Skin nerve pathology: Biomarkers of premanifest and manifest amyloid neuropathy.

OBJECTIVE: Small-fiber sensory and autonomic symptoms are early presentations of familial amyloid polyneuropathy (FAP) with transthyretin (TTR) mutations. This study aimed to explore the potential of skin nerve pathologies as early and disease-progression biomarkers and their relationship with skin amyloid deposits. METHODS: Skin biopsies were performed in patients and carriers to measure intraepidermal nerve fiber (IENF) density, sweat gland innervation index of structural protein gene product 9.5 (SGII[PGP9.5]) and peptidergic vasoactive intestinal peptide (SGII[VIP]), and cutaneous amyloid index. These skin pathologies were analyzed with clinical disability assessed by FAP stage score (stage 0-4) and compared to neurophysiological and psychophysical tests. RESULTS: There were 70 TTR-mutant subjects (22 carriers and 48 patients), and 66 cases were TTR-A97S. Skin nerve pathologies were distinct according to stage. In carriers, both skin denervation and peptidergic sudomotor denervation were evident: (1) IENF density was gradually reduced from stage 0 through 4, and (2) SGII(VIP) was markedly reduced from stage 1 to 2. In contrast, SGII(PGP9.5) was similar between carriers and controls, but it declined in patients from stage 2. Skin amyloids were absent in carriers and became detectable from stage 1. Cutaneous amyloid index was correlated with SGII(PGP9.5) and stage in a multivariate mixed-effect model. When all tests were compared, only IENF density, SGII(PGP9.5), and cutaneous amyloid index were correlated with stage, and IENF density had the highest abnormal rate in carriers. INTERPRETATION: Biomarkers of sensory and sudomotor innervation exhibited a stage-dependent progression pattern, with sensory nerve degeneration as the early skin nerve pathology. Ann Neurol 2019;85:560-573.

Sudomotor innervation in transthyretin amyloid neuropathy: Pathology and functional correlates.

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.

Patterns of target tissue reinnervation and trophic factor expression after nerve grafting.

BACKGROUND: Reinnervation of target tissues determines functional outcomes after nerve grafting, which is important in traumatic injury caused by accidents or consequences resulting from surgical removal of tumors. Previous studies documented the influences of nerve repair mainly based on nerve morphometry but rarely compared the final outcomes according to target reinnervation patterns by nerve fibers of different categories. METHODS: In a mouse model of nerve grafting, the authors analyzed the innervation indexes of different target tissues after transection-reimplantation on the sciatic nerve, which were defined as a parameter on the operated side normalized to that on the control side. RESULTS: Muscle reinnervation appeared to be the best compared with skin reinnervation (p < 0.0001) and sweat gland reinnervation (p < 0.0001) at postoperative month 3. The sudomotor reinnervation was relatively higher than the cutaneous reinnervation (p = 0.014). The abundance of trophin transcripts for brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin 3 (NT3) was higher in plantar muscles on the operated side than those on the control side. In contrast, transcripts of BDNF, GDNF, nerve growth factor, and NT3 were all similar in the footpad skin between the operated and control sides. CONCLUSIONS: The results suggested that, compared with the skin, muscles achieved the best reinnervation after nerve grafting, which was related to higher expression of BDNF, GDNF, and NT3 in muscles than in the skin.

Skin denervation and its clinical significance in late-stage chronic kidney disease.

