A Comparison On Bone Marrow Mobilization Vs Implantation Of Bone Marrow Mononuclear Cells For Treatmeating Ischemic Hindlimb In Rat

Objective:As the main reason of non-traumatic amputation,the morbidity rate of diabetic foot is higher year by year.Limb ischemia is the main reason of diabetic foot.The character of limb ischemia is out-flow tract occlusion, more severely more far.So it is too hard to recanalization by putting up bridge and intervention.In 2002,Tateishi-Yuyama etal reported that the autologous transplantation of bone marrow mononuclear cells was safe and effective for achievement of therapeutic angiogenesis in 43 patients with ischemic limbs and could significantly improve the clinical status in them whose legs received injections of bone marrow mononuclear cells.In 2006,Zhang nan etal reported the role of bone marrow mobilization on the angiogenesis in ischemic hindlimb of diabetic rats.The next year,a comparison of transplantation of endothelial progenitor cells and mononuclear cells on the treatment of ischemic disorder in the hind limb of diabetic rats was reported.But the Comparison on bone marrow mobilization vs implantation of bone marrow mononuclear cells for treatmeating ischemic hindlimb in rat has not been reported.We observed the effects of bone marrow mobilization vs implantation of bone marrow mononuclear cells by assaying the density of capillaries.In the present study,firstly,we made the model of ischemic hindlimbs of diabetic rats and compared the effects of bone marrow mobilization vs implantation of bone marrow mononuclear cells by assaying the density of capillaries.Secondly,we verificated the effects of implantation of bone marrow mononuclear cells again by assaying the density of capillaries,which supplied evidence for clinical treatment.Thirdly,we observed the effects of the two methods on the ischemic disease of diabetic rats by assaying vascular parameters.Methods:Healthy Sprague-Dawley rats(n=36,Hebei Medical University), male, weighing about 220g(8 weeks old).After hindlimb ischemia in diabetic rats model, The 36 rats were randomized to three groups,15 rats were proceeded with bone marrow mobilization(B group),15 rats were proceeded with implantation of bone marrow mononuclear cells (A group), 6 rats were in control group(C group),without any treating. The level of endothelin(ET) were assayed at before and after 3 days of bone marrow mobilization(before transplantation),2 and 4 weeks after transplantation. At the end of the experiment,ischemic muscles were harvested for making pathalodical examination,then the density of capillaries were assayed to compare the effects of bone marrow mobilization and implantation of bone marrow mononuclear cells.1 Making of diabetic ratsAfter the rats were abrosiaed for 12 hours, STZ was administered via intraperitoneal injection at 65mg/kg to make model. Blood was taken from the caudal vein at 8:00 to determinate of the fasting blood glucose(FBG). When FBG >16.65mmol/L , we took it as diabetic rat.2 Making of ischemic limbs in diabetic ratsAll of the diabetic rats were excised bilateral Femoral Arteries and all the side branches of them under anesthesia.3 days after,the operationed limbs started to appear atrophy and descented motoricity,but no gangrene.3 Bone marrow mobilization and transplantation of the mononuclear cellsAfter hindlimb ischemia in diabetic rats model, the rats from A group and B group were injected G-CSF(150ug.kg-1.d-1) subcutaneously for 3 days,at the same time, the rats from C group were injected saline 1ml/d,3 days.Then blood was taken from the rats of A group, mononuclear cells were isolated by density gradient centrifugation and injected into ischemic limbs of A group for 0.5cm×0.5cm distance.4 Counting of WBC and CD34+ cells in peripheral blood Blood was taken from the rats of A group before and after bone marrow mobilization, the rats of B and C group being done samely.Then counted the number of WBC and CD34+ cells by Flow Cytometry. 5 Assays of vascular parametersEndothelin(ET) was measured by radioimmunoassay on serum samples. Unit: pg/ml.6 Assays of the density of capillaries in musclesThe ischemic muscles were made by routine method for light microscope examination and proceed with vWF immunohistochemistry staining to assay the density of capillaries.7 Data analysis and statistical evaluation All data were treated with SPSS13.0,data presented as mean±SD( X±S).The statistic significance was determined by T-test between groups.The significance was indicated by P-value.The significant difference which had the significance was indicated by P<0.05.Results:1 Observing of experimental animalsAll of the diabetic rats were excised bilateral Femoral Arteries and all the side branches of them.3 days after,the operationed limbs started to appear atrophy and descented motoricity,but no gangrene until death.2 Counting of WBC and CD34+ cells by Flow CytometryIn A group, 3 days after mobilization,the percent of CD34+ cells(2.27±0.38)% and the number of WBC(22.53±2.44)×109/L were significantly higher than before(0.15±0.04)% and(7.37±1.36)×109/L (P<0.05).In B group, 3 days after mobilization,the percent of CD34+ cells(2.29±0.36)% and the number of WBC(23.09±2.44)×109/L were significantly higher than before(0.15±0.05)% and(7.83±1.15)×109/L (P<0.05) ,Although without mobilization,we can see the same phenomenon in C group.But the percent of CD34+ cells and the number of WBC were in A and B group were significantly higher than that in C group(0.45±0.10)% and (9.82±1.40)×109/L(P<0.05).3 Assays of vascular parametersThere was no significant difference in ET among the rats of each group before bone marrow mobilization.(P>0.05). There was no significant difference in ET beteen the rats of A group and B group at every stage.(P>0.05).The plasma levels of ET in rats of C group were obviously higher than those in A group and B group before bone marrow mobilization,after 3 days of bone marrow mobilization and in 2 and 4 weeks after transplantation (P<0.05).4 Assays of the density of capillaries in musclesThe vWF expression of the rats in B group was significantly higher than that of C group(P<0.05),and lower than that of A group(P<0.05).Conclusions:1 Therapeutic angiogenesis induced by bone marrow mobilization and implantation of bone marrow mononuclear cells could be an effective treatment for ischemic limb disease in diabetic rats. In present study,we found that the later was better than the former. 2 From the assay of CD34+ cells by Flow Cytometry we found that bone marrow mobilization could improce the percent of CD34+ cells and induce therapeutic angiogenesis.3 The assay of ET in peripheral blood told us that bone marrow mobilization and implantation of bone marrow mononuclear cells could also improve the situation of vascular disease in diabetic rats.