Damage of cutaneous peripheral nervous system evolves differently according to the disease phase and subset of systemic sclerosis

L. Ibba Manneschi, A. Del Rosso1, A. F. Milia1, A. Tani, D. Nosi, A. Pignone1, S. Generini1, R. Giacomelli2 and M. Matucci Cerinic1

Department of Anatomy, Histology and Forensic Medicine and 1 Department of Internal Medicine, Section of Rheumatology, University of Florence, Florence and 2 Department of Internal Medicine, University of L'Aquila, L'Aquila, Italy.

Correspondence to: L. Ibba Manneschi, Department of Anatomy, Histology and Forensic Medicine, Viale Morgagni, 85, 50134, Florence, Italy. E-mail: ibba{at}unifi.it


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. Evidence shows that peripheral nervous system (PNS) is involved in systemic sclerosis (SSc), but few morphological studies have assessed the ultrastructural pathological modifications. The aim was to study ultrastructural modifications of skin PNS fibres in SSc according to subsets [limited SSc (lSSc) and diffuse SSc (dSSc)] and phases (early and advanced) of the disease.

Methods. Skin biopsies were taken from the forearms of 23 SSc patients (11 lSSc and 12 dSSc) and 10 controls. Each biopsy was processed for transmission electron microscopy (TEM).

Results. At TEM, observation in skin from early lSSc, signs of inflammation were evident, while PNS fibres were not damaged. The microvascular wall showed hypertrophic endothelial cells bulging into the lumen. In advanced lSSc, fibrosis prevailed on inflammation and slight ultrastructural alterations of PNS fibres were evident in the papillary derma. In early dSSc, ultrastructural alterations of PNS fibres, similar to those observed in the advanced phase of lSSc, were found together with signs of inflammation and fibrosis. In advanced dSSc, in the papillary and reticular dermis PNS fibres were reduced and showed relevant ultrastructural alterations.

Conclusions. In SSc, PNS ultrastructure damage is linked to the progression and severity of skin involvement. The alterations evolve from the early to the advanced phase mainly in the diffuse subset. In particular, the severe PNS lesions found in advanced lSSc are already present and widely diffuse in early dSSc and the microvascular involvement in early lSSc seems to precede the modification of the PNS in the skin. Thus, an early therapeutic approach can be useful to reduce the progression of PNS and skin damage in SSc patients.

KEY WORDS: Systemic sclerosis, Peripheral nervous system, Transmission electron microscopy, Skin


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic sclerosis (SSc) is a connective tissue disorder of unknown aetiology characterized by microvascular and fibrotic changes in skin and internal organs [1]. In SSc, skin damage consists in progressive dermal thickening and epidermal thinning, ultimately leading to skin tightening.

In both limited and diffuse SSc (lSSc and dSSc) [2], the early phases are characterized by oedema and inflammatory infiltrate in the perivascular space, whereas in the advanced stages flattening of the dermo-epidermal papillae, abnormal deposition of collagen bundles and loss of skin appendages are present [3].

There are several morphological studies on microvascular changes in SSc skin [4–6], whereas few reports on peripheral nervous system (PNS) ultrastructure are available [3, 7–9].

The skin, as a sensory organ, shows a rich network of nerve fibres, nerve endings and specialized receptors. Normally, the nerve fibres run through the epidermis towards the corneal layer. A nerve network is found around skin appendages and blood vessels, with a relevant role in the regulation of artero-venous anastomosis of the dermis [10, 11].

In SSc, the involvement of cranial and spinal nerves is not unusual. Sometimes peripheral polyneuropathy may be the first symptom of the disease [12], and median nerve entrapment precedes disease onset by a number of years [9, 13–15].

In SSc, the distribution and morphology of dermal nerve fibres were described by light microscopy in 1952 [16]. Peptide-immunoreactive nerves in patients with Raynaud's phenomenon and SSc show a generalized decrease of PGP 9.5, CGRP and VIP fibres in digital skin [17, 18].

Autonomic nerve dysfunction (parasympathetic impairment and orthosympathetic overactivity) is involved in Raynaud's phenomenon [19], dysfunction of oesophageal and gastrointestinal motility [20], modification of cardiac rhythm (R–R interval at ECG) [21], pupillary function [22, 23] and the regulation of lung function [24].

Despite the involvement of the PNS in SSc, its role in the pathogenesis and progression of the disease is still a matter of debate. PNS involvement is considered by some authors as important in both the onset and maintenance of the disease, and by others just as a secondary phenomenon [12, 25].

The aim of our work was to identify the ultrastructural modifications of PNS in the skin of SSc patients, according to the different phases and subsets of the disease.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
SSc patients and controls
Twenty-three SSc patients (19 females and four males, mean age 54.8 ± 7.6 yr), attending the outpatient clinic of the Section of Rheumatology of the Department of Medicine at the University of Florence were enrolled.

