Density Of Small Nerve Fibers Research Articles

Reliable Method for Estimating Nerve Fiber Density in Epidermis Using Routine Histopathologic Tissue Preparation: A Promising Diagnostic Tool for Small Fiber Neuropathy.

Practical yet reliable diagnostic tools for small-fiber neuropathy are needed. We aimed to establish a histopathologic protocol for estimating intraepidermal nerve fiber density (eIENFD) on formalin-fixed, paraffin-embedded tissue (FFPE), evaluate its reliability through intraobserver and interobserver analyses, and provide normative reference values for clinical use. Sixty-eight healthy participants underwent nerve conduction studies and quantitative sensory testing. Skin biopsies from the distal and proximal leg were taken and processed using routine immunohistochemistry (anti-PGP9.5 antibodies) on thin 5 µm sections. eIENFD was assessed with a modified counting protocol. Interobserver and intraobserver reliabilities were excellent (ICC=0.9). eIENFD was higher in females than males (fibers/mm, 14.3±4.4 vs. 11.6±5.8, P <0.05), decreased with age ( r s =-0.47, P <0.001), and was higher proximally than distally (15.0±5.5 vs. 13.0±5.3, P =0.002). Quantile regression equations for the fifth percentile of distal and proximal eIENFD were presented: 13.125-0.161×age (y)-0.932×sex (male=1; female=0) and 17.204-0.192×age (y)-3.313×sex (male=1; female=0), respectively. This study introduces a reliable and reproducible method for estimating epidermal nerve fiber density through immunostaining on 5-µm thin FFPE tissue samples. Normative data on eIENFD is provided. Regression equations help identify abnormal decreases in small nerve fiber density.

Small fiber neuropathy and intractable scalp pruritus in dermatomyositis patients.

Scalp pruritus is a common symptom in Dermatomyositis (DM) patients. There are indications that small nerve fibers neuropathy could be involved in this symptom, however the etiology of scalp pruritus is not fully understood. To assess epidermal nerve fiber (ENF) density of dermatomyositis patients with scalp pruritus by biopsy by confocal microscopy and immunohistochemistry with subsequent imaging analysis. DM patients with severe scalp pruritus from the dermatology outpatient clinic were compared to healthy volunteers. Two 4-mm scalp skin biopsies were obtained above the right ear in the parietal region and below the occipital protuberance in the occipital region. Biopsy specimens were incubated with primary antibodies to protein gene product (PGP 9.5), calcitonin gene-related peptide (CGRP), substance P (SP) were used to visualize nerve fibers (ENF) and collagen IV was used to label the epidermal basement membrane. The number of ENFs per millimeter was counted and recorded as the mean of ± SD of counts in 16 images at two micrometer increments/sections, two from each of the samples. ENF densities were compared between groups and a multiple linear regression model was applied to associated factors with ENF density. Fifteen DM patients with severe scalp pruritus and 12 healthy volunteers were included in the study. The mean number of ENF/mm in occipital region of DM group was 16.0±13.9 while the control group in the same region was 99.8±33.1. In parietal region the number of ENF/mm of DM group was 18.0±20.7 while in control group was 50.4±17.4 (p<0.001). DM patients with pruritus could have some impairment of small nerve fiber density that could explain their recalcitrant scalp pruritus.

PD-1+ T-Cells Correlate with Nerve Fiber Density as a Prognostic Biomarker in Patients with Resected Perihilar Cholangiocarcinoma