OBJECTIVE: To investigate the skin innervation and its clinical significance in late-stage chronic kidney disease (CKD). DESIGN: Case series. SETTING: National Taiwan University Hospital, Taipei, Taiwan. PATIENTS: Forty consecutive nondiabetic patients with late-stage CKD (14 female and 26 male; mean [SD] age, 60.7 [12.3] years), including 2 cases with stage 3 CKD, 6 with stage 4 CKD, and 32 with stage 5 CKD, ie, end-stage kidney disease. INTERVENTIONS: Clinical evaluation of neurological deficits, nerve conduction study, autonomic function tests, and a 3-mm-diameter skin biopsy specimen taken from the distal leg. MAIN OUTCOME MEASURES: Quantitation of epidermal innervation, parameters of nerve conduction study, R-R interval variability, and sympathetic skin response. RESULTS: Clinically, 21 patients (52.5%) were symptomatic with paresthesia over the limbs or autonomic symptoms. The intraepidermal nerve fiber (IENF) density was markedly reduced in patients with CKD compared with age- and sex-matched controls (mean [SD], 2.8 [2.0] vs 8.6 [2.8] fibers/mm; P < .001). Skin denervation was observed in 27 patients (67.5%). Fifteen patients (37.5%) had abnormalities on nerve conduction studies, and 29 patients (72.5%) had abnormal results on autonomic function tests. By analysis with multiple regression models, the IENF density was negatively correlated with the duration of renal disease (P = .02). Additionally, the R-R interval variability at rest was linearly correlated with the IENF density (P = .02) and the absence of sympathetic skin responses at the soles was associated with reduced IENF density (P = .03). CONCLUSIONS: Small-fiber sensory and autonomic neuropathies constitute the major form of neuropathy in late-stage CKD. Furthermore, skin denervation was associated with the duration of renal disease.

Effects of 4-methylcatechol on skin reinnervation: promotion of cutaneous nerve regeneration after crush injury.

We assessed the effects of treatment with 4-methylcatechol (4MC), a known inducer of nerve growth factor, on peripheral nerve regeneration by analyzing cutaneous and muscular reinnervation in mice after sciatic nerve crush injury. At 3 months postinjury, the skin innervation index was significantly higher in the 4MC group than the control group (p=0.0002); there was also increased unmyelinated fiber density (p=0.0042) and unmyelinated fibers/Remak bundle (p = 0.001) in sural nerves, indicating unmyelinated nerve fiber regeneration. These changes were accompanied by increases of transcripts for nerve growth factor (p = 0.0026) and glial cell line-derived neurotrophic factor (p=0.03) in the 4MC group. In contrast, muscle innervation indices were similar in both groups and were higher than the skin innervation index (p < 0.0001). The regeneration of myelinated nerve fibers, as assessed by fiber density, diameter and g ratio analyses in sural nerves, and amplitudes of muscle action potential in sciatic nerves, was similar in both groups. Taken together, these data suggest that 4MC specifically promoted the regeneration of unmyelinated nerve fibers and reinnervation of the skin by increasing the expression of nerve growth factor and glial cell line-derived neurotrophic factor.

Depletion of peptidergic innervation in the gastric mucosa of streptozotocin-induced diabetic rats.

Autonomic neuropathy affecting the gastrointestinal system is a major presentation of diabetic neuropathy. Changes in the innervation of gastric mucosa or muscle layers can contribute to gastrointestinal symptoms. The present study investigated this issue by quantitatively analyzing the immunohistochemical patterns of the gastric innervation in rats with streptozotocin (STZ)-induced diabetes. In control rats, calcitonin gene-related peptide (CGRP) and substance P (SP) (+) nerve fibers appeared in the gastric mucosa and muscle layers. Double immunohistochemical staining showed that immunoreactivities for SP and CGRP were co-localized with a pan-neuronal marker protein gene product 9.5. Both SP (+) nerve fibers (p<0.001) and CGRP (+) nerve fibers (p<0.005) were decreased in the gastric mucosa within 4 weeks of diabetes; the reduction persisted throughout 24 weeks. Diabetic rats treated with insulin did not show decrease of SP or CGRP (+) fibers in the mucosa 4 weeks after STZ injection (p>0.05). There was no significant change in SP (+) nerve fibers (p>0.05) or CGRP (+) nerve fibers (p>0.05) of the gastric muscle layers. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression levels of SP and CGRP mRNA in the thoracic dorsal root ganglia were similar between diabetic and control animals (p>0.05). Qualitative and quantitative ultrastructural examinations on the gastric mucosa documented unmyelinated nerve degeneration. These results suggest the existence of gastric sensory neuropathy in STZ-induced diabetes, and this pathology provides a foundation for understanding diabetic gastropathy.

Glycemic control is related to the severity of impaired thermal sensations in type 2 diabetes.

