The role of skin biopsy in differentiating small-fiber neuropathy from ganglionopathy.

BACKGROUND AND PURPOSE: We aimed to test the clinical utility of the leg:thigh intraepidermal nerve-fiber (IENF) density ratio as a parameter to discriminate between length-dependent small-fiber neuropathy (SFN) and small-fiber sensory ganglionopathy (SFSG) in subjects with signs and symptoms of small-fiber pathology. METHODS: We retrospectively evaluated thigh and leg IENF density in 314 subjects with small-fiber pathology (173 with distal symmetrical length-dependent SFN and 141 with non-length-dependent SFSG). A group of 288 healthy subjects was included as a control group. The leg:thigh IENF density ratio was calculated for all subjects. We used receiver operating characteristic curve analyses to assess the ability of this parameter to discriminate between length-dependent SFN and SFSG, and the decision curve analysis to estimate its net clinical benefit. RESULTS: In patients with neuropathy, the mean IENF density was 14.8 ± 6.8/mm at the thigh (14.0 ± 6.9/mm in length-dependent SFN and 15.9 ± 6.7/mm in patients with SFSG) and 7.5 ± 4.5/mm at the distal leg (5.4 ± 3.2/mm in patients with length-dependent SFN and 10.1 ± 4.6/mm in patients with SFSG). The leg:thigh IENF density ratio was significantly (P < 0.01) lower in patients with length-dependent SFN (0.44 ± 0.23) compared with patients with SFSG (0.68 ± 0.28). The area under the curve of the receiver operating characteristic analysis to discriminate between patients with length-dependent SFN and SFSG was 0.79. The decision curve analysis demonstrated the clinical utility of this parameter. CONCLUSIONS: The leg:thigh IENF ratio represents a valuable tool in the differential diagnosis between SFSG and length-dependent SFN.

Small-fibre neuropathy in a patient with dermatomyositis and severe scalp pruritus.

Dermatomyositis (DM) is commonly associated with scalp pruritus that can be severe. In addition, significant crawling and burning sensations have been reported in these cases. The aetiology of these scalp sensations in the context of DM is not fully understood. We report a 42-year-old female with treatment-resistant DM and structural changes in scalp epidermal and dermal nerve fibres. The patient presented with characteristic skin manifestations (Gottron's papules and poikiloderma), severely pruritic scalp, intermittent muscle weakness on neurological exam with electrodiagnostically confirmed myositis, and joint pain. Structural changes in scalp epidermal and dermal nerve fibres were discovered in a skin biopsy, suggesting that small-fibre neuropathy associated with scalp pruritus may be a manifestation of the DM syndrome. Further clinical experience combined with selective skin biopsy in patients with DM and symptomatic scalp will help determine the frequency of coexistent small nerve fibre involvement. Based on our limited findings, we suggest that pruritus in DM may be associated with abnormal epidermal and dermal nerve fibre structure.

A multi-center, multinational age- and gender-adjusted normative dataset for immunofluorescent intraepidermal nerve fiber density at the distal leg.

BACKGROUND AND PURPOSE: Quantification of intraepidermal nerve fibers (IENFs) in skin biopsies is now the tool of choice to diagnose small fiber neuropathies. An adequate normative dataset, necessary to assess normality cutoffs, is available for brightfield microscopy but not for immunofluorescence. METHODS: Intraepidermal nerve fiber density data in distal leg skin samples processed with immunofluorescence were collected from 528 healthy individuals from four experienced laboratories worldwide. In all laboratories skin samples were collected, processed and analyzed according to standard procedures. Quantile regression analysis was employed to tailor the fit of the 5° percentile as the normal cutoff value and to test and measure the effect of age, gender, body mass index, race, biopsy site (lateral distal lower leg or medial posterior mid-calf) and participating laboratory as possible influential variables. RESULTS: Age, gender and biopsy site showed an independent linear correlation with IENF density. For each decade the 5° quantile IENF cutoff showed a 0.54 fibers/mm decrease, whilst females exhibited a 1.0 fiber/mm cutoff greater than males. Compared to the lateral distal lower leg, biopsies from the calf showed a 3.4 fibers/mm lower 5° percentile cutoff, documenting a variation linked by site. CONCLUSIONS: An age- and gender-adjusted normative dataset for IENF density at the lateral distal lower leg obtained with indirect immunofluorescence is presented for the first time by sharing data from four experienced laboratories worldwide. This dataset can be used as reference for laboratories processing skin biopsies with this technique.