Simple SummaryRecent studies have identified Nerve Fiber Density (NFD) as a prognostic biomarker for Cholangiocarcinoma (CCA). In the field of CCA treatment with checkpoint inhibitors (ICI) is increasing but not all patients respond. Good biomarkers to predict response to ICI are lacking. The present study investigates the immune cell composition and expression of checkpoint molecules in relation to NFD in perihilar cholangiocarcinoma (pCCA) patients. Our study identified NFD to correlate with PD-1+ T cells as a biomarker indicative for a good prognosis. Background and Aims: Perihilar cholangiocarcinoma (pCCA) is a hepatobiliary malignancy, with a dismal prognosis. Nerve fiber density (NFD)—a novel prognostic biomarker—describes the density of small nerve fibers without cancer invasion and is categorized into high numbers and low numbers of small nerve fibers (high vs low NFD). NFD is different than perineural invasion (PNI), defined as nerve fiber trunks invaded by cancer cells. Here, we aim to explore differences in immune cell populations and survival between high and low NFD patients. Approach and Results: We applied multiplex immunofluorescence (mIF) on 47 pCCA patients and investigated immune cell composition in the tumor microenvironment (TME) of high and low NFD. Group comparison and oncological outcome analysis was performed. CD8+PD-1 expression was higher in the high NFD than in the low NFD group (12.24 × 10−6 vs. 1.38 × 10−6 positive cells by overall cell count, p = 0.017). High CD8+PD-1 expression was further identified as an independent predictor of overall (OS; Hazard ratio (HR) = 0.41; p = 0.031) and recurrence-free survival (RFS; HR = 0.40; p = 0.039). Correspondingly, the median OS was 83 months (95% confidence interval (CI): 18–48) in patients with high CD8+PD-1+ expression compared to 19 months (95% CI: 5–93) in patients with low CD8+PD-1+ expression (p = 0.018 log rank). Furthermore, RFS was significantly lower in patients with low CD8+PD-1+ expression (14 months (95% CI: 6–22)) compared to patients with high CD8+PD-1+ expression (83 months (95% CI: 17–149), p = 0.018 log rank). Conclusions: PD-1+ T-cells correlate with high NFD as a prognostic biomarker and predict good survival; the biological pathway needs to be investigated.

Review and clinical experience exploring evidence, clinical efficacy, and safety regarding nonsurgical treatment of feminine rejuvenation

The use of energy-based devices for the treatment of vaginal laxity, orgasmic dysfunction, and stress incontinence, such as minimally ablative fractional laser and radiofrequency, is gaining momentum. This review aims to answer clinical questions on the application of energy-based devices for feminine genital rejuvenation. The target group includes physicians involved in esthetic medicine and feminine genital rejuvenation. A literature review was conducted on technologies in use for feminine rejuvenation to explore their safety, efficacy, tolerability, patient satisfaction, and clinical usability. A panel of physicians with clinical experience conducting these types of treatment reviewed and discussed the results of the literature search and gave clinical evidence-based recommendations. Energy-based devices may induce wound healing, stimulating new collagen, and elastin fiber formation. Radiofrequency treatment may also increase small nerve fiber density in the papillary dermis, improving nerve sensitivity, sexual function, including arousal and orgasmic dysfunction. Both minimally ablative fractional laser and radiofrequency has been shown to be effective when treating mild to moderate primary or secondary vulvovaginal laxity and associated secondary conditions. These treatments are reported to be safe, effective, and well tolerated with a rapid return to activities of daily living. As this is an evolving medical field, clinical evidence often lacks robustness. Studies and clinical experience suggest that feminine genital rejuvenation using energy-based devices seems an attractive option for patients with mild-to-moderate medical conditions. The treatment can be safely and effectively delivered by trained staff as part of the comprehensive care, that is, currently available to women.

Estrogen Replacement Regulates Vaginal Innervations in Ovariectomized Adult Virgin Rats: A Histological Study

Background. Our previous Gräfenberg spot findings confirmed that the distal-third areas of the anterior vaginal wall bore a significantly greater number of nerves and sexual hormone may have certain degree of influence on these significant differences. However, the role of estrogen in vaginal innervations remains controversial. Methods. To investigate whether hormonal-neural interactions occur in the vagina, sixty rats were randomly divided into six groups: Sham-operated, ovariectomy, and 4 treatment groups. After 2 weeks of treatment, vaginal biopsies were prepared with hematoxylin and eosin and PGP9.5 using immunohistochemistry. Results. The density of small nerve fibers was significantly higher in the distal-half areas of intact vaginal walls than the proximal-half areas (P = 0.001). In contrast, the overall PGP 9.5-ir fiber innervation density was significantly decreased in the OVX rats subjected to surgical menopause. Sustained estrogen administration for 2 weeks resulted in nerve fiber proliferation, with values reaching normal levels in the low-dose estradiol valerate group. Conclusion. Our findings indicate that systemic hormonal therapy with low-dose estradiol valerate is effective and safe for treating deficient vaginal innervation caused by low level of estrogen activity in menopausal women and may aid studies to identify an optimal estradiol dose to provide relief from vaginal discomfort.

