Neurovascular Factors in Wound Healing in the Foot Skin of Type 2 Diabetic Subjects

Twelve healthy control subjects (C group) and 12 subjects with type 2 diabetes and neuropathy (D group) were studied. Subjects were recruited on a consecutive basis from the diabetes outpatient clinics of the Ipswich Diabetes Centre. All subjects with diabetes selected for this study had peripheral neuropathy, as impaired wound healing is typically associated with this complication. Subjects with clinical features of peripheral vascular disease (ankle brachial pressure index <0.8) were excluded. The study was approved by the local ethical committee, and all subjects gave informed consent to take part in the study.

Neuropathy was assessed by measurement of the vibration perception threshold, using the ascending method of limits. A mean of three values was taken for analysis. The results were expressed in volts. A vibration perception threshold of ≥15 V (i.e., >95th percentile) for this age-group was considered abnormal (20).

In addition, sensation was assessed using the Neuropen (Owen Mumford, Oxford U.K.), which contains a 10-g monofilament to assess pressure perception and a Neurotip (Owen Mumford) for pinprick sensation. Ten-gram monofilaments were applied for 2 s on the plantar aspect of the first, third, and fifth metatarsal heads, and Neurotip was applied at the epinychium of the first toe (i.e., a total of four sites were tested, three for the 10-g monofilament and one for Neurotip). At sites where sensation was not felt, the test was repeated three times to confirm the abnormality. Subjects were assigned to have impaired sensation if they could not feel a stimulus on more than one of the tested sites. All diabetic subjects recruited had absent ankle reflexes, impaired sensation using the Neuropen, and impaired vibration perception threshold.

Subjects were allowed to acclimatize for 30 min in a temperature-controlled room, where the temperature was maintained at 25 ± 1°C. The foot temperature was measured proximal to the first and second metatarsal heads using an infrared thermometer (Linear Laboratories, Fremont, CA). Room temperature and relative humidity were monitored throughout. The axon-reflex–mediated LDI_flare was examined using an LDI (Moor Instruments, Devon, U.K.) (8). This uses a stable helium neon gas laser (λ = 632.8 nm) beam that is deflected by a moving mirror to create a raster pattern across the surface of the skin. The Doppler shifted light from moving blood, and nonshifted light from static tissue is directed back via the same mirror into two detectors. Fluctuations in the wavelength are processed to calculate the flux that is proportional to tissue blood flow. The data were recorded to a computer using MoorLDI (version 3.11) software, and a flux image was produced using a palette of 16 equally spaced colors in which dark blue represented lowest perfusion and red the highest perfusion.

The skin proximal to the first and second metatarsal heads on the dorsum of the foot was heated with a circular skin heater (diameter −0.9 cm) (Moor Instruments) to 44°C for 20 min. An area of 3.5 × 3.5 cm surrounding the heated skin was scanned with the LDI aligned to be perpendicular to the dorsum of the foot at a fixed distance of 30 cm, immediately after removing the heater probe. The scan images were stored in a computer and processed offline. On the flux image, the region of interest demarcated by the edge of the flare was drawn, and the area of the LDI_flare was calculated using MoorLDI (version 3.11) software. The results were expressed in centimeters squared.

The same flux image described above was also used to calculate the maximum hyperemia. A region corresponding exactly to the size of heater probe was defined, and the mean flux within that region was calculated using MoorLDI (version 3.11) software. This is the maximum hyperemic response that we have termed LDI_max. The results are expressed in arbitrary perfusion units.

Skin biopsies were performed using a sterile 3-mm biopsy punch (Stiefel Laboratories, Bucks, U.K.) in the same area in which the LDI_flare had been assessed on a separate day. No local anesthetic was applied and all subjects tolerated the biopsy. There was no infection or other adverse event.

Wound closure was assessed by digital microscopy at magnification ×50 immediately after biopsy, day 3, and day 10. Digital photographs were stored in the computer, and the wound area was analyzed offline using Mouseyes software. The computer monitor was calibrated and the region of interest drawn along the circumference of the wound to enable calculation of wound area expressed in millimeters squared.