In order to have two comparable groups according to disease subsets and phases, 12 patients affected by dSSc and 11 by lSSc [2] were chosen. Patients of both subsets (lSSc and dSSc) were further classified, according to disease duration, into early (five lSSc, six dSSc) and advanced (six patients) phases [26].

Each SSc patient was assessed according to the recent guidelines [27] by evaluating disease duration (assessed from the first symptom to appear after the onset of Raynaud's phenomenon), autoantibodies (ANA, ACA, anti-Scl70) and involvement of microvessels (by nailfold videocapillaroscopy, scored according to Cutolo et al. [28]), skin (Rodnan modified skin score) [29], lung, heart, kidney and oesophagus (evaluated in 15 out of 23 patients).

Before biopsy, all SSc patients underwent 15 days of drug washout, when only proton pump inhibitors and clebopride were allowed. No patients had nerve involvement according to symptoms, clinical examination and electromyography.

Ten age- and sex-matched subjects (eight females and two males; mean age 53.8 ± 6.5 yr), not affected by dermatological inflammatory or autoimmune diseases, undergoing surgery for traumatic lesions, were used as controls.

Enrolled subjects gave their written informed consent. The Italian law and the ethical guidelines of the Italian National Medical Council were followed throughout the clinical and laboratory procedures.

Biopsies and transmission electron microscopy
Full thickness biopsies of clinically involved skin (skin score ≥2 according to the Rodnan modified method), approximately 1 x 0.5 cm, were taken from the middle third of the forearm of SSc patients. Biopsies from controls were obtained from the same anatomical area.

Biopsies were immediately fixed in cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at room temperature and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, at room temperature. Specimens were routinely processed and embedded in Epon 812. Semithin sections, 2 µm thick, were cut and stained with toluidine blue–sodium tetraborate and observed under a light microscope (Nikon Eclipse E400). Ultrathin sections (1 µm) were obtained from pathological areas chosen after observation by light microscopy and stained with uranyl acetate and alkaline bismuth subnitrate for examination under a transmission electron microscope (TEM) (Jeol 1010, Tokyo, Japan).

Statistical analysis
Data from SSc patients and healthy controls are expressed as mean ± S.D. (for continuous variables) and as ratios (for binomial variables) and compared by Student's t and {chi}2 tests, respectively. Statistics are considered significant for P values <0.05.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The clinical characteristics of the patients are summarized in Table 1. Patients with dSSc presented higher skin scores than patients with lSSc (P<0.001). ACA were more frequent in lSSc than in dSSc, while anti-Scl70 were more frequent in dSSc than in lSSc (P<0.05 in both cases). lSSc and dSSc patients were not different according to any other laboratory and clinical variable.


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TABLE 1. Demographic and clinical characteristics of SSc patients and controls

 
Limited SSc
Early phase
Light microscopy
In involved areas, relevant dermal inflammatory infiltrate and oedema were observed, particularly around microvessels. Hair follicles and sebaceous and sweat glands were still innervated by an extensive network of nerve fibres.

Cutaneous changes had a patchy distribution: areas with pathological features were close, with abrupt transition, to healthy-looking areas that were not different from control biopsies.

TEM
Oedema and inflammatory cells (monocytes, neutrophils, mast cells, lymphocytes, macrophages and dendritic cells) were found in perivascular area (Fig. 1A). The lumen of microvessel was occluded by hypertrophic endothelial cells (Fig. 1B). At the dermo-epidermal junction level, lymphatic vessels were enlarged (Fig. 1C). In the papillary dermis, a relevant and diffuse oedema was visible. Healthy-looking areas did not differ from control skin. Skin innervation was maintained and no evident structural alterations were detected (Fig. 1C).



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FIG. 1. (A–C) Skin of an early lSSc patient. (A) Oedema and inflammatory cells are widely found in the perivascular area. Note the monocyte (M), active mast cells (MC) and dendritic cell (DC). (B) Two microvessels; hypertrophic endothelial cells occlude their lumen. (C) Lymphatic vessel with an enlarged lumen (asterisk) close to a well-preserved bundle of unmyelinated fibres (arrow). (D, E) Skin of an advanced lSSc patient. (D) Bundle of unmyelinated fibres; some of them show rarefied axoplasm (arrow). (E) Nerve with unmyelinated fibres. Note oedema in the endoneurium (asterisk) and in the perineurium (arrowhead). TEM; A, x2500; B, x5000; C, x5000; D, x10 000; E, x5000.

 
Advanced phase
Light microscopy
In affected areas, the thickness of epidermis was reduced, dermo-epidermal ridges were lost and papillae flattened. In the dermis, abundant collagen bundles were distributed irregularly. The patchy distribution of cutaneous changes was still evident.