Epidermal Nerve Fiber Quantification in Patients With Erythromelalgia

Erythromelalgia is a clinical diagnosis based on intermittent warmth, erythema, and pain in the distal extremities. One problem facing physicians is how to objectively test for this disease. Given that other painful conditions of the distal extremities (ie, neuropathy related to human immunodeficiency virus, diabetes, or Fabry disease) can be evaluated with a skin biopsy to visualize pathologically decreased densities of the small nerve fibers that innervate the epidermis, one hypothesis is that erythromelalgia could similarly be associated with a loss of epidermal nerve fiber density (ENFD). To examine whether erythromelalgia is associated with a structural loss of small fibers using the ENFD technique and to compare this with functional studies of small nerve fibers. In a retrospective study of 52 consecutive patients with erythromelalgia who were seen between September 1, 2010, and September 22, 2015, patients were interviewed and examined and their conditions clinically diagnosed by a board-certified dermatologist at a large tertiary referral center, where ENFD testing became a routine part of evaluating erythromelalgia in 2010. Thus, all 52 consecutive patients were included solely based on their clinical diagnosis of erythromelalgia. For quantification of ENFD, observers were masked to all patient information except for name and clinic number. The hypothesis that patients with erythromelalgia would have decreased ENFD was formulated before data collection. Epidermal nerve fiber density, the primary outcome, is a measurement of the density of small nerve fibers within the epidermis. Secondary measures included functional small fiber evaluation, such as autonomic (heart rate, blood pressure, and sweat testing) and subjective testing of pain. In this cohort study, 52 consecutively seen patients were identified (female, 42 [80%]; median age, 44 years; age range, 13-82 years). Whereas only 5 of 52 patients (10%) had ENFD at or below the fifth percentile of healthy control individuals, most patients had functional abnormalities of these small fibers; 29 patients (60%) had abnormal sweat test results, 21 (42%) had abnormal pain thresholds, and 20 (38%) had abnormal blood pressure or heart rate control. Unlike other diseases of the small nerve fibers that cause acral pain syndromes, erythromelalgia is not characterized by loss of ENFD. However, most patients have impaired function of these small fibers. Physicians would benefit from performing functional rather than structural small fiber studies when evaluating erythromelalgia.

Increased cutaneous miR-let-7d expression correlates with small nerve fiber pathology in patients with fibromyalgia syndrome.

Fibromyalgia syndrome (FMS) is a chronic widespread pain condition probably comprising subgroups with different underlying pathomechanisms. There is increasing evidence for small nerve fiber impairment in subgroups of patients with FMS. MicroRNAs (miRNAs) regulate molecular factors determining nerve de- and re-generation. We investigated whether systemic and cutaneous miRNA expression in patients with FMS is related to small nerve fiber pathology. We confirmed previous findings of disturbed small fiber function and reduced intraepidermal nerve fiber density in subgroups of patients with FMS. We found 51 aberrantly expressed miRNAs in white blood cells of patients with FMS, of which miR-let-7d correlated with reduced small nerve fiber density in patients with FMS. Furthermore, we demonstrated miR-let-7d and its downstream target insulin-like growth factor-1 receptor as being aberrantly expressed in skin of patients with FMS with small nerve fiber impairment. Our study gives further evidence of small nerve fiber pathology in FMS subgroups and provides a missing link in the pathomechanism that may lead to small fiber loss in subgroups of patients with FMS.

Immunohistochemical analysis of axillary skin biopsies for the detection of adrenergic innervation of sweat glands in normal subjects and Parkinson’s disease patients