The skin biopsy specimen was immediately fixed in 4% paraformaldehyde for 18–24 h, routinely processed (Citadel 2000 Processor; ThermoElectron, Waltham, MA), and embedded in paraffin wax. Serial 5-μm tissue sections were cut from each block (Microtome Leitz Wetzlas 1512) and mounted onto positively charged slides (Fisher Scientific, Loughborough, U.K.). Sections were dewaxed in xylene and gradually rehydrated through decreasing ethanol dilutions. Epidermal melanin was bleached with 0.25% KMnO₄ followed by 5% oxalic acid. Series selected for blood vessel density assessment by CD31/vWF immunolocalization underwent trypsinization. For VEGF-A and VEGF-R2, sections were microwaved to disclose the antigen, and, for HIF-1α, optimal visualization was obtained using a tyramide amplification reagent (CSA I; Dako). Sections were incubated overnight at 5°C with mouse monoclonal antibodies to CD31 and vWF (diluted 1:100 and mixed) (both from Dako) and to VEGFR-2 (1:50; Santa Cruz Biotechnology) and with rabbit polyclonal antibodies to VEGF-A (1:300; Santa Cruz Biotechnology) and to HIF-1α (1:300; Abcam). For nerve fiber density, sections were incubated overnight with 1:1,200 biogenesis polyclonal rabbit anti-human antibody (Serotec, Oxford, U.K.). Biotinylated swine anti-rabbit secondary antibody (1:300, 1 h) was then applied; sections were quenched with 1% H₂O₂ in 30% MeOH-PBS (30 min) before incubation for 1 h with 1:500 horseradish peroxidase streptavidin (Vector Laboratories, Peterborough, England). The reactions were demonstrated using the following, listed sequentially: biotinylated secondary antibodies, streptavidin horseradish peroxidase, and the chromogenic substrate 3′-3′diaminobenzidine (DAB; Sigma-Aldrich, Dorset, U.K.).

Patterns of immunostaining were examined by light microscopy. To quantify the amount of VEGF-A, VEGF-R2, and HIF-1α staining, microphotographs were taken using a Nikon digital camera mounted on a Leitz DM RB microscope. Percentages of stained area were quantified separately in the epidermis and in the upper dermis with the computer program Leica QWin Standard, version 2.4 (Leica Microsystem Imaging, Cambridge, U.K.), set to detect color intensities within a fixed, constant range. Blood vessel and nerve fiber cross-sections in the papillary dermis were counted manually and their density expressed as number per millimeter squared.

Descriptive statistics (median and interquartile range) were used to describe subject characteristics. Mann-Whitney U test was used to determine the differences between the groups. Mean ± SD for each variable is described, and a P value of <0.05 was considered significant. SPSS (version 11.0) software package was used for statistical analysis.

Clinical characteristics of diabetic and control subjects are shown in Table 1. All subjects were Caucasian, and there was no significant difference in age between groups C and D.

Wound closure (Fig. 1) determined by area change (mean ± SD) did not differ between diabetic patients (in mm²: day 0, 6.17 ± 0.5; day 3, 4.63 ± 0.4; and day 10, 2.93 ± 0.5) and control subjects (day 0, 6.28 ± 0.3; day 3, 4.89 ± 0.8; and day 10, 3.01 ± 0.7). There were no complications, and all wounds were fully reepithelialized by day 10.

LDI_max expressed as perfusion units (PU) was significantly reduced in the diabetic group (C: 577.4 ± 125.3 vs. D: 310.33 ± 97.3; P < 0.0001), whereas dermal blood vessel density (per mm²) did not differ between control subjects (116.5 ± 21.0 mm²) and diabetic patients (116.8 ± 27.8 mm²) (P = 0.96). LDI_flare was significantly reduced in diabetic patients compared with control subjects (in cm²: C: 5.2 ± 1.8 vs. D: 1.8 ± 0.7; P < 0.0001) as was dermal nerve fiber density (per mm²: C: 456.3 ± 160.1 vs. D: 216.0 ± 144.0; P = 0.001). LDI_flare was significantly associated with nerve fiber density (r = 0.6; P < 0.0001).

The expression of HIF-1α in epidermal vessels (C: 6.42 ± 6.32 vs. D: 8.68 ± 11.74; P = 0.63) and dermal vessels (C: 16.99 ± 15.98 vs. D: 10.22 ± 12.55; P = 0.14) did not differ significantly between control subjects and diabetic patients (Table 2). However, there was a significant difference in the expression of epidermal VEGF-A (C: 0.36 ± 0.30 vs. D: 0.16 ± 0.18; P = 0.03) and dermal VEGF-A (C: 0.04 ± 0.07 vs. D: 0.01 ± 0.004; P = 0.04). Also, epidermal blood vessel VEGFR-2 (C: 21.58 ± 25.99 vs. D: 9.66 ± 12.91; P = 0.05) and dermal blood vessel VEGFR-2 (C: 7.94 ± 6.88 vs. D: 3.45 ± 3.14; P = 0.04) expression were significantly reduced in diabetic patients compared with control subjects (Table 2, Fig. 2).

