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Distal degeneration of sensory and autonomic cutaneous nerve fibres in systemic sclerosis
  1. V Provitera1,
  2. M Nolano1,
  3. N Pappone2,
  4. C di Girolamo3,
  5. A Stancanelli1,
  6. F Lullo1,
  7. C Crisci4,
  8. L Santoro5
  1. 1Neurology Division, “Salvatore Maugeri” Foundation, IRCCS, Centre of Telese Terme, Italy
  2. 2Rheumatology Division, “Salvatore Maugeri” Foundation, IRCCS, Centre of Telese Terme, Italy
  3. 3Rheumatology Unit, “G Rummo” Hospital, Benevento, Italy
  4. 4Clinic Centre, Private Hospital for Rehabilitation, Naples, Italy
  5. 5Department of Neurological Sciences, University of Naples “Federico II”, Italy
  1. Correspondence to:
    Dr V Provitera
    Neurology Division, “Salvatore Maugeri” Foundation, IRCCS, Via Bagni Vecchi 1, 82037, Telese Terme (BN), Italy; vproviterafsm.it

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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).

Table 1

 Clinical and morphological data in sclerodermic patients compared with mean values in the control group

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.

Figure 1

 Confocal micrographs of cutaneous innervation in thigh skin samples. (A) Normal skin, (C) affected, and (E) apparently unaffected skin from a patient with SSc: samples are triple stained to visualise nerve fibres (rPGP in yellow-green), basement membrane and vessels (mColIV in red), and endothelium and epidermis (ULEX Europaeus in blue). Sweat gland images in a healthy subject (B) and in sclerodermic patients from affected (D) and apparently normal (F) skin, are double stained to visualise nerve fibres (rPGP in yellow-green) and vessels (mColIV in red). A derangement of dermal architecture, of subepidermal neural plexus, and a marked reduction of ENF density are evident in affected and, although to a lesser extent, in apparently normal skin of patients with SSc (C and E compared with A). A loss of nerve fibres innervating dermal annexes such as arrector pilorum muscles (E compared with A) and sweat glands (D and F compared with B) in skin from sclerodermic patients is also evident. Bar  =  50 µm. E, epidermis; ENFs, epidermal nerve fibres; BM, basement membrane; V, blood vessels; SNP, subepidermal neural plexus; APM, arrector pilorum muscle.

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.

DISCUSSION

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.

View Abstract

Footnotes

  • Competing interest statement: No author has any competing interest regarding this report.

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