Transplantation Of Bone Marrow-Derived Endothelial Progenitor Cells To Treat Peripheral Neuropathy In Diabetic Rats

Background:Diabetes mellitus is one kind of endcrine metabolic disorder diseases which is caused by a number offactors, and it’s complications are harmful to human body. Diabetic neuropathy, especially diabeticperipheral neuropathy (DPN) is one of the most common complications of diabetes. Oxidative stress andblood vessel damage play important roles in the development of DPN. Many researches showed thatimproving oxidative stress and repairing the damaged blood vessels are beneficial to the recovery ofneuropathy. Endothelial progenitor cells (EPCs) as one kind of stem cell, have the function of differentiateinto vascular endothelial cells and involve in new blood vessel formation. Many studies revealed that thedecreasing number and impaired function of EPCs are closely related to the abnormality of blood vessel.Transplantation of EPCs is effective in the treatment of ischemic diseases. But the report whethertransplanting EPCs to the treatment of DPN can improve oxidative stress and be conducive to neuralrestoration is rare. At present, it is a hot topic for domestic and overseas scholars.Objective:Using bone marrow-derived EPCs from rats to treat sciatic neuropathy in diabetic rats, by measuringthe sciatic nerve motor nerve conduction velocity (MNCV), observing the expression of pathologic changesof sciatic nerve, nuclear transcription factor-kappa B (NF-κB) and myelin basic protein (MBP) before andafter transplantation in order to evaluate the therapeutic effect of EPCs on DPN and study the mechanism.Methods:1The separation, cultivation and identification of rat bone marrow-derived EPCs1.1The separation and cultivation of rat bone marrow-derived EPCsMononuclear cells were isolated from rat bone marrow using density gradient centrifugation method.Then the cells were cultured in flask coated with human fibronectin.1.2The identification of rat bone marrow-derived EPCs.EPCs were identified by double fluorescent staining and the experiment of microvascular formation.2Modellbau of DPN rats Male SD rats with a body weight of220-240g were fed for7days to adapt the experimental conditions.1%STZ by a single intraperitoneal injection were conducted to induce diabetes rats after overnight fast.The levels of fasting blood glucose were measured72hours later by acupuncturing tail tip, and rats withfasting glucose levels≥16.7mmol/L were regarded as diabetic rats. We measured sciatic nerve MNCV of10rats with normal blood glucose before the injection of STZ, the diabetic rats were measured at week4,week8and week12after the attack of diabetes. Compared to the normal rats,when the sciatic nerveMNCV of diabetic rats was slower and there was a statistical difference, then the DPN model rats wereobtained.3Groups of experimentGroup NC: the rats with normal blood glucose (10rats)group A: DPN model rats (10rats)group B: DPN model rats+saline (10rats)group C: DPN model rats+EPCs (10rats)4Transplantation of the vitro culture of EPCsAfter12weeks of diabetes for the rats of group C, we collected the EPCs which separated from thebone marrow of normal rats and cultured for7days, and then injected1ml (containing1×106cells) to thesciatic nerve after dissolved by saline. Same volume of saline were injected into the rats of group B after12weeks of diabetes.5Measurement of MNCV of the sciatic nerve4weeks after transplantation, we measured the MNCV of the group B and group C, the group NC andgroup A were measured after12weeks of diabetes. Method:10%chloral hydrate (3ml/kg) were used toanesthetize the rats, then exposed the right sciatic nerve and measured the MNCV with a electromyographyand trigger potentiometer NTS-2000instrument.6Preparation of sciatic nerve pathologic specimensWe made pathologic specimens of the group NC and group A after12weeks of diabetes, group B andgroup C after4weeks of transplantation. Method: HE staining and electron microscopy specimens wereprepared, then observed the pathological changes of sciatic nerve using optical microscope and electronmicroscope. 7Measurement of NF-κB and MBP in the sciatic nerveThe group NC and group A with12weeks of diabetes, group B and group C in4weeks oftransplantation were measured. Method: measure the expression of NF-κB and MBP in the sciatic nervewith the immunohistochemical method.Results:1We successfully obtained plenty of EPCs by using density gradient centrifugation in vitro.2Compared to the normal rats, the sciatic nerve MNCV of12weeks of diabetic rats was slowerremarkably, and there was a statistically significant difference (p＜0.01).3MNCV Compared with group NC, group A and group B had slower sciatic nerve MNCV (p＜0.01). The sciatic nerve MNCV of group C and group NC revealed no statistical differences (p＞0.05).4HE staining results Group A: the number of nerve fibers was reduced, myelin became uneven anddegeneration, axon became thin; group B: the number of nerve fibers was seriously decreased, myelinbecame more uneven and degeneration was aggravated, axon became rather thin and even disappeared;group C: demyelination and axonal degeneration of group C were more relieved than those of group B, butwere more serious than those of group A.5Electron microscope results Group A: myelin became uneven, part of the myelin layer wereseparated, some grew into the axon, axon became thin; the organelles of schwann cells were damaged,chromatic agglutination was observed and nuclear membrance was incomplete. Group B: myelin becamemore uneven, the scope of plate layer disappear was larger, axon became thin and even atresia; theorganelles of schwann cells were badly damaged and even disappeared, chromatic agglutination and evenkaryopyknosis was observed and nuclear membrance was incomplete. Group C: the unevenness of myelinwas not serious, most of the plate layers were existing; the organelles of schwann cells were normal,nuclear membrance was complete, whereas chromatic agglutination was observed.6Expression of NF-κB Compared with group NC, the expression of NF-κB was increased in thesciatic nerve of group A (p＜0.01). The expression of NF-κB in the sciatic nerve of group C was lower thangroup A (p＜0.05), but still higher than group NC (pPA＜0.01,pIOD＜0.05).7Expression of MBP Compared with group NC, the expression of MBP was less in the sciatic nerveof group A (p＜0.01). There was no statistical difference for the expression of MBP between group C and group A (p＞0.05). Compared with group B, the expression of MBP was higher in group C (p＜0.01).Conclusions:1After feed for12weeks, diabetic rats could progress to DPN rats;2Transplantation of EPCs could improve MNCV, demyelination and axonal degeneration pathologicalchanges of sciatic nerver of the DPN rats;3Transplantation of EPCs could increase the expression of NF-κB and inhibit the expression of MBP,this might be conducive to the repairs of nerve injury.