The pathophysiological mechanisms contributing to delayed wound healing in diabetes are complex and may be mediated by vascular, neuronal, cellular, and immune factors. Our study is unique, as we have quantified the wound-healing response and related it to neurovascular integrity and the expression of vascular factors central to the wound-healing response.

Against expectation and in contrast to findings in animal models and the observation of poor healing in diabetic patients with foot ulceration, the rate of wound closure was identical in diabetic and control subjects. It is important to note that we studied the healing response of an acute wound on the dorsum of the foot in an area that is not exposed to continued high pressure that occurs in chronic diabetic plantar foot ulcers. Whether acute wounds on the plantar surface behave differently or trigger factors such as infection, which could turn such wounds into chronic ulcers, remains to be determined. None of the wounds in the present study became infected, and great care was taken to ensure that the wounds were well protected.

Impaired hyperemic response to tissue injury and iontophoresis of acetylcholine in the presence of normal vascular density has led previous investigators to implicate functional microvascular defects in delayed wound healing in diabetic patients (21–23). However, this mechanism has only been inferred and never previously directly assessed.

While Veves et al. (24) previously demonstrated a reduction in endothelial nitric oxide synthase expression, few studies have explored in detail other molecular alterations that may be relevant to the wound-healing response following injury in diabetic patients. We believe such studies are essential if we are to gain an understanding of any perturbation in the wound-healing response following injury and development of an ulcer. It is known that VEGF expression is normally increased during the granulation phase of wound healing, and this response is diminished in diabetic mice (16). Furthermore, topical application of VEGF or overexpression of VEGF by an adenoviral vector markedly accelerates wound healing in diabetic animals (17,25). While adenovirus-mediated gene transfer of a soluble form of VEGFR-2 (Flk-1) reduces angiogenesis, it does not delay wound closure in db/db mice (26). Although tissue hypoxia, a typical feature of healing wounds, is thought to increase the expression of VEGF through HIF-1α (27), the role of HIF-1α in diabetic wounds has not been explored in experimental studies and in particular in diabetic patients. In the present study, we demonstrate no difference in HIF-1α expression per se between diabetic patients and control subjects.

Thus, despite an impaired maximal hyperemic response, wound healing was normal in our diabetic patients. Blood vessel density was similar in the control and diabetic groups, consistent with our previous findings in those with type 1 (28) and type 2 (24) diabetes. The normal vascular density may well have maintained skin oxygenation, as evidenced by comparable HIF-1α expression in both groups. Despite lower expression of VEGF and VEGFR-2 in diabetic skin, wound closure did not differ between diabetic patients and control subjects. This suggests that VEGF may play a limited role in acute wound healing in diabetic patients.

With regard to neuropathy, it may contribute to the development of foot ulceration via a loss of protective sensation and reduced axon reflex–mediated vasodilatation. Impaired expression and regulation of nerve growth factor and reduction in skin nerve density have been speculated to delay healing (29). We demonstrate a marked reduction in both dermal nerve fiber density and the axon reflex as assessed by LDI_flare. However, despite significant abnormalities in both parameters there was no impact on wound healing.

One of the perceived limitations of this study is that of studying an acute wound and expression of neuronal and vascular integrity at baseline, with healing by secondary intention compared with chronic wounds in a typical diabetic foot ulcer. However, this is no different than all experimental studies where wounds are also acute and yet the wound healing response is delayed. Thus, we believe our study has provided important translational insights and questioned established concepts of wound healing mainly derived from studies in experimental animals. This study also establishes the safety of distal skin biopsies in the assessment of diabetic neuropathy. Due to the relatively small number of study subjects, further larger studies may be needed to confirm the findings.

In conclusion, this study suggests that wound closure in subjects with type 2 diabetes is not delayed despite significant alterations in neurovascular function and structure. This reiterates the importance of pressure relief in those with neuropathic ulcers, restoration of adequate blood flow in those with ischemic ulceration, and aggressive treatment of wound infection as the principal strategies to successfully heal diabetic wounds.