Morphological features of nerves in skin biopsies.

Skin biopsy is an effective test for diagnosis of peripheral nerve disorders. The most commonly reported indication of abnormality in a skin biopsy is reduction of epidermal nerve density. Morphological changes of epidermal nerves and the underlying subepidermal nerve plexus provide added evidence for the presence of neuropathy. We determined the prevalence of epidermal axon swellings, dermal axon swellings, and a unique type of epidermal nerve that we call a crawler, in a group of normal subjects, diabetic subjects, and patients with idiopathic small fiber neuropathy. Other morphologic features examined include thinning of the subepidermal nerve plexus, sprouts at nerve terminals, encapsulated endings, and immunoreactive basal cells.

Effects of cell density and transformation on the formation of a fibronectin extracellular filamentous matrix on human fibroblasts.

We have studied normal and transformed cultured human fibroblasts by ultrastructural immunocytochemistry with antibodies to fibronectin (the large external transformation-sensitive glycoprotein). Human fibroblasts at low density have very little extracellular material organized into a filamentous form. A diffuse membrane component of low-density fibroblasts reacts with fibronectin antibodies. As normal fibroblasts grow to confluence, an extensive extracellular filamentous matrix forms. The filaments are 15 to 20 nm in diameter, lack periodicity, and frequently occur in a meshwork. Antibody to fibronectin reacts with these filaments as well as with a diffusely distributed membrane component. Cultures of SV40-transformed human fibroblasts lack the extracellular filamentous matrix reacting with fibronectin antibodies; however, fibronectin is detected on plasma membranes. These studies indicate that a major difference between normal and transformed human fibroblasts is the failure of transformed fibroblasts to form a fibronectin-containing extracellular filamentous matrix.

Reversal by glucocorticoid hormones of the loss of a fibronectin and probollagen matrix around transformed human cells.

Confluent cultured human skin fibroblasts had an extracellular fibrillar matrix of fibronectin and procollagen. Human skin fibroblasts transformed by SV40 did not have such a matrix. Treatment of transformed fibroblasts with 10(-5) to 10(-8) M dexamethasone and 10(-5) to 10(-7) M cortisol, but not testosterone or progesterone, caused partial restoration of the matrix. Glucocorticoid-treated transformed human fibroblasts can serve as a model for partial reversion toward normal or differentiation of transformed human fibroblasts.

Functional interactions between tumor and peripheral nerve: changes in excitability and morphology of primary afferent fibers in a murine model of cancer pain.

We used a murine model to investigate functional interactions between tumors and peripheral nerves that may contribute to pain associated with cancer. Implantation of fibrosarcoma cells in and around the calcaneus bone produced mechanical hyperalgesia of the ipsilateral paw. Electrophysiological recordings from primary afferent fibers in control and hyperalgesic mice with tumor revealed the development of spontaneous activity (0.2-3.4 Hz) in 34% of cutaneous C-fibers adjacent to the tumor (9-17 d after implantation). C-fibers in tumor-bearing mice exhibited a mean decrease in heat threshold of 3.5 +/- 0.10 degrees C. We also examined innervation of the skin overlying the tumor. Epidermal nerve fibers (ENFs) were immunostained for protein gene product 9.5, imaged using confocal microscopy, and analyzed in terms of number of fibers per millimeter of epidermal length and branching (number of nodes per fiber). Divergent morphological changes were linked to tumor progression. Although branching of ENFs increased significantly relative to control values, in later stages (16-24 d after implantation) of tumor growth a sharp decrease in the number of ENFs was observed. This decay of epidermal innervation of skin over the tumor coincided temporally with gradual loss of electrophysiological activity in tumor-bearing mice. The development of spontaneous activity and sensitization to heat in C-fibers and increased innervation of cutaneous structures within the first 2 weeks of tumor growth suggest activation and sensitization of a proportion of C-fibers. The decrease in the number of ENFs observed in later stages of tumor development implicates neuropathic involvement in this model of cancer pain.

Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.

We have reported previously that human keratinocytes synthesize and secrete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study defines the location of muscarinic acetylcholine receptors in human epidermis and describes some pharmacologic and molecular properties of these receptors. Confocal microscopy employing the anti-muscarinic receptor monoclonal antibody M35 visualized the receptors in the intercellular areas of normal human epidermis. Using immunoelectron microscopy, the receptors appeared to be attached to the keratinocyte plasma membranes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and high-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by saturable binding of the reversible radioligand [3H]quinuclidinyl benzilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An apparent isoelectric point of pH 4.3 was determined in immunoblots of sodium-cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, were visualized in immunoblots of membrane-bound or solubilized receptors separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The covalent, irreversible ligand [3H]propylbenzilylcholine mustard confirmed these results. Thus, human keratinocytes express a heterogeneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenously secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic receptors.

Immunohistochemical study of skin reinnervation by regenerative axons.

The time sequence of sensory and sudomotor nerve regeneration to the mouse footpad was studied between one and seven weeks after crush or section of the sciatic nerve. Protein gene product 9.5, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide were localized in thick sections by using indirect immunofluorescence techniques and imaged by confocal microscopy. Nerve regeneration was visually assessed in all nerves and quantified in sweat glands. After denervation, protein gene product 9.5 immunoreactivity remained as dim fluorescence within thick fibers of dermal nerve trunks, whereas thin nerve fibers to sweat glands and to epidermis disappeared. By 14 days postcrush and 35 days postsection, the first protein gene product 9.5 immunoreactive regenerating axons appeared in large nerve trunks, quickly extending to epidermis and sweat glands. Reinnervation of Meissner's corpuscles occurred nearly simultaneous with return of epidermal free nerve endings and sudomotor network. Calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P immunoreactivity disappeared completely one week after denervation, then reappeared at 17-18 days postcrush and 35 days postsection. Fewer nerve fibers were immunoreactive to these peptides than to protein gene product 9.5. The overall density of reinnervation, although reduced, more closely resembled normal in the sweat glands and Meissner's corpuscles than in the epidermis. Reinnervation was more successful after crush than after nerve section. The time course for functional return of sweating paralleled the return of protein gene product 9.5 immunoreactivity, whereas appearance of vasoactive intestinal peptide was delayed by several days.

Quantitation of epidermal nerves in diabetic neuropathy.

We describe methods to quantify epidermal nerve fibers (ENFs) in skin biopsy specimens from diabetic candidates for pancreas transplantation and control subjects. ENFs and the dermal-epidermal basement membrane were stained by immunohistochemical methods, imaged with a confocal microscope, and quantified using a neuron tracing system. The number of ENFs per surface of epidermis was diminished in diabetic subjects. ENF number and summed length of all ENFs per volume of epidermis examined were also decreased. Length and number of branch points of single surviving ENFs were similar in skin of control and diabetic subjects. The methods and results constitute a basis for continued study of the effects of the euglycemia that attends successful pancreas transplantation and the effects of therapy in patients with various types of polyneuropathy.

Topical capsaicin in humans: parallel loss of epidermal nerve fibers and pain sensation.

Capsaicin applied topically to human skin produces itching, pricking and burning sensations due to excitation of nociceptors. With repeated application, these positive sensory responses are followed by a prolonged period of hypalgesia that is usually referred to as desensitization, or nociceptor inactivation. Consequently, capsaicin has been recommended as a treatment for a variety of painful syndromes. The precise mechanisms that account for nociceptor desensitization and hypalgesia are unclear. The present study was performed to determine if morphological changes of intracutaneous nerve fibers contribute to desensitization and hypalgesia. Capsaicin (0.075%) was applied topically to the volar forearm four times daily for 3 weeks. At various time intervals tactile, cold, mechanical and heat pain sensations were assessed in the treated and in contralateral untreated areas. Skin blisters and skin biopsies were collected and immunostained for protein gene product (PGP) 9.5 to assess the morphology of cutaneous nerves and to quantify the number of epidermal nerve fibers (ENFs). Capsaicin resulted in reduced sensitivity to all cutaneous stimuli, particularly to noxious heat and mechanical stimuli. This hypalgesia was accompanied by degeneration of epidermal nerve fibers as evidenced by loss of PGP 9.5 immunoreactivity. As early as 3 days following capsaicin application, there was a 74% decrease in the number of nerve fibers in blister specimens. After 3 weeks of capsaicin treatment, the reduction was 79% in blisters and 82% in biopsies. Discontinuation of capsaicin was followed by reinnervation of the epidermis over a 6-week period with a return of all sensations, except cold, to normal levels. We conclude that degeneration of epidermal nerve fibers contributes to the analgesia accredited to capsaicin. Furthermore, our data demonstrate that ENFs contribute to the painful sensations evoked by noxious thermal and mechanical stimuli.

