Modifying quantitative sensory testing to investigate behavioral reactivity in a pediatric global developmental delay sample: Relation to peripheral innervation and chronic pain outcomes.

Early tactile and nociceptive (pain) mechanisms in children with global developmental delay at risk for intellectual and developmental disability are not well understood. Sixteen children with global developmental delay (mean age = 5.1 years, SD = 1.4; 50% male) completed a modified quantitative sensory testing (mQST) protocol, an epidermal (skin) punch biopsy procedure, and parent-endorsed measures of pain. Children with reported chronic pain had significantly greater epidermal nerve fiber density (ENFd) compared to children without chronic pain. Based on the mQST trials, ENFd values were associated with increased vocal reactivity overall and specifically during the light touch and cool thermal stimulus trials. The findings support the feasibility of an integrative biobehavioral approach to test nociceptive and tactile peripheral innervation and behavioral reactivity during a standardized sensory test in a high-risk sample for which there is often sensory dysfunction and adaptive behavior impairments.

Pathophysiology of Gastroparesis Syndromes Includes Anatomic and Physiologic Abnormalities.

BACKGROUND: Factors underlying gastroparesis are not well defined. AIMS: We hypothesized that multiple systems may be involved in patients with gastroparesis symptoms and performed a comparative physiologic study. METHODS: We studied 43 consecutive eligible patients with gastroparetic symptoms categorized by GI symptoms, metabolic status, illness quantification, and gastric physiology. Patients were evaluated by two methods in each of five core areas: inflammatory, autonomic, enteric, electrophysiologic, and hormonal with abnormalities examined by correlations. RESULTS: Patients had similar GI symptoms regardless of baseline gastric emptying or diabetic/idiopathic status, and all patients demonstrated abnormalities in each of the 5 areas studied. Nearly all patients presented with elevated markers of serum TNFα (88%) and serum IL-6 (91%); elevated cutaneous electrogastrogram frequency (95%); and interstitial cells of Cajal count abnormalities (inner: 97%, outer: 100%). Measures of inflammation correlated with a number of autonomic, enteric anatomy, electrophysiologic and hormonal abnormalities. CONCLUSIONS: We conclude that patients with the symptoms of gastroparesis have multiple abnormalities, when studied by traditional, as well as newer, diagnostic assessments. Inflammation appears to be a fundamental abnormality that affects other organ systems in symptomatic patients. Future work on gastroparetic syndromes and their treatment may benefit from a focus on the diffuse nature of their illness, diverse pathophysiologic mechanisms involved, especially the possible causes of underlying inflammation and disordered hormonal status. TRAIL REGISTRY: This study is registered with Clinicaltrials.gov under study # NCT03178370 https://clinicaltrials.gov/ct2/show/NCT03178370 .

Fast-adapting mechanoreceptors are important for force control in precision grip but not for sensorimotor memory.

Sensory feedback from cutaneous mechanoreceptors in the fingertips is important in effective object manipulation, allowing appropriate scaling of grip and load forces during precision grip. However, the role of mechanoreceptor subtypes in these tasks remains incompletely understood. To address this issue, psychophysical tasks that may specifically assess function of type I fast-adapting (FAI) and slowly adapting (SAI) mechanoreceptors were used with object manipulation experiments to examine the regulation of grip force control in an experimental model of graded reduction in tactile sensitivity (healthy volunteers wearing 2 layers of latex gloves). With gloves, tactile sensitivity decreased significantly from 1.9 ± 0.4 to 12.3 ± 2.2 µm in the Bumps task assessing function of FAI afferents but not in a grating orientation task assessing SAI afferents (1.6 ± 0.1 to 1.8 ± 0.2 mm). Six axis force/torque sensors measured peak grip (PGF) and load (PLF) forces generated by the fingertips during a grip-lift task. With gloves there was a significant increase of PGF (14 ± 6%), PLF (17 ± 5%), and grip and load force rates (26 ± 8%, 20 ± 8%). A variable-weight series task was used to examine sensorimotor memory. There was a 20% increase in PGF when the lift of a light object was preceded by a heavy relative to a light object. This relationship was not significantly altered when lifting with gloves, suggesting that the addition of gloves did not change sensorimotor memory effects. We conclude that FAI fibers may be important for the online force scaling but not for the buildup of a sensorimotor memory.

Epidermal innervation in healthy children and adolescents.

INTRODUCTION: Epidermal nerve fiber (ENF) density, morphology, and epidermal innervation patterns were examined in children using 2 different techniques, punch biopsy and suction blister. METHODS: Healthy children without symptoms or history of peripheral neuropathy and normal by neurologic examination were studied. Punch biopsy and suction blister specimens were collected from the lateral thigh and distal leg. ENFs were traced from confocal images of immunohistochemically stained samples. Statistical methods included repeated-measures analysis of covariance. RESULTS: Blister and biopsy nerve counts were associated. ENF density in children was dense, lower for older children (P<0.01) and with no difference between boys and girls (P=0.92). Many ENFs appeared multibranched and elongated. CONCLUSIONS: Epidermal innervation in the pediatric population is dense and age-dependent. Blister specimens are less invasive and may provide an alternative to punch biopsy for determining ENF density in children at risk for neuropathy.