BACKGROUND: Small-fibre sensory neuropathy of diabetes presenting as impaired thermal sensations is associated with ominous consequences, such as foot ulcer and amputation, but there is a lack of systematic studies on its occurrence in large cohorts. We investigated (1) the impact of glycemic control on thermal thresholds, (2) the frequencies and patterns of sensory deficits, and (3) the contribution of sensory nerve abnormalities to neuropathic symptoms. METHODS: Quantitative sensory testing and nerve conduction studies were performed to measure warm and cold thresholds of extremities, and amplitudes of nerve action potentials on 498 type 2 diabetic patients and 434 control subjects with similar age and gender distributions, enrolled during the same period. RESULTS: The diabetic patients had higher thermal thresholds than control subjects (p < 0.0001). Thermal thresholds of the lower and upper extremities were linearly correlated with HbA1C on multiple linear regression analysis (p < 0.01). By the multivariate logistic regression analysis, HbA(1C) and age were the most important risk factors independently associated with elevated thermal thresholds (p < 0.01). Elevated warm threshold in the big toe was the most frequent abnormality (60.2%) compared to abnormal cold threshold in the big toe (39.6%) and abnormal sural nerves on nerve conduction studies (12.9%). Elevated thermal thresholds were risk factors for neuropathic symptoms independent of HbA(1C). CONCLUSION: Small-fibre neuropathy with the impairment of thermal sensations is the most frequent sensory deficit in diabetes, and HbA1C is significantly associated with the elevated thermal thresholds.

Skin denervation and cutaneous vasculitis in systemic lupus erythematosus.

To understand the clinical significance and mechanisms of cutaneous denervation in systemic lupus erythematosus (SLE), we assessed intraepidermal nerve fibre (IENF) density of the distal leg in 45 SLE patients (4 males and 41 females, aged 38.4 +/- 13.6 years) and analysed its correlations with pathology, lupus activity, sensory thresholds and electrophysiological parameters. Compared with age- and gender-matched control subjects, SLE patients had lower IENF densities (3.08 +/- 2.17 versus 11.27 +/- 3.96 fibres/mm, P < 0.0001); IENF densities were reduced in 38 patients (82.2%). Pathologically, 11 patients (24.4%) were found to have definite cutaneous vasculitis; the severity and extent of cutaneous vasculitis were correlated with IENF densities. Patients with active lupus had even lower IENF densities than those with quiescent lupus (1.86 +/- 1.37 versus 4.15 +/- 2.20 fibres/mm, P = 0.0002). By linear regression analysis, IENF densities were negatively correlated with the SLE disease activity index (r = 0.527, P = 0.0002) and cumulative episodes of lupus flare-up within 2 years before the skin biopsy (r = 0.616, P = 0.0014). Clinically, skin denervation was present not only in the patients with sensory neuropathy but also in the patients with neuropsychiatric syndrome involving the CNS. SLE patients had significantly elevated warm threshold temperatures (P = 0.003) and reduced cold threshold temperatures (P = 0.048); elevated warm threshold temperatures were associated with the reduced IENF densities (P = 0.032). In conclusion, cutaneous vasculitis and lupus activities underlie skin denervation with associated elevation of thermal thresholds as a major manifestation of sensory nerve injury in SLE.

Skin denervation in type 2 diabetes: correlations with diabetic duration and functional impairments.