Ultrastructural localization of viral antigens using the unlabeled antibody-enzyme method.

Employing the unlabeled antibody enzyme method at the ultrastructural level, a comparison was made between preembedding staining and postembedding staining for the detection of viral antigens. The bacteriophage P1 absorbed to the surface of Shigella dysenteriae was used as a model system. Preembedding staining resulted in the specific deposition of peroxidase-antiperoxidase (PAP) complexes as an electron-dense coating around the viral heads. Disadvantages of the preembedding staining method included the agglutination of cells by the primary antiserum which produced a gradient of specific staining and the "bleeding" or migration of electron dense reaction product away from the sites of attached PAP complexes. The postembedding staining method had distinct advantages over the preembedding staining in that PAP complexes were deposited directly over exposed viral heads within the thin section. In addition, the specific immunostaining of viruses was uniform through the section and no artifactual migration of reaction product was observed.

Fibronectin presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization.

Fibronectin is a major constituent of the fibroblast extracellular matrix. Fibronectin binds to collagen, mediates fibroblast adhesion to collagen, and is synthesized and secreted into the medium of cultured fibroblasts. Affinity-purified antibodies to fibronectin and collagen were localized using the peroxidase-antiperoxidase method or with ferritin-coupled secondary antibodies. Using human fibroblasts cultured under routine conditions, fibronectin and procollagen I react in a nonperiodic manner with: 1) approximately 10 nm extracellular fibrils, 2) cell membrane, and 3) membrane-associated vesicles. All fibrils react with both antibodies, suggesting some form of codistribution of fibronectin and collagen in these fibrils. Treatment with ascorbate leads to the development of a larger diameter extracellular fibril, approximately 40 nm in diameter. These large diameter fibrils are clearly collagen fibrils as documented by the procollagen antibody reaction. Importantly, fibronectin is bound to or a constituent of these "native" or cellular made collagen fibrils. Fibronectin and procollagen antibodies localized with the peroxidase-antiperoxidase method have a 70 nm axial repeat of reaction product on ascorbate-treated fibroblasts. Localization of antibodies with ferritin-labeled secondary antibodies is less satisfactory, but supports the basic observations made with the unlabeled antibody enzyme method. This observation rules out any potential criticisms. Although it is more difficult to observe with immunoferritin, there is an indication that antibodies to fibronectin react with an axial periodicity on cellular produced collagen fibrils.

Absent innervation of skin and sweat glands in congenital insensitivity to pain with anhidrosis.

OBJECTIVES: A case of a 10-year-old girl with congenital insensitivity to pain with anhidrosis (CIPA) is reported. METHODS AND RESULTS: Parents referred several hyperpyretic episodes without sweating occurring since birth, and insensitivity to pain, noticed when the child was 2 years old. Her body had many bruises and scars, bone fractures and signs of self-mutilation. Neurological examination was normal except for insensitivity to pain. Her IQ was 52. Electrical and tactile sensory nerve conduction velocities were normal. The patient was unable to detect thermal stimuli. Histamine injection evoked a wheal but not a flare; pilocarpine by iontophoresis did not induce sweat. Microneurography showed neural activity from A-beta sensory fibers while nociceptive and skin sympathetic C fiber nerve activity was absent. No small myelinated fibers and very rare unmyelinated fibers were found in the sural nerve. Immunohistochemistry showed a lack of nerve fibers in the epidermis and only few hypotrophic and uninnervated sweat glands in the dermis. CONCLUSIONS: The lack of innervation of the skin (C and A-delta fibers) appears to be the morphological basis of insensitivity to pain and anhidrosis, and is consistent with the loss of unmyelinated and small myelinated fibers in the sural nerve biopsy.

