Male Sprague-Dawley (SD) rats at 6 weeks of age were obtained from Chubu Kagakushizai (Nagoya, Japan). For the induction of diabetes, STZ (Sigma Chemical Co., St. Louis, MO, USA) (60 mg/kg body weight) was intraperitoneally injected into rats. Blood glucose levels greater than 14 mmol/l were identified as diabetes at 1 week after STZ injection and were used in the experiments. Small doses of insulin pellet (LinShin Canada, Inc. Toronto, Canada) were administered subcutaneously once a month from 8 weeks after STZ injection to avoid excess hyperglycemia (Fig. 2a). Age-matched male SD rats were used as control. Rats were housed in a room maintained under controlled temperature (24 ± 1.0 °C) on a 12-h light/dark cycle and were given standard laboratory rat chow with water ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committees of Aichi Gakuin University (AGUD 059) and were conducted in accordance with the United States Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering.
Isolation and culture of DPSCs
Dental pulp tissue was excised from the incisor teeth of 6-week-old male SD rats, and DPSCs were isolated and cultured in an alpha modification of the Eagle’s medium (α-MEM) (GIBCO Lab Inc., Grand Island, NY) with 20% fetal bovine serum (FBS; GIBCO) as previously reported . DPSCs from passage 3 or 4 were used for all experiments.
Characterization of DPSCs
Cells were stained with the R-PE-conjugated antibodies against rat CD29 (Becton Dickinson, Franklin Lakes, NJ, USA), CD90, CD45 (Becton Dickinson), and CD34 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the FITC-conjugated antibody against rat CD49d to characterize the DPSCs by flow cytometry (Miltenyi Biotec, Bergisch Gladbach, Germany). Isotype-identical antibodies served as the controls. Data were analyzed with MACSquant software (Miltenyi Biotec). The multi-differentiability of DPSCs was assessed by their differentiation into osteoblasts, chondrocytes, and adipocytes according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).
Transplantation of DPSCs
Forty-eight weeks after the STZ induction of diabetes, the rats were anesthetized with pentobarbital (50 mg/kg, intraperitoneally) and underwent transplantation of DPSCs into the hind limb skeletal muscles. The DPSCs (1 × 106 cells) were suspended in 1.0 ml saline and injected into 10 points in the unilateral gastrocnemius, soleus, and biceps femoris muscles of both the normal and the diabetic rats using a 26-gauge needle. Saline was injected into the opposite side of hind limb skeletal muscles as the control. The parameters discussed below were measured 4 weeks after transplantation.
Sciatic nerve conduction velocities
Rats were anesthetized by isoflurane inhalation, and the near nerve temperature was maintained at 37 °C by a warming pad using a multipurpose thermometer (Bioresearch Co., Nagoya, Japan). The motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) in the sciatic nerve were measured using a Neuropak MEB-9400 (Nihon-Koden, Osaka, Japan).
Sciatic nerve blood flow
Rats were deeply anesthetized by isoflurane inhalation and sciatic nerve blood flow (SNBF) was measured using a Laser Doppler Blood Flow Meter (FLO-N1; Omega Wave Inc., Tokyo, Japan) as previously described . During the procedure, the rats were laid out on a heated pad and the near nerve temperature was maintained at 37 °C using a thermometer (Bioresearch Co.).
Current perception threshold using a Neurometer
The current perception threshold (CPT) of the sensory nerve fibers was measured in diabetic and normal rats using a CPT/LAB neurometer (Neurotron, Denver, CO, USA). Each rat was kept in an awake state in a Ballman cage (Natsume, Tokyo, Japan). The plantar surfaces of the rats were stimulated by 5, 250, and 2000 Hz sine-wave pulses. The intensity of each stimulation was gradually increased automatically. The minimum intensity when startled was defined as the current perception threshold of each rat.
Capillary density in the hind limb skeletal muscles
Rats were killed with an overdose of pentobarbital (150 mg/kg), and the hind limb skeletal muscles were fixed in a 4% paraformaldehyde solution. The fixed materials were embedded in paraffin and cut into 5-μm sections. The sections were incubated overnight at 4 °C with the anti-von Willebrand factor polyclonal antibody (DAKO Japan, Tokyo, Japan) and subsequently stained using the Simplestain rat system (Nichirei, Tokyo, Japan). The capillary endothelial cells were counted under light microscopy (Leica Microsystems, Wetzlar, Germany).
Laser Doppler perfusion image of the hind limb blood flow
Rats were anesthetized and placed on a heating pad to keep a constant rectal temperature of 37 °C. Hind limb blood flow was visualized using a laser Doppler perfusion image (LDPI) analyzer (Moor Instruments, Devon, UK). Low to no flow was displayed as dark blue, whereas high flow was displayed as red.