Sensory neuropathy is a prominent component of diabetic neuropathy. It is not entirely clear how diabetes influences skin innervation, and whether these changes are correlated with clinical signs and laboratory findings. To investigate these issues, we performed skin biopsies on the distal leg of 38 consecutive type 2 diabetic patients with sensory symptoms in lower limbs (25 males and 13 females, aged 56.2 +/- 9.4 years) and analysed the correlations of intraepidermal nerve fibre (IENF) densities in skin with glycaemic status (duration of diabetes, HbA1C, and fasting and post-prandial glucose levels), and functional parameters of small fibres (warm and cold thresholds) and large fibres (vibratory threshold and parameters of nerve conduction studies). Clinically, 23 patients (60.5%) had signs of small-fibre impairment, and 19 patients (50.0%) had signs of large-fibre impairment. IENF densities were much lower in diabetic patients than in age- and gender-matched controls (1.794 +/- 2.120 versus 9.359 +/- 3.466 fibres/mm, P < 0.0001), and 81.6% (31/38) of diabetic patients had reduced IENF densities. IENF densities were negatively associated with the duration of diabetes (standardized coefficient: -0.422, P = 0.015) by analysis with a multivariate linear regression model. Abnormal results of functional examinations were present in 81.6% (warm threshold), 57.9% (cold threshold), 63.2% (vibratory threshold) and 49% (amplitude of sural sensory action potential) of diabetic patients. Among the three sensory thresholds, the warm threshold temperature had the highest correlation with IENF densities (standardized coefficient: -0.773, P < 0.0001). On nerve conduction studies in lower-limb nerves, there were abnormal responses in 54.1% of sural nerves, and 50.0% of peroneal nerves. Of neurophysiological parameters, the amplitude of the sural sensory action potential had the highest correlation with IENF density (standardized coefficient: 0.739, P < 0.0001). On clinical examination, 15 patients showed no sign of small-fibre impairment, but seven of these patients had reduced IENF densities. In conclusion, small-fibre sensory neuropathy presenting with reduced IENF densities and correlated elevation of warm thresholds is a major manifestation of type 2 diabetes. In addition, the extent of skin denervation increases with diabetic duration.

Effects of aging on human skin innervation.

To understand the effect of aging on human skin innervation, we investigated intraepidermal nerve fiber (IENF) density of skin biopsies. IENF densities of the distal leg were lower in elderly (> or = 60 years of age) than in young adults (19-39 years of age; 7.80 +/- 0.79 vs 13.55 +/- 0.85 fibers/mm, p < 0.01). A similar trend was also observed in the distal forearm (11.67 +/- 1.55 vs 19.39 +/- 1.60 fibers/ mm, p < 0.001). IENF densities were negatively correlated with age according to multiple linear regression analysis on the entire study population (age range: 19-78 years) with standardized coefficients of -0.462 (p < 0.001) in the distal leg and -0.335 (p = 0.005) in the distal forearm. These findings suggest a significant reduction in IENF densities with age.

Cutaneous innervation in Guillain-Barré syndrome: pathology and clinical correlations.

Guillain-Barré syndrome (GBS) is traditionally considered to be a large-fibre neuropathy. However, the presence of hypo-aesthesia, dysaesthesia and dysautonomia in GBS patients raises the possibility that small-diameter sensory and autonomic nerves may also be affected. To investigate small-fibre neuropathy in GBS, we performed a skin biopsy from the distal leg of 20 patients with the demyelinating form of GBS. Skin sections were immunohistochemically stained with antiserum against protein gene product 9.5 (PGP 9.5), a ubiquitin C-terminal hydrolase. Cutaneous innervation was evaluated by measuring epidermal nerve density (END), and END was further correlated with various clinical and electrophysiological parameters. In GBS patients, END values were much lower than in age- and gender-matched control subjects (5.03 +/- 1.18 versus 10.16 +/- 0.87 fibres/mm, P < 0.001). Eleven patients (55%) had reduced epidermal innervation with pathological evidence of active nerve degeneration in the dermis: fragmentation of subepidermal nerve plexuses and a beaded appearance of dermal nerves. GBS patients had significantly elevated thermal thresholds with higher warm threshold temperatures (44.54 +/- 1.04 versus 39.00 +/- 0.35 degrees C, P < 0.001) and lower cold threshold temperatures (25.57 +/- 1.11 versus 29.05 +/- 0.21 degrees C, P = 0.032). Reduced END values were associated with an elevated warm threshold (P = 0.027), ventilatory distress (P = 0.037) and dysautonomia (P = 0.001). END values were negatively correlated with disability grade on a scale of 1-6 (slope -0.134 +/- 0.038, P = 0.0018). Patients with reduced END values tended to have a slower recovery than those with normal END values (P = 0.013, median time 12 versus 2 weeks). Patho logically, sudomotor innervation of the skin was reduced in five of 17 (29.4%) GBS patients in whom sweat glands could be recognized. These findings suggest that small-fibre sensory and autonomic neuropathies exist in a significant proportion of GBS patients, and that END values are correlated with functional disabilities. In summary, GBS should be considered a global neuropathy instead of a pure large-fibre neuropathy.