Investigation Of Nerve Regeneration And Sensate Recovery After Harvest Of Flap On The Back Of Rats

Objective: Skin flap is the most common way to repair tissue defects.After flap harvest,one of the complications is the loss of sensation in the flap,which can cause disturbance to the daily of patients if the flap is located in places such as heel,female breast,oral cavity and perineum.Therefore,it is of clinical importance to investigate the way that can promote flap reinnervation.To begin the strategy for intervention,the first step is to investigate whether the axons could spontaneously grow into the flap and the sensation could subsequently follow.SD rats were used in this study as the animal model,and gross dissection,HE staining and Sihler staining were adopted to investigate the layers of skin,the origin and distribution pattern of nerves that innervate the dorsal skin of rat.After harvest of completely denervated flaps on the dorsum of rats,tissues from the proximal and distal ends of the flaps were harvested,and panaxonal marker(NF-200),myelin marker(P75),sympathetic marker(TH),and sensory nerve marker(CGRP)were used to identify the change patterns of the various components of the nerves in the flap.Meanwhile,transmission electron microscope was used to the axonal changes within the flap at the ultrastructural level.Furthermore,approaches using a clamp and a therm-controlled heat transmitting device were explored to test the extent of recovery of nociceptive and thermic sensations after flap harvest.Methods 1.The origin of nerves entering the skin on the back of the rats were explored using microsurgical tools under a stereomicroscope,the detailed course of the nerves was demonstrated using Sihler's staining and the layers of the skin were observed using HE staining.After anatomical dissection,denervated flaps were established on the backs of 35 rats,which were then divided equally into the following time points: 0d,5d,7d,14 d,30d,60 d and 90 d after surgery.At each time point,tissues from the proximal and distal ends of the flaps were harvested for immunostaining using anti-NF-200,anti-P75,anti-TH,and CGRP antibodies for the purpose of identifying the changing pattern of nerves within the flaps.Then Kruskal Wallis test was used to analyze the statistical difference between different groups of data.P less than 0.05 was considered statistically significant.Meanwhile,part of the tissues were processed for ultrastructural observation using a transmission electron microscope.2.Denervated flaps were established on the back of 10 rats,and a surgical clamp and a self-made therm-controlled heat transmitting device were used to determine extent of recovery of the nociceptive and thermal thermic sensations 14 d,30d,40 d,50d,60 d,70d and 90 d after surgery.Results 1.The skin of the back had two sources nerve supply: one was the 12 pairs of cutaneous nerves from the intercostal nerves that travelled obliquely,the other was 6 branches from the bracheal plexus named as the longitudinal nerve that traveled longitudinally from in head-to-tail direction.HE staining showed that the dorsal skin consisted of four layers: epidermis,dermis,panniculus carnosus and subcarnosus fascia.2.The outcomes of the immunostaining for NF-200,P75,TH,and CGRP showed that the amount and intensity of immunopositivity for NF-200 was strong at day 0,and faded and disappeared quickly at 5d,7d and 14 d.At 30 d after surgery,the immunopositivity of NF-200 returned and intensified at 60 d and 90 d.There were significant differences in the positive expression scores among groups(F = 33.147,P < 0.01),and there were significant differences in the fluorescence intensity scores among groups(F = 30.143,P < 0.01).The intensity of P75 immunopositivity was weak at 0d,but intensified at 5d,14 d,14d,and 30d;However,immunopositivity weakened at 60 d and 90 d.The expression pattern of TH and CGRP was similar with NF-200.There were significant differences in the positive expression scores among groups(F = 32.588,P < 0.01),and there were significant differences in the fluorescence intensity scores among groups(F = 32.252,P < 0.01).The only difference was that the reappearance of their immunopositivity was 2w earlier.There were significant differences in the scores of positive expression among TH groups(F = 33.156,P < 0.01).There were significant differences in the scores of fluorescence intensity among groups(F = 33.272,P < 0.01).There were significant differences in the scores of positive expression among CGRP groups(F = 33.081,P < 0.01).There were significant differences in the scores of fluorescence intensity among groups(F = 33.130,P < 0.01).3.The ulatrastructural observation revealed that laminar and concentric arrangement of myelin could be observed around an axon,and wallarian degeneration could be observed 5d later.14 d later,an axon with a relatively thin myelin could be observed.30 d later,an unmyelinated axon could be observed.60 d later,the myelin of the axon returned to normal.4.30 d after flap harvest,nociceptive sensation returned and intensified over time.The reemergence of thermic sensation was 20 days later than the nociceptive sensation,but intensified at a faster pace.Conclusions: The skin on the back of the rats are innervated by cutaneous nerves from the intercostal nerves and the longitudinal nerve.After flap harvest,the ingrowth of axons into the flap can occur spontaneously,and the sensation can partially return.