Diabetic neuropathy, which is the most frequent and earliest diabetes complication, decreases the quality of life and increases the morbidity of diabetic patients (1) and occurs because of the impairment of nerve blood flow and metabolic imbalances in the neural compartment of the peripheral nerve. Disruption of the vasa nervorum has been reported in peripheral nerves in experimental diabetic neuropathy (2), and numerous studies indicate that reduced blood flow directly caused symptoms of diabetic neuropathy (3,4). We and others have demonstrated that vasodilatory and anticoagulant treatments, such as prostaglandin [E.sub.1]/[I.sub.2], niceritrol, and cilostazol, improved nerve function and symptoms with diabetic neuropathy (5-8). Although these treatments ameliorate the symptoms of diabetic neuropathy, more effective treatments are needed clinically.
Since Asahara et al. (9) first demonstrated the existence of circulating endothelial progenitor cells (EPCs) in adult peripheral blood, the concept of adult vasculogenesis has been developed. When local ischemia occurs, EPCs are mobilized from bone marrow to the peripheral blood. Then EPCs are differentiated in the area of local ischemia, which induces neovascularization (10). This paradigm is termed "postnatal vasculogenesis." From the viewpoint of vasculogenesis, the ischemic diseases may be caused by an insufficient supply of EPCs. A number of experimental and clinical studies have revealed that ischemic heart diseases and arteriosclerosis obliterans can effectively be treated by EPC transplantation, causing postnatal neovascularization (11-14).
Schratzberger et al. (15) showed that vascular endothelial growth factor (VEGF) gene transfer significantly increased nerve conduction velocity and nerve blood flow as well as the amount of vasculature in the muscles and nerves, suggesting that the induction of local angiogenesis ameliorates experimental neuropathy. Therefore, we hypothesized that the transplantation of EPCs may promote local vasculogenesis and reverse diabetic neuropathy.
EPCs can be isolated from the bone marrow, cord blood, and peripheral blood. We and others have reported that EPCs isolated from cord blood have a greater proliferative potential and a higher cell cycle rate than EPCs from other sources, suggesting that cord blood-derived EPCs may more effectively contribute to therapeutic vasculogenesis (11,12,16). In this study, we examined whether transplantation of human umbilical cord blood-derived EPCs into the hindlimb skeletal muscles may prevent the development of diabetic neuropathy in streptozotocin (STZ)-induced diabetic immunodeficient nude rats.
RESEARCH DESIGN AND METHODS
Human umbilical cord blood. Human umbilical cord blood (50-120 ml) was obtained from each donor after the baby's delivery. Written informed consent was obtained from all mothers before labor and delivery. Protocols for sampling human umbilical cord blood were approved by the Institutional Review Board.
Cell culture. Mononuclear cells (MNCs) were isolated from umbilical cord blood using the Histopaque-density centrifugation method (9). The MNC layer was collected, washed twice with 1 mmol/l EDTA in PBS, and suspended in degassed PBS with 0.5% BSA and 2 mmol/l EDTA. Total MNCs were cultured in M-199 medium with 20% fetal bovine serum (Sigma, St. Louis, MO) and bovine brain extract on human fibronectin-coated plastic plates. EPCs from attached cells and cell clusters were expanded under the condition described above. To confirm that these cells had the character of endothelial cells, separate coverslips were incubated with 10 mg/ml 3,3'-dioctadecylindocarbocyanine (DiI)-acetylated LDL for 4 h at 37[degrees]C and observed under fluorescent microscope.
Tube formation in basement matrix gel. At day 7 of culture, cord blood-derived EPCs were transferred to basement membrane matrix gel (Matrigel; BD Biosciences, San Jose, CA). At days 3-7 of culture, the formations of angiogenesis-like endothelial cell networks were seen under a fluorescence microscope.
Flow cytometry. Cord blood-derived EPCs at day 7 of culture were subjected to flow cytometric analysis as described previously (17). Cells were then stained with phycoerythrin-conjugated CD34 (clone 8G12; BD Biosciences) or fluorescein isothiocyanate-conjugated CD45 (clone 2D1; BD Biosciences) antibody. Isotype-identical antibodies served as control. After staining, cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry.
