The distal end of the right ulnar nerve was transferred to the left supraclavicular fossa through the subcutaneous tunnel. It was sutured to the left whole cervical 7 nerve root.
The second stage of cC7 transfer
Eight weeks after the first stage of cC7 transfer, the rats in Group A, B and C underwent the second stage of cC7 transfer. The procedure in the second stage of cC7 transfer in each group was as follows:
Group A (cC7 --- median nerve): A right longitudinal incision was made on the inner side of the upper arm. The ulnar and median nerves were identified at the retracing point of the ulnar nerve. (Figure 1) Both of them were cut off. The proximal end of the ulnar nerve was sutured to the distal end of the median nerve without tension.
Group B (cC7 --- axillary nerve): A Z shape incision was made in the right axilla. The axillary nerve was found at the quadrilateral foramen . The ulnar nerve was exposed at the retracing point. The axillary nerve was separated and cut off.The ulnar nerve was cut in the axilla. (Figure 2) Then the proximal end of ulnar nerve was sutured to the distal end of the whole axillary nerve without tension.
Group C (cC7 --- median and axillary nerves): AZ shape incision was made to expose the ulnar nerve, median nerve and axillary nerve in the right axilla . The axillary nerve was cut off at the quadrilateral foramen. The ulnar and median nerves were separated to enough length for suture in theaxilla. Then they were cut off. The proximal end of ulnar nerve was sutured to the distal ends of the axillary and median nerves without tension at the same time. (Figure 3)
In the experiment, the nerves were sutured with 11-0 nylon (Ethicon, Johnson & Johnson, New Brunswick, New Jersey) under 10 times microscope.
Evaluation methods
All the examinations and evaluations were carried out 12 weeks after the second stage of cC7 transfer. First, the behavioral tests were made in the rats. Then we used electromyogram (EMG) examinations (Neuromatic 2000M electrophysiological apparatus, Dantec, Les Ulis, France) to evaluate nerve regeneration. The rats were anesthetized by 1% pentobarbital sodium intraperitoneal injection before EMG examinations. . After EMG examinations, the median and axillary nerves were biopsied for nerve fibers count. Flexor Carpi Radialis (FCR) and deltoid (DEL) were biopsied for calculating the mean cross-sectional areas of muscle fibers. Gene expressions related to muscle atrophy were assayed by RT-qPCR (Agilent Technologies, Inc., Santa Clara, CA, USA).
Behavioral test
In Group A, the grasping test was performed. The rat was put on a wire mesh. Under normal circumstances, its paw would do the grasp action to catch the wire. We divided the results into M0-M2. M0: no digit flexion was observed. M1: digit flexion was observed without grasping. M2: grasping was observed. We defined M1 and M2 as effective recoveries for digit flexion.
In Group B, water spray test was performed. The rat’s posterior skull was continuously sprayed with a syringe. The rat would do shoulder abduction and external rotation to touch the wet position if the axillary nerve recovered.. We recorded the number of rats with shoulder abduction and external rotation. Then we calculated the effective rate.
The grasping test and water spray test were carried out in Group C.
EMG examination
The rats were anesthetized with sodium pentobarbital injected intraperitoneally. A transverse incision was made on the left supraclavicular fossa to expose the bridging ulnar nerve. A stimulating electrode was put on the bridging ulnar nerve. A recording electrode was inserted into the target muscle. A current of 1 Hz, 0.5mA was used for stimulating the nerve.
Group A: the recording electrode was inserted into the FCR.
Group B: the recording electrode was inserted into the DEL.
Group C: the recording electrodes were inserted into FCR and DEL.
The emergence of compound muscle action potential (CMAP) was regarded as an effective recovery for motor function.
Cross-sectional area of the muscle fiber
FCR and DEL were biopsied from the affected side in Group A and B, respectively. Both of the two muscles were obtained in Group C and D. All the muscles were fixed in 10% paraformaldehyde (Shanghai Reagent Company, Shanghai, China). Then they were fixed in paraffin (Shanghai Reagent Company, Shanghai, China). The muscles were stained with hematoxylin and eosin（HE）(Shanghai Reagent Company, Shanghai, China). The mean cross-sectional area of the muscle fiber was measured and calculated by Image J system (National Institutes of Health, USA).
Nerve fiber count
Median and axillary nerves were biopsied from the affected side in Group A and B, respectively. Both of the two nerves were obtained in Group C and D. All nerve sections were taken near their entry points into the target muscles. The samples were fixed in 25% glutaraldehyde (Shanghai Reagent Company, Shanghai, China) and embedded with paraffin for slices. Then they were stained with 5% toluidine blue (Shanghai Reagent Company, Shanghai, China). The average number of nerve fibers in unit area was measured and calculated by the Image J system.
Gene expressions related to skeletal muscle atrophy
A previous study was reported5that Muscle RING Finger 1（MuRF1）and Muscle Atrophy F-box （MAFbx）were the genes related to skeletal muscle atrophy. They were positively correlated with muscle atrophy. In Group A and B, FCR and DEL were biopsied respectively. Both of the two muscles were biopsied in Group C and D. Muscle tissue samples were dissolved in TRIzol™ (1600 Faraday Ave, Carlsbad CA92008 USA). Then DNA was separated, precipitated and extracted from the samples. The purity of the DNA was determined. Expression of each gene was assayed by RT-qPCR (Agilent Technologies, Inc., Santa Clara, CA, USA). The relative expression levels of the two genes were determined using 2-ΔΔCt method.
After the completion of the biopsy, each group was euthanized using CO2. The flow rate for CO2 euthanasia displaced 20% of the chamber volume/min according to the 2013 edition of the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.
Date analysis
Comparisons of the effective rates among different groups were performed using Fisher’s exact test in behavioral test and EMG. One-way ANOVA (analysis of variance) was used in the aspects of cross-sectional area of the muscle fiber, nerve fiber count and gene expressions. The p-values were two-tailed and p-values <0.05 were considered significant. All analyses were performed using Statistical Package for Social Sciences (SPSS, Chicago, IL, USA) 24.0 software.