OBJECTIVE—Peripheral neuropathy is the most frequent neurological complication in diabetic patients. The diagnosis is established by both clinical neurological examination and demonstration of reduced epidermal nerve fibers in skin biopsies (1). Whereas the decrease of free nerve endings has been extensively studied in diabetic patients (2,3), no data are available on possible changes of terminal Schwann cells. Besides their role as scaffold for peripheral nerves, they also play an important role in supporting survival and function of peripheral nerves (4).

RESEARCH DESIGN AND METHODS—We analyzed the subepidermal nerve plexus in dermal sheet preparations of deceased diabetic and nondiabetic patients by immunostaining for detection of the neural cell adhesion molecule and quantification of the subepidermal nerve plexus.

RESULTS AND CONCLUSIONS—The subepidermal nerve plexus, comprising nerve fibers and ensheathing Schwann cells, was significantly reduced in diabetic patients. Whether the reduction in terminal Schwann cells is cause or consequence of the loss of peripheral nerve fibers remains to be investigated.

We have recently established a novel method for analyzing the skin nerve plexus in its entirety (5). Using this method, we have investigated skin derived from the forefoot of deceased patients with diabetes and demonstrate that both axons and terminal Schwann cells are greatly reduced compared with those in nondiabetic control subjects.

Skin biopsies were performed at autopsy on 12 patients with type 2 diabetes (seven male and five female) aged 50–98 years (mean ± SD age 75.6 ± 12.8 years). Disease duration ranged from 13 to 24 years. No data on the presence of clinically manifest diabetic polyneuropathies were available. The control group consisted of nine patients (four male and five female) aged 60–99 years (75.6 ± 12.3 years) without diabetes. Punch biopsies (6 mm) were taken from the back of the left foot 30 mm proximal to the second toe.

The dermal sheets were prepared as previously described (5). Immunohistochemical staining was performed with anti-neural cell adhesion molecule (NCAM/CD56) (Becton Dickinson, San Jose, CA) as primary antibody, detecting an antigen expressed by Schwann cells and unmyelinated axons (6). As secondary antibody, a biotinylated sheep anti-mouse (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) was used, followed by incubation with the StreptABComplex (DAKO, Glostrup, Denmark) and with DAB Chromogen tablets (DAKO, Carpinteria, CA). As negative control, an isotype-matched mouse antibody (Coulter Corporation, Miami, FL) was used. Three computerized image analysis stacks of visual fields were acquired per sample with a spot camera RT-slider 2000 (Diagnostic Instruments, Sterling Heights, MI) attached to an Olympus Provis AX70 microscope (Olympus Austria, Vienna, Austria) with a 20× objective. In the 2-dimensional projection of the stacks, all nerve fibers were marked manually with Adobe Photoshop CS (Adobe Systems Austria, Vienna, Austria) and the total axonal length of the subepidermal nerve plexus per millimeter squared of skin was measured with Meta Morph Software Version 4.5 (Universal Imaging Corporation, West Chester, PA). In the same images, Schwann cells, as defined by a nucleus within the immunostained boundaries of a nerve fiber, were counted manually. Statistical analysis was done with StatView software (SAS Institute, Cary, NC).

Immunohistochemical staining of dermal sheets for NCAM/CD56 depicted a dense and regular nerve fiber network in dermal sheets of seven of nine control patients (Fig. 1A). In two of nine, the subepidermal nerve plexus was less dense but nevertheless regular. By contrast, in all diabetic patients, the supepidermal nerve network was more loosely and irregularly distributed, with fewer ramifications overall (Fig. 1B). Over wide stretches, the plexus was only composed of fragments and was even completely missing in some areas (Fig. 1B). The mean ± SD nerve fiber length was 48.52 ± 7.15 mm/mm2 (Fig. 1C) with a range from 35.07 ± 3.32 to 58.35 ± 1.50 mm/mm2 in the control patients. In diabetic patients, nerve fiber length was considerably lower at only 26.38 ± 7.78 mm/mm2 (Fig. 1C) with a range from 10.33 ± 1.17 to 35.70 ± 5.53 mm/mm2. Statistical analysis by Mann-Whitney U test comparing both groups revealed a highly significant difference with a P value of 0.0002. Neither age (Fig. 1E) nor sex showed an influence on the length of the subepidermal nerve plexus in both groups.

The mean number of Schwann cells/1 mm2 dermis was 216 ± 34 (range 170 ± 6–278 ± 8) in control patients (Fig. 1D). The subepidermal nerve plexus of diabetic patients contained less than half as many Schwann cells, with a mean of 107 ± 29 per millimeter squared (Fig. 1D) (68 ± 3 to 162 ± 20). The difference was statistically significant. with a P value of 0.0001. Age had no influence on the Schwann cell number in either group (Fig. 1F). In the control group, men had significantly fewer Schwann cells (192 ± 16) than women (235 ± 33) (P value = 0.0275). This difference might be attributed to the small size of the group. No sex-related differences were seen in diabetic patients.

In contrast to conventionally used thick sections of skin for analysis and quantification of intraepidermal nerve endings (2,3), dermal sheet preparations as proposed here allow for the evaluation of much larger parts of the skin's nerve plexus, since the surface of a dermal sheet from a 6-mm punch biopsy comprises 28.27 mm2 compared with ∼0.6 mm2 in the case of a thick section. Using this approach, we were able to demonstrate a significant reduction in the length of nerve fibers and, for the first time, a distinct loss of Schwann cells in the subepidermal nervous plexus of diabetic patients compared with nondiabetic control subjects.