In affected skin areas, nerve fibres were sparse, discontinuous, and rarely reached and crossed the dermo-epidermal junction. The nervous network around the remaining appendages was discontinuous. In spared areas, the innervation was normal.

TEM
Dermal inflammatory infiltrate was absent and replaced by an abnormal deposition of collagen bundles and elastin. Some mast cells, with evident and regular granules in their cytoplasm, were still found. The lumen of capillaries was occluded by hypertrophic endothelial cells. Basal membranes were often delaminated and thickened.

Nerve endings were reduced but not altered. The bundles of unmyelinated fibres were present in the middle part of the papillary dermis and showed ultrastructural alterations, such as rarefaction of the axoplasm and scarcity of neurotubules (Fig. 1D). In small nerves, the oedema separated both the fibres in the endoneurium and the cells in perineurium (Fig. 1E).

Diffuse SSc
Early phase
Light microscopy
In dSSc, pathological skin changes were diffuse and homogeneously distributed. The thickness of epidermis was reduced, the dermo-epidermal ridges were lost and the papillae flattened. In the dermis, inflammatory infiltrate accompanied by oedema was observed. The concomitant presence of collagen bundles, irregularly distributed, indicates that the fibrotic process, differently from lSSc, starts in the early phase. Hair follicles, sebaceous and sweat glands were sparse. Fibres innervating these structures, although scant, appeared dotted, similar to those observed in healthy controls.

TEM
Most of microvessels were occluded in their lumen by hypertrophic endothelial cells and showed delaminated basal membranes (Fig. 2A). In the dermis, activated and degranulating mast cells, mononuclear perivascular inflammatory cells, oedema and diffuse abnormal bundles of collagen were detected.



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FIG. 2. Skin of an early dSSc patient. (A) Microvessel with an occluded lumen shows delaminated basal membrane (arrow). (B) Bundle of unmyelinated nerve fibres. Some of them show oedematous axoplasm and loss of neurotubules and neurofilaments (arrows). (C) Nerve with unmyelinated fibres is surrounded by oedematous connective tissue. The nerve shows oedema in the endoneurium (asterisk) and between the perineurium layers (arrowhead). (D) Nerve enwrapped by a considerable amount of collagen fibres. Note the oedema in the perineurium (arrowhead). TEM; A, x2500; B, x10 000; C, x5000; D, x6000.

 
Nerve endings were present at the dermo-epidermal junctions and showed electron-dense bodies. In the papillary dermis, unmyelinated nerve fibres were severely altered, with oedematous axoplasm and loss of neurotubules and neurofilaments (Fig. 2B). Occasionally, dense bodies were present in the axoplasm. Some fibre bundles were surrounded by oedematous connective tissue (Fig. 2C) and others were already enwrapped by a considerable amount of collagen. Nerves had oedema in the endoneurium and within the perineurium layers (Fig. 2D).

Advanced phase
Light microscopy
A worsening of the lesions observed in the epidermis in the early phase and a decrease of dermal thickness were observed. Fibrosis was severe and extended throughout the dermis. Most of blood microvessels were enwrapped by fibrosis and occluded. Sweat and sebaceous glands and hair follicles were considerably reduced.

In this phase, the innervation was severely compromised. Nerve fibres were lost in the papillary dermis and altered in the reticular dermis. In particular, nerve fibres were scarce in perivascular areas and around the few remaining appendages. Small nerves were swollen and surrounded by fibrosis.

TEM
Most of the vessels showed thickened and delaminated wall, and were surrounded by exuberant collagen. Nerve endings were rare at the dermo-epidermal junction and nerve fibres were scarce in the papillary dermis, which was reduced in thickness. The remaining nerve fibres showed pale axoplasm occupied by dense bodies and pseudomyelinic structures (Fig. 3A). Schwann cells often appeared hypertrophic with a voluminous nucleus, an enlarged endoplasmic reticulum and hyperplastic basal membrane (Fig. 3A). In the reticular dermis, small nerves showed Schwann cells and their axons embedded in oedematous endoneurial matrix with sclerotic packed collagen fibres (Fig. 3B). In the perineurium, the space between the concentric layers of flattened cells was enlarged by oedema and abnormal collagen fibres (Fig. 3B). Occasionally, myelinic fibres showed a delaminated myelin sheath (Fig. 3C). Luse bodies (long spacing collagen fibrils) were found in both the endoneurium and perineurium spaces in the nerves of reticular dermis (Fig. 3D).



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FIG. 3. Skin of an advanced dSSc patient. (A) Bundle of unmyelinated fibres. Dense body and pseudomyelinic structures within the oedematous axoplasm of nerve fibres (arrow). Arrowhead indicates a Schwann cell with enlarged endoplasmic reticulum and hyperplastic membrane. (B) Nerve with a myelinated fibre and unmyelinated fibres embedded in oedematous endoneurial matrix (asterisk). Oedema and abnormal collagen separate the perineurium layers (arrowheads). (C) Nerve containing a fibre with delaminated myelinic sheets (arrow). (D) Longitudinal section of nerve. Note Luse bodies (arrows). TEM; A, x16 500; B, x10 000; C, x10 000; D, x10 000.

