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We studied innervation and dermal vasculature in affected and apparently normal skin of sclerodermic patients to evaluate the involvement of different nerve fibre groups and to determine a possible correlation with vascular damage in this disease. Immunohistochemical analysis and confocal microscopic examination of skin biopsy samples were used.
METHODS AND RESULTS
We obtained 3 mm punch skin biopsy samples from the distal thigh and distal leg in 11 consecutive 34–70 year old female patients with systemic sclerosis (SSc), identified by the American College of Rheumatology classification criteria.1 We excluded patients who had been exposed to potentially neurotoxic exogenous or endogenous conditions. The skin appeared sclerotic in 4/11 patients in the leg and in 3/11 in the thigh (table 1). In four patients a further skin sample from fingertip was taken to evaluate myelinated fibres. None of the patients complained of sensory disturbances, and neurological and neurophysiological evaluations were normal except in two patients, in whom a conduction velocity study showed the presence of an entrapment syndrome. Patient morphological findings were compared with data from a group of 16 healthy volunteers (nine male, seven female, age range 34–65 years).
Skin biopsy specimens were processed according to previously published procedures.2 Floating sections were immunostained using a panel of primary antibodies, including the pan-neuronal marker anti-protein gene product (PGP) 9.5, anti-myelin basic protein for myelinated fibres, anti-vasoactive intestinal peptide (VIP) to mark autonomic nerve fibres, and anti-collagen IV to visualise basement membrane and blood vessels.
We quantified, as previously described, epidermal nerve fibres (ENFs) per linear millimetre,3 Meissner corpuscles (MCs), and myelinated papillary endings per square millimetre4 on confocal images using image analysis software (Neurolucida, Microbrightfield Inc, Colchester VT, USA; ScionImage, Scion Corporation, Frederick, MD, USA). On the same images used to quantify ENF density, we measured blood vessel density in µm2/100 µm2 of dermal tissue within 250 µm below the basement membrane.
We found a significant loss of ENFs in sclerodermic patients in all the examined sites (table 1) without a distal-proximal gradient, a poor subepidermal neural plexus, and a reduced innervation of sweat glands, blood vessels, and arrector pilorum muscles compared with controls. These findings, evident in apparently unaffected areas (figs 1E and F compared with 1A and B), were more severe in clinically involved skin (figs 1C and D compared with 1A and B) and affected both sensory and autonomic unmyelinated nerve fibres as demonstrated by PGP and VIP immunostainings.
The mean (SD) density of blood vessels measured in µm2/100 µm2 of dermal tissue, was 6.4 (2.9) and 8.7 (4.7), respectively, in the thigh and leg of patients with SSc. These values significantly correlated with the density of epidermal nerve fibres in both sites (r2 = 0.51; p<0.05 at the thigh and r2 = 0.58; p<0.05 at the leg). In glabrous skin we found a significant reduction of MC density compared with controls, with a number of intrapapillary myelinated fibres still within the normal range. Moreover, evident structural abnormalities of the surviving mechanoreceptors and predegenerative aspects of myelinated fibres, such as swellings or vacuolisation, were present.
Our data indicate that the cutaneous nerves in SSc are impaired. This mainly involves the unmyelinated sensory and autonomic nerve fibres, but does not completely spare the large fibres. The observation that the loss of ENFs was more significant in subjects with an evident reduction of vascular bed suggests that ischaemia may have a role in determining the neuropathic process. However, we cannot rule out the possibility that early biohumoral changes, demonstrated in apparently unaffected skin,5,6 may induce both neural and vascular damage. We speculate that the abnormalities of terminal innervation seen in the skin may be present in multiple organs in SSc. This neuropathic process, affecting primarily unmyelinated nerve fibres, may contribute to the production of abnormalities that are common in SSc, like visceral dysmotility and cardiac arrhythmias.
Competing interest statement: No author has any competing interest regarding this report.
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