In the subepidermal nerve plexus, the bulk of nerve fibers are classified as small fibers comprising nonmyelinating C-fibers (7) and A-δ fibers. An abnormal function of C-fibers has recently been demonstrated by microneurographic recordings in patients with diabetic neuropathy (8). The extensive nerve fiber loss in the subepidermal nerve plexus seen in our sample of diabetic patients might represent abnormal function of C-fibers’ morphologic counterpart.

Pathogenetically, the extensive loss of terminal Schwann cells that we observed in the subepidermal nerve plexus of diabetic patients could occur as a consequence of axonal degeneration or, alternatively, as an effect of the metabolic dysregulations in diabetes (9). In such a scenario, Schwann cell damage might even precede axonal damage. Because the rate of reinnervation is remarkably slower in the absence of dermal Schwann cells (10), their loss might also have a considerable impact on impaired nerve fiber regeneration in diabetes.

Figure 1—

Depiction of subepidermal nerve fibers and Schwann cells in dermal sheet preparations by immunostaining for NCAM/CD56. A: The subepidermal nerve plexus in a nondiabetic control patient is represented as a dense and regular network of nerve fibers ensheathed by Schwann cells. Branching of thick nerve fibers into thinner fibers can easily be discerned. Protrusions of Schwann cell nuclei (arrow) were present at regular intervals. The total nerve fiber length was 50.68 mm/mm2; 211 Schwann cells were detected per millimeter squared. B: In a representative diabetic patient, the subepidermal nerve plexus consisted of a sparse nerve fiber network dominated by thicker fibers with few ramifications. Schwann cell nuclei were sporadically present. The total nerve fiber length amounted to 22.26 mm/mm2 and 89 Schwann cell nuclei/mm2 were found. Scale bar: 100 μm. One protrusion containing a Schwann cell nucleus is shown as an example in A and B (arrow). C: Statistical analysis of the nerve fiber length of the subepidermal nerve plexus per millimeter squared of skin in diabetic patients compared with that of nondiabetic patients. The mean nerve fiber length for both groups is shown in box diagrams. In the diabetic group, the mean ± SD nerve fiber length was 26.38 ± 7.77 mm/mm2, which was significantly lower than that in the control group (48.52 ± 7.15 mm/mm2) (P = 0.0002; Mann-Whitney U test). D: Statistical analysis of the Schwann cell number contained in the subepidermal nerve plexus per millimeters squared of skin in diabetic patients compared with that in nondiabetic patients. In the diabetic group, the mean Schwann cell number was 107 ± 29 per millimeters squared compared with 216 ± 34 per millimeters squared in the control group (P = 0.0001; Mann-Whitney U test). E: Regression analysis of the nerve fiber length per millimeters squared with respect to the age of the individuals. No correlation could be found between these two parameters in the diabetic group (P = 0.2174) or the control group (P = 0.3716). F: Regression analysis of the Schwann cell number per millimeters squared with respect to age. No correlation could be found between these two parameters in the control group (P = 0.8774) or the diabetic group (P = 0.6672).

Figure 1—

Depiction of subepidermal nerve fibers and Schwann cells in dermal sheet preparations by immunostaining for NCAM/CD56. A: The subepidermal nerve plexus in a nondiabetic control patient is represented as a dense and regular network of nerve fibers ensheathed by Schwann cells. Branching of thick nerve fibers into thinner fibers can easily be discerned. Protrusions of Schwann cell nuclei (arrow) were present at regular intervals. The total nerve fiber length was 50.68 mm/mm2; 211 Schwann cells were detected per millimeter squared. B: In a representative diabetic patient, the subepidermal nerve plexus consisted of a sparse nerve fiber network dominated by thicker fibers with few ramifications. Schwann cell nuclei were sporadically present. The total nerve fiber length amounted to 22.26 mm/mm2 and 89 Schwann cell nuclei/mm2 were found. Scale bar: 100 μm. One protrusion containing a Schwann cell nucleus is shown as an example in A and B (arrow). C: Statistical analysis of the nerve fiber length of the subepidermal nerve plexus per millimeter squared of skin in diabetic patients compared with that of nondiabetic patients. The mean nerve fiber length for both groups is shown in box diagrams. In the diabetic group, the mean ± SD nerve fiber length was 26.38 ± 7.77 mm/mm2, which was significantly lower than that in the control group (48.52 ± 7.15 mm/mm2) (P = 0.0002; Mann-Whitney U test). D: Statistical analysis of the Schwann cell number contained in the subepidermal nerve plexus per millimeters squared of skin in diabetic patients compared with that in nondiabetic patients. In the diabetic group, the mean Schwann cell number was 107 ± 29 per millimeters squared compared with 216 ± 34 per millimeters squared in the control group (P = 0.0001; Mann-Whitney U test). E: Regression analysis of the nerve fiber length per millimeters squared with respect to the age of the individuals. No correlation could be found between these two parameters in the diabetic group (P = 0.2174) or the control group (P = 0.3716). F: Regression analysis of the Schwann cell number per millimeters squared with respect to age. No correlation could be found between these two parameters in the control group (P = 0.8774) or the diabetic group (P = 0.6672).

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Published ahead of print at http://care.diabetesjournals.org on 10 March 2008. DOI: 10.2337/dc07-1832.

C.M.R. and H.T. contributed equally to this study.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.