 
The ultrastructural findings are summarized in Table 2.


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TABLE 2. Score of PNS damage in the SSc skin

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Our data clearly show that PNS involvement in SSc is a progressive event evolving from the early to the advanced phase. This is the first report describing the severity of ultrastructural changes of cutaneous PNS according to disease phases (early or advanced) and subsets of SSc (lSSc or dSSc).

In early dSSc, cutaneous PNS fibres are diffusely damaged and surrounded by both oedematous connective tissue and abnormal collagen. The alterations progress from the dermo-epidermal junction to the reticular dermis, and in the advanced phase they also affect nerves containing myelinated fibres.

Ultrastructural damage of PNS fibres is present only in advanced lSSc, with features similar to early dSSc, although not as severe as in that subset.

In dSSc, PNS changes are homogeneously diffuse and differ from those in lSSc, in which PNS damage has a patchy distribution concordant with the dermal fibrosis and oedema, similar to those described in internal organs, such as the lung and heart [24, 30, 31].

Our study confirms the presence of PNS ultrastructural damage in SSc, already observed in the gastrointestinal tract innervation [32, 33] and the sural nerve [8, 25, 34].

In the anorectal wall from recent-onset lSSc, ultrastructural signs of axonal degeneration in unmyelinated fibres have been found in the absence of significant fibrosis [32]. In the stomach wall from longstanding lSSc, we have described severe nervous alterations, similar to those observed in skin, and diffuse fibrosis [33]. Taken together, these studies indicate that in the gastrointestinal tract the damage of PNS fibres is present from the early phase of lSSc and it worsens in the advanced phase.

Ultrastructural PNS damage in SSc has been described in sural nerve biopsies from patients with symptomatic lower limb neuropathy [8], whereas changes in cutaneous nerves have been shown in just one study [7]. In the sural nerve, the disappearance of myelinated fibres and splitting of the myelinic lamellae and degeneration of axons and dense material within unmyelinated axons have been described [8, 25, 34, 35]. Also, abnormal hyperplasia of basal membrane of Schwann cell is described [8]. Microangiopathic [34] and vasculitic alterations [36] have also been reported in the sural nerve vasa nervorum.

In SSc skin, some changes in PNS myelinated and unmyelinated fibres have been observed previously, such as disintegration of myelinic sheaths, irregular thickening of basal membranes, oedema of the axoplasm, and, rarely, reduction in the number of neurofibrils and microtubules [7].

Our results are in agreement with literature [7, 8, 24, 34, 35] but the PNS alterations that we observed differ according to the phases and subsets of SSc. Only in advanced dSSc our data are concordant with the TEM findings already described in cutaneous PNS fibres [7].

In advanced dSSc, we have also found Luse bodies (fibrous long spacing collagen), also observed in the perineural connective tissue [37, 38], pachydermoperiostosis [39], PNS neoplasms [37] and ageing processes [40]. In dSSc, they are probably linked to the compression of PNS fibres by abnormal quantity of collagen [41, 42].

It is noteworthy that in the early phase of lSSc, when PNS damage is not yet detectable, the microvascular involvement is already present, suggesting that it might precede the PNS changes.

Our observations confirm that the development of pathological changes in SSc skin represents a continuum over time through a sequence of events [5]. Moreover, we have demonstrated that the onset of the PNS lesions is similar in its order, but different in its timing, between dSSc and lSSc. The progression in cutaneous PNS changes is earlier and more severe in dSSc than in lSSc. The observed alterations parallel the lesions in the epidermis, dermis and microvessels, and are also more precocious and worse in the advanced than in the early phase and in the diffuse than in the limited subset.

Our findings were obtained by TEM and thus they are based on morphological, not functional, studies. However, the PNS damage that we observed may parallel the significant decrease in peptide-immunoreactive nerves described in the digital skin in SSc [18], which may suggest an altered activity of PNS fibres.

In conclusion, our findings show that PNS damage is linked to the progression and severity of skin involvement in SSc. We provide evidence that the ultrastructure of PNS fibres is altered in SSc skin, particularly in the diffuse subset, evolving from the early to the advanced phase. Furthermore, the severity of the lesions observed in advanced lSSc is already evident and widely distributed in early dSSc.

Our results suggest that an early therapeutic intervention can be useful to reduce the progression of PNS and skin damage in SSc patients.


    Acknowledgments
 
This work was supported by the Ministero della Ricerca Scientifica e Tecnologica (MIUR 40% No. 2001062925) to L.I.M. and M.M.C.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 13 September 2004; revised version accepted 5 January 2005.



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