Intra-epidermal nerve fiber density
After the fixation of the footpads, tissues were immersed in an OCT compound (Sakura Finetechnical, Tokyo, Japan) containing liquid nitrogen and isopentane. Three longitudinal 25-μm thick footpad sections from each rat were cut on a cryostat (Leica CM 1510 S). The sections were incubated overnight at 4 °C with the primary antibody (anti-PGP9.5 antibody; Millipore, Tokyo, Japan). Alexa Fluor 594-coupled goat anti-mouse IgG antibody (Invitrogen, Carlsbad, CA, USA) was applied as the second antibody. IENFD was assessed as previously reported . Nerve fibers were counted blindly by three independent investigators under an FV10i confocal system (Olympus, Tokyo, Japan) and the average values were used.
mRNA expression of the hind limb skeletal muscles
Total RNA was extracted from the frozen samples of hind limb skeletal muscles and purified using an RNeasy kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan). Primers and probes for fibroblast growth factor 2 (FGF2; also known as bFGF), nerve growth factor (NGF), neurotrophin 3 (NT-3) and β 2microglobulin (Applied Biosystems, Foster City, CA, USA) were purchased. Real-time quantitative polymerase chain reaction (PCR) was performed using the ABI Prism 7000 (Applied Biosystems) and calculated by the ΔΔCt method.
Morphometrical analysis of the sural nerves
The sural nerves were fixed in 2% glutaraldehyde followed by osmium tetroxide and were embedded in Epon. Semi-thin sections (0.5-μm thick sections) were stained with toluidine blue and examined by light microscopy (Leica microsystems). The total complement of sural nerve myelinated fibers was assessed using the analysis software ImageJ (Research Services Branch of the National Institutes of Mental Health, Bethesda, MD, USA). The investigator was blinded to the group identity throughout the morphometric process.
Preparation of DPSC-conditioned media (DPSC-CM)
When DPSCs reached 70% confluence in 10-cm dishes, they were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing penicillin and streptomycin in a 5% CO2 humidified atmosphere at 37 °C. After 24 h, the culture media were collected, concentrated 10 times using 10-kDa centrifugal filters (Amicom Ultra-15, Nihon Millipore, Tokyo, Japan), and frozen at −20 °C until use.
Primary culture of dorsal root ganglion (DRG) neurons and evaluation of neurite outgrowth
DRG neuron cultures were prepared from 8-week-old male SD rats as previously described . DRG neurons were cultured for 24 h in serum-free medium (DMEM/F12 supplemented with B27 (Invitrogen)) for use in neurite outgrowth with DPSC-CM. DRG neurons were immunostained with rabbit polyclonal anti-neurofilament heavy-chain antibody (Millipore). Alexa Fluor 488-coupled goat anti-rabbit IgG antibody (Invitrogen) was applied as the second antibody. Coverslips were counterstained with 4’ ,6-diamidino-2-phenylindole (DAPI; Millipore). Neurite outgrowth was observed in 40–50 neurons per cover slip, and the total length and joint number of neurites was calculated by a computed image analysis system (Angiogenesis Image Analyzer Ver. 2, KURABO Industries, Osaka, Japan).
Cell viability assay in immortalized adult Fischer rat Schwann cells, IFRS1
IFRS1 cells, immortalized adult rodent Schwann cells, were seeded on plastic dishes and were maintained in DMEM containing 5% FBS, 20 ng/ml recombinant human heregulin-β (Upstate, Lake Placid, NY, USA), and 5 μM forskolin (Sigma) [16, 17]. IFRS1 cells were seeded in 96-well plates (5 × 103 cells/well), and were maintained in DMEM containing 1% FBS media. After 12 h, the cells were incubated with DPSC-CM, and the cell viability in 1-, 3-, and 5-day culture was assessed by the CCK-8 assay (Dojin Laboratories, Kumamoto, Japan). The absorbance at 450 nm of each well was read on a spectrophotometer. Three independent experiments were performed in quadruplicate.
Analysis of myelin-related protein formation in IFRS1 cells
IFRS1 cells were cultured with DPSC-CM in DMEM containing 1% FBS supplemented with 50 μg/ml ascorbic acid for 5 days. Western blot analyses were performed with the rabbit anti-myelin protein zero (MP0) polyclonal antibody (Abcam) and rabbit anti-β-actin monoclonal antibody (Cell Signaling Technologies, Beverly, MA, USA).
For immunostaining, 4% PHA-fixed cells were incubated with a 1:400 dilution of the MP0 substrate antibody. Alexa Fluor 568-coupled donkey anti-rabbit IgG antibody (1:200; Invitrogen) was applied at room temperature for 1 h. Conventional microscopy images were taken using the FV10i confocal system (Olympus).
All group values are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were made by Student’s t test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by the Bonferroni correction for multiple comparisons.