Induction of diabetes and wounding
All the procedures involving animals and their care were conducted in accordance with international guidelines, laws and policies and with the National Institutes of Health Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985). The protocol was authorised by our local institutions. Ten- to twelve-week-old C57BL6 mice (in-house colony) were used. Diabetes was induced by a single intraperitoneal injection of 150 mg/kg streptozotocin (STZ) in pH 4.5 citrate buffer. Hyperglycaemia was confirmed after 4 and 7 days by testing blood glucose using a commercially available glucose meter (Glucocard G-Sensor; Menarini, Florence, Italy). Mice with blood glucose >16.7 mmol/l (300 mg/dl) were used. To rule out a persistent effect of STZ on immune/inflammatory blood cells that might bias results from the present study, in separate experiments (n = 6) we analysed white blood cell subpopulations, CD34+ cells and apoptotic CD34+ cells at baseline, 3, 5, 7 and 30 days after STZ injection.
After 4 weeks of hyperglycaemia, skin wounds were created on the dorsal surface of the right hindlimb using a 4 mm punch biopsy device (H-S Medical, Colton, CA, USA). At the time of ulceration, capillary glucose was measured and was confirmed to be >16.7 mmol/l. Wounds were monitored by taking high resolution frontal photos every 2–3 days using a digital camera (Coolpix; Nikon, Torino, Italy) and the area was calculated relative to a millimetre reference using the image processing software ImageJ (Research Services Branch, National Institute of Mental Health, Bethesda, MD, USA). Diabetic and control animals were used either to determine wound closure times (n = 8 per group) or for histological analyses (n = 6 per group). At baseline, 1 and 4 days after wounding, blood samples were drawn for determination of EPC levels by flow cytometry (n = 6 per group). Based on preliminary data, we expected this sample size to be sufficient to detect a 25% difference in mid-time wound healing, as well as a 30% difference in granulation tissue thickness. We chose the limb wound in a previously validated type 1 diabetic animals model , proven suitable to study the altered diabetic wound healing process.
Bone marrow transplantation
In separate experiments, wild-type mice (n = 6) were myeloablated with a sublethal dose of intraperitoneal cyclophosphamide (350 mg/kg). Meanwhile, BM cells were prepared from C57BL/6 mice constitutively producing green fluorescent protein (GFP) (in-house colony) by flushing femurs and tibia. Twenty-four hours after myeloablation, GFP+ cells were intravenously infused in myeloablated mice. Four weeks after BM transplantation, recovery and chimerism were assessed by flow cytometry on a peripheral blood sample, by looking at the GFP signal. Then, animals were either injected with STZ (n = 3) or vehicle (n = 3). Four weeks later, wounds were created as described above and tissues collected on day 1 and 4 and after complete healing. We calculated that this sample size had 80% power to detect a significant 35% difference in the number of tissue GFP+ cells after wounding in diabetes vs. controls.
Half-closed time was calculated as the time (in days) after which the wound area was reduced to 50%. Wound tissue and the surrounding 2–4 mm tissues, as well as deep planes, were then removed en bloc for histological analyses and immunofluorescence. The wound tissue block was transversally sectioned at the equator of the wound to standardise the wound area to be analysed. Cryosections, 7 μm thick, were stained with haematoxylin and eosin, and Masson’s trichrome, using commercially available kits (Bio-Optica, Milan, Italy) according to the manufacturer’s instructions. Within each section, wound edges and the granulation tissue were identified under low magnification; within the granulation tissue of each section, measurements were performed based on 10 random microscopic fields to minimise operator-dependent selection of areas. Thickness of the granulation tissue was measured in cross-sections, as the connective tissue stained blue with Masson’s trichrome between epithelium and the underlying muscle fascia. Vascular density within the granulation tissue was determined by staining with GSL I-isolectin B4 (Vector Labs, Burlingame, CA, USA). Apoptosis of granulation tissue cells was detected with Apoptag Green Plus In situ Apoptosis Detection Kit (Millipore, Vimodrone, Milan, Italy) and counted in ten high-power random fields per slice.
Wound tissues excised from GFP+ BM transplanted mice were stained with a primary anti-von Willebrand factor (vWf) rabbit polyclonal antibody (dilution 1:150; Abcam, Cambridge, MA, USA) and a secondary TRITC-conjugated anti-rabbit Ig. BM-derived cells in the wound tissue were identified based on the endogenous GFP fluorescence. Total GFP+ cells (all BM-derived cells) and GFP+vWf+ cells (BM-derived endothelial cells) were counted in 10 randomly selected high-power fields per slice, and normalised for the degree of chimerism. The percentage of GFP+ cells co-expressing vWf were also counted. Apoptosis of GFP+ cells was assessed in histological sections using the Apoptag Red Plus In situ Apoptosis Detection Kit (Millipore) and identified as double (red and green) fluorescent nucleated cells. Nuclei were counterstained in blue with Hoechst 33258 (Sigma-Aldrich, Schnelldorf, Germany). Proliferating cells were identified in histological sections by the presence of histone H3 phosphorylated at serine 10, using a rabbit polyclonal histone H3 phospho S10 primary Ab, with dilution 1:250 (ab47297; Abcam, Cambridge, UK) and a secondary swine anti-rabbit TRITC-conjugated polyclonal Ab (Dako Cytomation, Glostrup, Denmark) with dilution 1:100. We counted the number of total proliferating cells (labelled in red) in each section, as well as cells showing double fluorescence for GFP and H3S10, representing proliferating BM-derived cells, in ten random high-power fields per slice.
Quantification of circulating EPCs
Mouse circulating EPCs were quantified using flow cytometry on the basis of the expression of the stem cell antigen CD34 and of the endothelial antigen fetal liver kinase (FLK)-1. This phenotype is reminiscent of the CD34+KDR+ cell population, which is considered the best human EPC phenotype . Peripheral blood was collected before wounding and 4 days later. After erythrocyte lysis, 150 μl of blood was incubated with 10 μl of Alexa Fluor 647 rat α-mouse CD34 Ab (Beckton Dickinson, Franklin Lakes, NJ, USA) and 10 μl of Alexa Fluor 488 rat α-mouse FLK-1 Ab (BioLegend, San Diego, CA, USA). The frequency of peripheral blood cells positive for the above reagents was determined by a two-dimensional side scatter fluorescence dot plot analysis, after appropriate gating to exclude granulocytes. Initially we gated CD34+ peripheral blood cells and then examined the resulting population for dual expression of FLK-1. Mouse white blood cell subpopulations were assessed at different time points after STZ administration by FACS according to side scatter and forward scatter properties of lymphocytes, monocytes and granulocytes. Apoptosis of CD34+ cells was analysed after gating on CD34+ events based on Annexin V (Becton Dickinson) binding to externalised phosphatidylserine. Data were processed using the Macintosh CELLQuest software programme (Beckton Dickinson).
Data are expressed as mean ± standard error. Differences between means were assessed using two-tailed unpaired Student’s t test for two independent groups and paired Student’s t test for two repeated measures. When there were more than two repeated measures, we used repeated measures ANOVA with the post hoc Hochberg procedure. Statistical significance was accepted at p < 0.05.