Negative pressure for diabetic foot:Evaluation based on calcitonin gene-related peptide in the limb skin of dogs

摘要

BACKGROUND: Very good therapeutic effect has been obtained for limb negative pressure in treating limb ischemic diseases.But its mechanism in treating diabetic foot needs further investigation.OBJECTIVE:To observe the changes of calcitonin gene related peptide(CGRP)in sensory nerve fiber of skin of dogs with diabetic foot before and after limb negative pressure treatment,and make a comparison with normal dogs,then analyze the mechanism of limb negative pressure in treating diabetic foot.DESIGN:A randomized controlled animal experiment.SETTING:Third Department of General Surgery,Xijing Hospital,Fourth Military Medical University of Chinese PLA.MATERIALS:Fifteen healthy hybrid dogs,of either gender,weighing 12-18 kg,were provided by Animal Room,Xijing Hospital of the Fourth Military Medical University of Chinese PLA.After being numbered,the involved dogs were randomly assigned into 3 groups:treatment group,model group and normal control group,5 dogs each.Limb negative pressure therapeutic machine(self-made);Anti-CGRP serum (primary antibody,Watpa Company,England);rabbit anti-BigG(second antibody),ABC compound solution,DAB reagent,glucose oxidase reagent,Tdxon-X100 solution(Sigma company,USA),ammonium nickelous sulfate reagent(Xi'an Chemical Reagent Factory),neutral gum-solution(import,Shanghai Reagent Co.,Ltd.);Leitz1400 sliding freezing microtome(Leitz Company,Germany);Leica 1800 cryostat microtome(Leica Company,Germany);Quantimet570 image analyzer(Leica Company,Germany).METHODS:This experiment was carried out in the Animal Room,Xijing Hospital,Fourth Military Medical University of Chinese PLA from October 2005 to June 2006.①After the dogs in the treatment and model groups were anesthetized,their pancreatic tissues were removed completely and animal models of type I diabetes mellitus were developed.All the femoral artery branches including deep femoral artery were ligated and cut off.A helical wire was inserted into the distal femoral artery cavity for narrowing femoral artery 1/2,then contributing to ischemic status of this limb,thus,dog models of diabetic foot of posterior limbs were developed.When the dogs in the treatment group were successfully modeled,negative pressure treatment was conducted on their affected limbs after 14 days.When the dogs were lightly anesthetized,depilation was performed on the base of left posterior limb in ring form.Then,their depilated limbs were placed in self-made negative pressure cabin for 15 minutes of-12 kPa negative pressure treatment every day within 10 days successively.Negative pressure treatment on affected limbs was omitted in the model group.The dogs in the normal control group were not modeled or given negative pressure treatment.②After 10 days of treatment,3 groups of animals all subjected to immunohistochemical staining of toe skin of affected limb for detecting CGRP-immunoreactive positive nerve fibers.Analysis of variance and LSD-t test were used for comparison of measurement data.MAIN OUTCOME MEASURES:Changes of CGRP in sensory nerve fibers of skin of 3 groups of animals.RESULTS:CGRP-immunoreactive positive nerve fibers in the skin of animals of model group were(53.24±5.6)μm/mm2 ,which were significantly more than those of control group[(11.73±2.3)μm/mm2,t=16.94,P＜0.01].CGRP-immunoreactive positive nerve fibers in the skin of animals of treatment group were(32.49±2.9)μm,mm2,which were less than those of model group(t=8.47,P＜0.01),but were still more than those of normal control group(t=8.47,P＜0.01).CONCLUSION:①Following the onset of diabetic foot,CGRP-immunoreactive positive nerve fibers in the skin of distal limb are obviously increased,which may be a self-protective mechanism of body.②Limb negative pressure treatment can treat diabetic foot.which attributes to that the release of CGRP in the sensory nerve fibers can promote vasodilatation.