The innervation of human epidermis.

Using immunohistochemical procedures numerous nerve fibers have been found in all cell layers of human epidermis. These nerves originate from nerve trunks in the dermis, enter the epidermis, then divide distally to eventually end in small enlargements, near the surface of the skin and in deeper areas. Some endings may be external to stratum granulosum cells. Epidermal nerves appear to have a three-dimensional territorial distribution in relationship to the skin's surface. The presence of epidermal nerve fibers was confirmed by electron microscope studies. The nerves are presumed to be sensory in nature. The existence of epidermal nerve fibers will necessitate changes in present theory of structure and function of peripheral sensation.

A new device to quantify tactile sensation in neuropathy.

OBJECTIVE: To devise a rapid, sensitive method to quantify tactile threshold of finger pads for early detection and staging of peripheral neuropathy and for use in clinical trials. METHODS: Subjects were 166 healthy controls and 103 patients with, or at risk for, peripheral neuropathy. Subjects were screened by questionnaire. The test device, the Bumps, is a checkerboard-like smooth surface with 12 squares; each square encloses 5 colored circles. The subject explores the circles of each square with the index finger pad to locate the one circle containing a small bump. Bumps in different squares have different heights. Detection threshold is defined as the smallest bump height detected. In some subjects, a 3-mm skin biopsy from the tested finger pad was taken to compare density of Meissner corpuscles (MCs) to bump detection thresholds. RESULTS: The mean (±SEM) bump detection threshold for control subjects was 3.3 ± 0.10 µm. Threshold and test time were age related, older subjects having slightly higher thresholds and using more time. Mean detection threshold of patients with neuropathy (6.2 ± 0.35 µm) differed from controls (p < 0.001). A proposed threshold for identifying impaired sensation had a sensitivity of 71% and specificity of 74%. Detection threshold was higher when MC density was decreased. CONCLUSIONS: These preliminary studies suggest that the Bumps test is a rapid, sensitive, inexpensive method to quantify tactile sensation of finger pads. It has potential for early diagnosis of tactile deficiency in subjects suspected of having neuropathy, for staging degree of tactile deficit, and for monitoring change over time.

Gastric mucosal nerve density: a biomarker for diabetic autonomic neuropathy?

BACKGROUND: Autonomic neuropathy is a frequent diagnosis for the gastrointestinal symptoms or postural hypotension experienced by patients with longstanding diabetes. However, neuropathologic evidence to substantiate the diagnosis is limited. We hypothesized that quantification of nerves in gastric mucosa would confirm the presence of autonomic neuropathy. METHODS: Mucosal biopsies from the stomach antrum and fundus were obtained during endoscopy from 15 healthy controls and 13 type 1 diabetic candidates for pancreas transplantation who had secondary diabetic complications affecting the eyes, kidneys, and nerves, including a diagnosis of gastroparesis. Neurologic status was evaluated by neurologic examination, nerve conduction studies, and skin biopsy. Biopsies were processed to quantify gastric mucosal nerves and epidermal nerves. RESULTS: Gastric mucosal nerves from diabetic subjects had reduced density and abnormal morphology compared to control subjects (p < 0.05). The horizontal and vertical meshwork pattern of nerve fibers that normally extends from the base of gastric glands to the basal lamina underlying the epithelial surface was deficient in diabetic subjects. Eleven of the 13 diabetic patients had residual food in the stomach after overnight fasting. Neurologic abnormalities on clinical examination were found in 12 of 13 diabetic subjects and nerve conduction studies were abnormal in all patients. The epidermal nerve fiber density was deficient in skin biopsies from diabetic subjects. CONCLUSIONS: In this observational study, gastric mucosal nerves were abnormal in patients with type 1 diabetes with secondary complications and clinical evidence of gastroparesis. Gastric mucosal biopsy is a safe, practical method for histologic diagnosis of gastric autonomic neuropathy.