Rats. Male immunodeficient nude rats (F344/N rnu/rnu) at 6 weeks of age were provided by Cler Japan (Tokyo, Japan). All protocols were approved by the Nagoya University Institutional Animal Care and Use Committee.
Induction of diabetes. Diabetes was induced after an overnight fast with a single intraperitoneal injection of STZ (60 mg/kg in 0.9% sterile saline). Serum glucose levels were measured every week, and rats with serum glucose 12.5 mmol/l were used as the diabetic rats. Age- and weight-matched nude rats were used as control animals.
Transplantation of ex vivo expanded EPCs for therapeutic neovascularization. Eight weeks after the induction of diabetes, control and diabetic anesthetized nude rats (5 mg/100 g pentobarbital i.p.) were injected with EPCs (1 x [10.sup.6] cells/rat) intramuscularly in the unilateral femoral quadriceps muscle, the femoral biceps muscle, and the soleus muscle using a 26-gauge needle. Saline was injected into the contralateral hindlimb skeletal muscles in both normal and diabetic nude rats as controls. Four weeks after the treatments, physiological and histological assessments were performed under anesthesia as follows.
Motor nerve conduction velocity. Rats were anesthetized with pentobarbital (5 mg/100 g) by intraperitoneal injection and placed on a heated pad in a room maintained at 25[degrees]C to ensure a constant rectal temperature of 37[degrees]C. Motor nerve conduction velocity (MNCV) in sciatic nerves between the ankle and sciatic notch was measured as described previously (18). MNCV was determined with a Neuropak NEM-3102 instrument (Nihon-Koden, Osaka, Japan) by methods described previously (6).
Sciatic endoneurial nutritive blood flow. Rats were anesthetized with pentobarbital (5 mg/100 g) by intraperitoneal injection and placed on a heated pad in a room maintained at 25[degrees]C to ensure a constant rectal temperature of 37[degrees]C. Sciatic endoneurial nutritive blood flow (SNBF) was measured by the hydrogen clearance technique with an analog recorder BW-4 (Biochemical Science, Kanawasa, Japan) and electrolysis tissue blood flow meter RBA-2 (Biochemical Science), as described previously (15), and calculated with the equation of Koshu et al. (19).
Immunohistological staining. At the end of the experiments, rats were killed with an overdose of pentobarbital. After perfusion-fixation in 4% paraformaldehyde, the soleus muscles from both sides of eight rats (four normal rats and four diabetic rats) were immersed in 4% paraformaldehyde overnight. All of the samples were embedded in paraffin and cut into 5-[micro]m sections for hematoxylin-eosin staining and immunohistochemical staining with primary antibody. The sections were cleared of paraffin in xylene and rehydrated through decreasing concentrations of ethanol. The slides were treated with a solution of 0.3% hydrogen peroxide ([H.sub.2][O.sub.2]) in methanol for 30 min at room temperature to abolish endogenous peroxidase activity. Sections were then incubated overnight at 4[degrees]C with the primary antibody (anti-von Willebrand factor (vWF) polyclonal antibody, DAKO Japan, Tokyo, Japan) diluted 1:600. Sections were subsequently stained by Simplestain rat system (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. Negative control was performed by omitting anti-factor VIII antibody. The capillary endothelial cells were counted under light microscopy (x200) to determine the capillary density. Five fields from the muscle samples were randomly selected for the capillary counts. To avoid overestimating the capillary density because of muscle atrophy or underestimating it because of interstitial edema, the capillary density was expressed as the capillary-to-muscle fiber ratio.
Statistical analysis. All group values were expressed as means [+ or -] SE. Statistical analyses were made by a one-way ANOVA with the Bonferroni correction for multiple comparisons.
RESULTS
Body weights and blood glucose concentrations of nude rats. Diabetic nude rats showed significant decrease in body weights and significant increase in blood glucose concentrations compared with normal nude rats (Table 1). The transplantation of EPCs into unilateral hindlimb skeletal muscles did not change body weights and blood glucose concentrations compared with untreated nude rats (data not shown).
