Sudomotor innervation in transthyretin amyloid neuropathy: Pathology and functional correlates

Abstract

Objective: Autonomic neuropathy is a major component of familial amyloid polyneuropathy (FAP) due to mutated transthyretin, with sudomotor failure as a common manifestation. This study aimed to investigate the pathology and clinical significance of sudomotor denervation.

Methods: Skin biopsies were performed on the distal leg of FAP patients with a follow-up duration of 3.8 ± 1.6 years. Sudomotor innervation was stained with 2 markers: protein gene product 9.5 (PGP 9.5), a general neuronal marker, and vasoactive intestinal peptide (VIP), a sudomotor nerve functional marker, followed by quantitation according to sweat gland innervation index (SGII) for PGP 9.5 (SGIIPGP 9.5) and VIP (SGIIVIP).

Results: There were 28 patients (25 men) with Ala97Ser transthyretin and late onset (59.9 ± 6.0 years) disabling neuropathy. Autonomic symptoms were present in 22 patients (78.6%) at the time of skin biopsy. The SGIIPGP 9.5 and SGIIVIP of FAP patients were significantly lower than those of age- and gender-matched controls. The reduction of SGIIVIP was more severe than that of SGIIPGP 9.5 (p = 0.002). Patients with orthostatic hypotension or absent sympathetic skin response at palms were associated with lower SGIIPGP 9.5 (p = 0.019 and 0.002, respectively). SGIIPGP 9.5 was negatively correlated with the disability grade at the time of skin biopsy (p = 0.004), and was positively correlated with the interval from the time of skin biopsy to the time of wheelchair usage (p = 0.029).

Interpretation: This study documented the pathological evidence of sudomotor denervation in FAP. SGIIPGP 9.5 was functionally correlated with autonomic symptoms, autonomic tests, ambulation status, and progression of disability.

Figure 1

Congo red staining and polarized microscopic examinations of sural nerve and skin biopsies from patients with familial amyloid polyneuropathy (FAP). Sections of sural nerve (A, B) and skin (C–F) biopsies were stained with Congo red (A, C, and E) and observed under a polarized microscope (B, D, and F). (A, B) Congo red–stained materials in an arteriole of the sural nerve exhibited apple‐green birefringence under polarized microscopy. (C, D) Secretory coils of sweat glands were stained with Congo red (arrow) but did not show birefringence under polarized microscopy. (E, F) In a dermal nerve bundle from an FAP patient, Congo red⁺ materials were detected surrounding a small arteriole (arrow), which showed birefringence under polarized microscopy. Scale bar = 50μm for A and B, 200μm for C and D, and 25μm for E and F.

Figure 2

Transthyretin (TTR) expression in sural nerve and skin biopsies in familial amyloid polyneuropathy (FAP). Sections of sural nerves (A, B) were stained with hematoxylin and eosin (H&E; A) and immunohistochemistry of TTR (B), respectively. Immunofluorescent staining with protein gene product 9.5 (PGP 9.5; labeled with green fluorescence in C) and TTR (labeled with blue fluorescence in E) followed with Congo red counterstaining (with red autofluorescence in D) was performed on the skin biopsy from the same FAP patient in Figure 1C to F. (A, B) There was TTR immunoreactivity in amyloid with eosinophilic amorphous appearance surrounding an arteriole from sural nerve biopsy. (C–F) Scanty PGP 9.5⁺ nerve fibers were detected surrounding sweat glands, which exhibited red autofluorescence after Congo red staining. In these Congo red–stained sweat glands, there was no TTR immunoreactivity. Scale bar = 100μm in B applied to A and B, 50μm in F applied to C–F.

Figure 3

Sweat gland and skin innervation in familial amyloid polyneuropathy (FAP) due to transthyretin Ala97Ser mutation. Skin sections from control subjects (A and C) and patients with FAP (B and D) were immunostained with protein gene product 9.5. (A) In the control skin, the sudomotor nerve fibers appeared as dense immunoreactivities with a circular pattern surrounding the secretary coils of sweat glands. (B) The nerve fibers surrounding sweat glands in FAP patients were markedly reduced, and only some loose and fragmented immunoreactivities could be observed. (C) In the control skin, intraepidermal nerve fibers (arrow) with a varicose appearance ascended from the subepidermal nerve plexus (snp) at the border of the epidermis (epi) and dermis (derm). Dermal nerve fascicles (d.n.) exhibited a pattern of linear and dense immunoreactivities in the deep dermis. (D) Intraepidermal nerve fibers disappeared in the skin of FAP patients and dermal nerves became fragmented, indicating nerve degeneration. Scale bar = 45μm for A and B, 20μm for C and D.

Figure 4

Sweat gland innervations in familial amyloid polyneuropathy (FAP). Skin sections were immunostained with protein gene product 9.5 (PGP 9.5) antiserum and then counterstained with Congo red. (A) In the control skin, sweat glands appeared as red coils and were surrounded by the linear and varicose sudomotor nerve fibers of dense PGP 9.5 immunoreactivities. (B) The sweat gland innervation was markedly reduced in FAP, and only some scattered and fragmented immunoreactivities surrounding a small portion of the sweat gland could be observed, indicating sudomotor nerve degeneration. Scale bar = 50μm.

Figure 5

Sweat gland innervations in familial amyloid polyneuropathy (FAP). Skin sections were immunostained with vasoactive intestinal peptide (VIP; green) for sudomotor nerves (A and D), and Na‐K‐Cl cotransporter (NKCC1, red) for sweat glands (B and E). (A–C) In the control skin, the sudomotor nerve fibers were labeled by VIP immunoreactivities with linear and circular patterns (A) and the sweat gland was labeled by NKCC1 immonoreactivities as red coils (B). The sudomotor nerve fibers encircled the secretary coils of the sweat gland (C). (D–F) In FAP patients, the innervation of sweat glands by VIP⁺ nerves (D) surrounding NKCC1⁺ sweat glands (E) was significantly reduced. Only scattered VIP immunoreactivities surrounded the secretary coils of the sweat gland (F). Scale bar = 100μm.

Figure 6

Quantitation of sweat gland and skin innervation in familial amyloid polyneuropathy (FAP). Quantitative data of sweat gland and skin innervation were analyzed according to: (A) the sweat gland innervation index by protein gene product 9.5 immunostaining (SGIIPGP 9.5), (B) the sweat gland innervation index by vasoactive intestinal peptide immunostaining (SGIIVIP), and (C) the intraepidermal nerve fiber (IENF) density. All 3 parameters were significantly reduced in the FAP group compared to the age‐ and gender‐matched control subjects. **p < 0.005.

Figure 7

The clinical significance of sudomotor innervation in familial amyloid polyneuropathy. (A) The comparison of sudomotor innervation was made between patients with and without orthostatic hypotension. The sweat gland innervation index by protein gene product 9.5 immunostaining (SGIIPGP 9.5) was lower in patients with orthostatic hypotension (Ortho Hypo) than those without this symptom. (B) The SGIIPGP 9.5 was lower in patients with absent sympathetic skin response at the palm than in those with present response, reflecting the relationship between the pathology and functional consequence of sudomotor denervation. (C, D) SGIIPGP 9.5 was negatively correlated with the disability grade at the time of skin biopsy (C) and positively correlated with the time from skin biopsy to loss of ambulation ability (D), indicating the lower sudomotor innervation, the higher grade, and the more rapid progression of disability. SSR = sympathetic skin response at palm; + = presence; − = absence. *p < 0.05, **p < 0.005.