Beside the typical motor symptoms Parkinson’s disease (PD) is characterized, with varying severity, by autonomic dysfunction. Several studies have shed light on the anatomical and molecular changes that underlie the peripheral neuronal degeneration associated with PD and other Lewy body (LB) diseases (LBDs). By using skin biopsies from LBDs patients it was possible to detect misfolded phospho-α-synuclein (p-syn) deposits within dermal nerve fibers and correlate them with a reduced density of small nerve fibers. (1, 2). The skin biopsy approach is an inexpensive and minimally invasive technique. To date, there is not a standardized procedure for sampling site, tissue processing and nerve fibre assessment, so the goal of a diagnostic instrument for an early diagnosis of (LBDs) still remains a challenge. We have carried out a retrospective study setting up a novel protocol based on 10 µm thick serial sections from FFPE axillary skin biopsies. This choice take advantage from the presence of apocrine glands in the axillary region, as they receive a dense sympathetic adrenergic innervation, exploitable for a clear nervous fibers tracking. The biopsies were taken from 14 individuals who had been, in the first instance, diagnosed with various traits of motor and neurological dysfunction and two control subjects. Serial tissue sections were analysed by IHC (DAB chromogen) and by immunofluorescent labelling, using anti-p-α-synuclein (S129), anti- α -synuclein, anti-PGP9.5 and anti-tyrosine hydroxylase antibodies. This particular setting has proven useful to well highlight the adrenergic fibers surrounding the apocrine sweat glands and to visualize the fibers α -synuclein deposition. Our results enabled us to support the first diagnosis in various cases with probable PD but gave a negative p-Syn-S129 immunoreactivity results for samples from vascular Parkinson, multiple system atrophy, essential tremor and frontotemporal dementia. Our methodological setting is able to detect the adrenergic innervation of sweat apocrine glands and both the presence of Lewy bodies and Lewy neurites in axillary skin biopsies.

Small fiber neuropathy in women with fibromyalgia. An in vivo assessment using corneal confocal bio-microscopy

ObjectiveA consistent line of investigation suggests that fibromyalgia is a neuropathic pain syndrome. This outlook has been recently reinforced by several controlled studies that describe decreased small nerve fiber density in skin biopsies of patients with fibromyalgia. The cornea receives the densest small fiber innervation of the body. Corneal confocal bio-microscopy is a new noninvasive method to evaluate small nerve fiber morphology. Our objective was to assess corneal small nerve fiber morphology in patients with fibromyalgia, and to associate corneal nerve microscopic features with neuropathic pain descriptors and other fibromyalgia symptoms. MethodsWe studied 17 female patients with fibromyalgia and 17 age-matched healthy control subjects. All the participants completed different questionnaires regarding the symptoms of fibromyalgia, including a neuropathic pain survey. A central corneal thickness scan was obtained with a confocal microscope. Nerve measurements were made by a single ophthalmologist without knowledge of the clinical diagnosis. Stromal nerve thickness was defined as the mean value between the widest and the narrowest portion of each analyzed stromal nerve. Corneal sub-basal plexus nerve density was also assessed. ResultsPatients with fibromyalgia had stromal nerve thickness of 5.0 ± 1.0µm (mean ± standard deviation) significantly different from that of control׳s values (6.1 ± 1.3) p = 0.01. Patients also had decreased sub-basal plexus nerve density per square millimeter (85 ± 29) vs. 107 ± 26 of controls p = 0.02. When controls and patients were grouped together, there was an association between stromal nerve slenderness and neuropathic pain descriptors (Fisher׳s exact test p = 0.007). ConclusionWomen suffering from fibromyalgia have thinner corneal stromal nerves and diminished sub-basal plexus nerve density when compared to healthy controls. Nerve scarcity is associated with neuropathic pain descriptors. Small fiber neuropathy may play a role in the pathogenesis of fibromyalgia pain. Corneal confocal microscopy could become a useful test in the study of patients with fibromyalgia.

ARA 290 Improves Symptoms in Patients with Sarcoidosis-Associated Small Nerve Fiber Loss and Increases Corneal Nerve Fiber Density

Small nerve fiber loss and damage (SNFLD) is a frequent complication of sarcoidosis that is associated with autonomic dysfunction and sensory abnormalities, including pain syndromes that severely degrade the quality of life. SNFLD is hypothesized to arise from the effects of immune dysregulation, an essential feature of sarcoidosis, on the peripheral and central nervous systems. Current therapy of sarcoidosis-associated SNFLD consists primarily of immune suppression and symptomatic treatment; however, this treatment is typically unsatisfactory. ARA 290 is a small peptide engineered to activate the innate repair receptor that antagonizes inflammatory processes and stimulates tissue repair. Here we show in a blinded, placebo-controlled trial that 28 d of daily subcutaneous administration of ARA 290 in a group of patients with documented SNFLD significantly improves neuropathic symptoms. In addition to improved patient-reported symptom-based outcomes, ARA 290 administration was also associated with a significant increase in corneal small nerve fiber density, changes in cutaneous temperature sensitivity, and an increased exercise capacity as assessed by the 6-minute walk test. On the basis of these results and of prior studies, ARA 290 is a potential disease-modifying agent for treatment of sarcoidosis-associated SNFLD.

