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Retrograde labelling
For double labelling study, the combination of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate
(DiI) (Molecular Probes, Leiden, The Netherlands) and True Blue (TB) (Molecular probes, Leiden, The Netherlands)
were used 1. 3 months postoperatively, The left MN trunk was re-exposed, cut at the elbow level, and placed into a
plastic well containing 2% TB saline solution. Similarly, 2% DiI was administered to the MCN stump. To avoid
tracer leakage, the base of the well was sealed by petroleum gel. One hour later, nerve stumps were removed out of
the well and rinsed. Care was taken to prevent the tracer from contaminating the surrounding tissue.
One week later, rats were re-anesthetized, transcardially perfused and their C5-T1 spinal cord as well as dorsal root
ganglions (DRGs) were harvested and prepared according to previous study2. Cryostat serial cross-sections of spinal
cord (40 um) and DRG (10 um) were collected. Labeled neurons were screened and counted in every tenth section
of C5-T1 DRGs and spinal cord. All specimens were evaluated under LEICA DMLB2 epi-fluorescence microscope
(Leica, Wetzlar, Germany) using BP 340-380 excitation filter (TB) and BP 535/50 excitation filter (DiI).
Nerve Fluorescence Studies
Three month postoperatively, nerve segment samples including the junction of Mh/ Lh of MN, together with a
segment of the sutured MCN was harvested from the transgenetic GFP-fluorescent rats, and prepared as previously
described. The nerve tissue was mounted outstretched on a plastic stick, and then immersed in 4% paraformaldehyde
in phosphate-buffer (PB) at 4°C overnight. They were cryoprotected by sequential incubation in phosphate buffer
(PB) containing 5%/10%/ 20%/30% sucrose. Longitudinal 20-um-thick slices was mounted onto glass slides,
coverslipped to be examined under Olympus FV1000 confocal microscope (Olympus, Shinjuku, Japan).
Median Nerve Topography
To study the intra-neural topography of rat MN, the proximal part of the MN together with Mh/Lh junction (about
1.0 cm) was taken from the left upper limb of 5 naive rodents. A marker was made on each of the MN to signify the
location where Lh and Mh were epineurially merged together. Tissues were then mounted outstretched on a plastic
stick, immersed in 4% paraformaldehyde in phosphate-buffer (PB) at 4°C overnight and incubated in phosphate
buffer (PB) containing 5%/10%/ 20%/30% sucrose. After embedded in OCT compound, cryostat serial cross
sections were sectioned starting from the aforementioned marker downward, at 40 um thickness, with 0.16 mm
interval. A total of 30 specimens, i.e. 4.8 mm, for each nerve were collected.
For double immunofluorescent staining, all specimens were washed three times in PBS, blocked with 10%
donkey serum plus 0.3% Triton X-100 for 2 hours at RT, and then treated with primary antibodies for large axons
(mouse monoclonal antibody to the 200 MW neurofilament protein, NF200, 1:2000 dilution, 48 hours, Abcam, San
Francisco, CA), and for perineurium (rabbit polyclonal antibody to Glucose Transporter-1 protein, GLUT1, 1:200
dilution, 24 hours, Abcam, San Francisco, CA). After rinsing five times with PBS for 3 minutes, the specimens were
incubated with Cy3 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse immunoglobulin G antibodies (1:500,
Invitrogen, Grand Island, NY) for 120 minutes at RT. All slides were then rinsed and coverslipped before
examination using A1R confocal microscopy (Nikon, Tokyo, Japan).
Reference
1.
Zele, T., Sketelj, J., Bajrovic, F. F. Efficacy of fluorescent tracers in retrograde labeling of cutaneous
afferent neurons in the rat. J Neurosci Methods 2010;191:208-214.
2.
Sanapanich, K., Morrison, W. A., Messina, A. Physiologic and morphologic aspects of nerve
regeneration after end-to-end or end-to-side coaptation in a rat model of brachial plexus injury. The
Journal of hand surgery 2002;27:133-142.