The prophylactic release procedures were performed at 6 weeks of age in all rats before the onset of diabetes mellitus, which was confirmed by blood sampling. The surgery day was regarded as day zero for all rats.

Pin prick test, toe-spread test and walking tract analysis were used for functional evaluation and Somatosensory evoked potential test (SSEP) for electrophysiological evaluation.

This test was used in all rats at 12 weeks and in groups 4, 5, and 6 at 24 weeks. The pin-prick test was performed by applying pinching stimuli to the hind-limb skin from the knee to the toes until a withdrawal reflex of the extremity was obtained in response to the pain stimulus [7]. The withdrawal reflex was graded as 1 when obtained above the knee, as 2 when obtained distal to ankle and proximal plantar region and 3 when obtained at the toe level. The grade was accepted as 0 in the absence withdrawal reflex.

The TS test was performed at the same time as the PP testing to evaluate motor recovery. The following scores were used to note the rats’ response when they were raised from their tails; the absence of any movement was graded as “0”, the presence of any sign of toe-spread was accepted as grade “1”; toe abduction as grade “2”, and both abduction and extension of the toes were accepted as grade “3” [7].

WTA was performed at 12th week for the short-term and 24th week for the long-term follow-up groups before sacrifice. The animals were placed on an 8.2 × 42 cm walking track that was darkened at one end. White paper was laid on the track. The hind paws of the rats were dipped in black ink, and the footprints were left on the papers as they walked through the track. The sciatic function index (SFI) was calculated for each animal on the normal and experimental sides using the parameters as the distance from the first to the fifth toe (toe spread), the distance from the second to the fourth toe (the intermediary toe spread), and the distance from the heel to the third toe (the print length) by Bain – Mackinnon – Hunter SFI formula [8].

Electrophysiological assessment was performed by somatosensory evoked potential (SSEP) evaluation at postoperative week 12 at short-term follow up groups and 24 at long-term follow-up groups under general anesthesia. A Bio-Logic A-PAC 486 computer (Bio-Logic Systems Corp, Chicago, IL) was used for testing. Two stimulating electrodes were placed, one being at the first web space and the other posterior to the lateral malleolus in a subcutaneous plane at the limb to be evaluated (Fig. 55.4). The ground electrode was placed posterior to the lateral malleolus on the contra-lateral limb. Two burr holes were drilled on both sides of the sagittal suture over the parietal bone, and recording electrodes were placed over the parietal cortex (the active electrode over the contra-lateral cortex). The cortical SSEP responses were recorded bilaterally for the operated and non-operated sides.

The positive and the negative potentials of the waveform morphology in the SSEP measurement were obtained and the initial negative wave was marked as N1 and the following positive wave as P1. The second negative potential was marked as N2. P1 and N2 potentials were used for comparison of sensory recovery between the groups as P1 and N2 potentials are the most robust and consistent potentials (Fig. 55.4). The operated (left) to non-operated (right) side ratios were obtained for P1 and N2 waves in each animal. Higher ratios indicated impaired neurosensory functions.

Gastrocnemius muscles were harvested from both legs from rats of short-term follow up groups at 12 weeks and from long-term follow up groups at 24 weeks following sacrifice and wet-muscle weight measurements were taken immediately (Fig. 55.5). The wet weight of the operated site was compared with the non-operated contralateral side and the ratio is calculated as the gastrocnemius muscle index (GMI).