Diagnosis of endometrial nerve fibers in women with endometriosis

Recent studies indicated that there is a high density of small nerve fibers in the functional layer of the endometrium in women with endometriosis and that it can be used as a marker to detect endometriosis. In this study, the efficacy assessment of small nerve fibers' density as a diagnostic marker was compared in patients with and without endometriosis. In this study, women with history of pelvic pain and/or infertility who were candidates for laparoscopy or laparotomy in Rassoul hospital (2007-2009) were enrolled. Histological sections of endometrial tissue were prepared from endometrial biopsy from women with endometriosis (n = 12) (1) and without endometriosis (n = 15) (2). Protein gene product 9.5 and neurofilament were evaluated as marker from endometrial biopsies by immunohistochemical methods. There was no statistically significant difference between two groups according to age, body mass index. Nerve fibers were detected in all endometrial biopsies from all women with endometriosis but detected only in three women without endometriosis. The mean density of nerve fibers was 2.2 ± 4.7 mm(-2) in group without endometriosis and) 13.1 ± 3.3 (in group with endometriosis (p < 0.001). Women with endometriosis had significantly higher nerve fiber density in comparison with women without endometriosis. Our findings indicated that endometrial biopsy for detecting density of nerve fibers by usage of protein gene product 9.5, provided a reliable marker for diagnosis of endometriosis.

Innervation of Vagina: Microdissection and Immunohistochemical Study

The aim of this study is to elucidate the innervation of human vagina by performing a microdissection and PGP immunohistochemistry study. Seven fresh Korean cadavers were used. In five cadavers, the site at which the nerve entered the vaginal wall was observed by microdissection. Two vaginas were stained with trichrome and protein gene product 9.5 (PGP) immunostain. Terminal nerve branches in the vaginal wall were most dense at the second 1/5 partition from the inferior anterior wall (1.49 ± 0.51/cm 2 ). Its density started decreasing (0.57 ± 0.09/cm 2 −0.89 ± 0.46/cm 2 ) and became scarce at the fourth and fifth 1/5 partitions from the bottom (0.37 ± 0.14/cm 2 /3–0/cm 2 ). The mucosa of the anterior vaginal wall at the distal 1/3 point (designated 1) was 3.50 ± 2.06 mm in thickness and was 1.18 ± 0.20 mm at the proximal point (designated 2). It was 1.57 ± 0.78 mm at the distal 1/3 point (designated 3) of the lateral wall and 1.43 ± 0.39 mm at the proximal 1/3 point (designated 4) of the posterior wall. Point 1 was thicker than 2, 3, and 4. The vaginal muscle of 1 (5.59 ± 2.74 mm) was thicker than 2 (3.19 ± 0.62 mm), 3 (3.31 ± 1.31 mm), and 4 (2.98 ± 0.33 mm). Small nerve fibers were most dense at 1 (6.5 ± 1.1/mm 2 ) and scarce at 4 (3.8 ± 1.5/mm 2 ). Large nerve fibers were most dense at 1 (1.9 ± 0.9/mm 2 ) and scarce at 2 (0.7 ± 0.7/mm 2 ) and 4 (0.7 ± 1.0/mm 2 ). The density of small nerve fibers is 2 ∼ 2.9/mm 2 in the muscle and 0.7∼ 1.5/mm 2 in large nerve fibers. The second 1/5 partition of the distal anterior wall had significantly richer innervation than the surrounding areas. Therefore, we think this 1/5 partition of the distal anterior wall could be so called “G-spot.”

Histopathologic findings in primary erythromelalgia are nonspecific: Special studies show a decrease in small nerve fiber density

The histopathology of primary erythromelalgia has been poorly characterized. A total of 33 skin biopsy specimens from 29 patients with a diagnosis of primary erythromelalgia were re-examined. Histopathologic findings were nonspecific. Vascular thrombi were not identified. A relative decrease in small nerve fiber density was noted in specimens from 13 of 16 patients.