Skin and self-injury: a possible link between peripheral innervation and immune function?

The aim of this preliminary case study series was to investigate epidermal innervation in pediatric patients with significant neurological impairment and self-injurious behavior. We enrolled four pediatric patients with self-injury (two males, two females; mean age 12y, range 9-14y) and used archival specimens from healthy, age-matched children with typical development for comparison purposes. Epidermal nerve fiber density, peptide content, and mast cell degranulation patterns from non-damaged skin were tested between the patients and the comparison group. The male patients with self-injury had significantly increased epidermal nerve fiber densities, increased substance P positive fiber count and extensive mast cell degranulation compared with sex- and age-matched individuals with typical development. Our case series shows for the first time altered peripheral innervation from non-damaged tissue in children with significant self-injury and developmental disability compared with a healthy comparison group. Establishing the role of peripheral nociceptive and immune modulatory neural pathways may offer new treatment avenues for this devastating neurobehavioral disorder.

Evidence for neural progenitor cells in the human adult enteric nervous system.

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.

Second-order spatial analysis of epidermal nerve fibers.

Breakthroughs in imaging of skin tissue reveal new details on the distribution of nerve fibers in the epidermis. Preliminary neurologic studies indicate qualitative differences in the spatial patterns of nerve fibers based on pathophysiologic conditions in the subjects. Of particular interest is the evolution of spatial patterns observed in the progression of diabetic neuropathy. It appears that the spatial distribution of nerve fibers becomes more 'clustered' as neuropathy advances, suggesting the possibility of diagnostic prediction based on patterns observed in skin biopsies. We consider two approaches to establish statistical inference relating to this observation. First, we view the set of locations where the nerves enter the epidermis from the dermis as a realization of a spatial point process. Secondly, we treat the set of fibers as a realization of a planar fiber process. In both cases, we use estimated second-order properties of the observed data patterns to describe the degree and scale of clustering observed in the microscope images of blister biopsies. We illustrate the methods using confocal microscopy blister images taken from the thigh of one normal (disease-free) individual and two images each taken from the thighs of subjects with mild, moderate, and severe diabetes and report measurable differences in the spatial patterns of nerve entry points/fibers associated with disease status.

Degranulated mast cells in the skin of adults with self-injurious behavior and neurodevelopmental disorders.

The role of nociceptive processes in relation to chronic, tissue-damaging self-injury among individuals with neurodevelopmental disorders is poorly understood. Scientific investigation has been limited, in part, by the clinical reality that the majority of individuals with severe intellectual impairments have co-morbid communicative impairments making it difficult to ascertain information regarding pain. Recently, we found abnormal patterns of peripheral epidermal nerve fiber (ENF) innervation and increased neuropeptide (substance P; SP) content among a subset of individuals with chronic self-injury. Here, we provide initial evidence for peripheral neuro-immune activity specific to self-injury. Skin samples from non-injury body-matched sites were compared between non-verbal adults with and without self-injury matched on gender and disability level. Relative to disability-matched controls, individuals with chronic self-injury had significantly more degranulated mast cells and were more responsive to tactile stimulation during a sensory testing procedure. Thus, nociceptive mechanisms and peripheral afferent sensitization may play a part in mediating and maintaining chronic self-injury.

A clinical case-control comparison of epidermal innervation density in Rett syndrome.

INTRODUCTION: Rett syndrome (RTT), a rare neurodevelopmental disorder occurring primarily in females (1:10-15,000 female live births), is most often caused by loss-of-function mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2). Clinical observations and preclinical findings indicate apparent abnormal sensory and nociceptive function. There have been no direct investigations of epidermal sensory innervation in patients with RTT. METHODS: We compared 3 mm epidermal punch biopsy specimens from adolescent female RTT patients (N = 4, aged 12-19 years) against an archived approximate age-, sex-, body-site matched comparison sample of healthy adolescent females (N = 8, ages 11-17). RESULTS: Confocal imaging revealed, on average, statistically significant increased epidermal nerve fiber (ENF) peptidergic (co-stained calcitonin gene-related protein [CGRP]) innervation density compared with healthy female control individuals. CONCLUSIONS: Given the clinical phenotype of disrupted sensory function along with diagnostic criteria specific to cold hands/feet and insensitivity to pain, our preliminary observations of ENF peptidergic fiber density differences warrants further investigation of the peripheral neurobiology in RTT.

Gastroparesis syndromes: Response to electrical stimulation.

BACKGROUND AND AIMS: Factors underlying gastroparesis are not well defined, nor is the mechanism of action of gastric electrical stimulation (GES). We hypothesized that GES acts via several mechanisms related to underlying disordered pathophysiology. METHODS: We studied 43 consecutive eligible patients with gastroparetic symptoms, previously evaluated by two methods in each of five core areas: inflammatory, autonomic, enteric, electrophysiologic, and hormonal; and also categorized by GI symptoms, metabolic status, illness quantification, and gastric physiology. We then studied 41 patients who underwent temporary GES for 5-7 days. Thirty-six of those patients were implanted and 30 were followed up at 6 months after permanent GES. RESULTS: In previous but separately reported work, patients had similar GI symptoms regardless of baseline gastric emptying or diabetic/idiopathic status and all patients demonstrated abnormalities in each of the five areas studied. After GES, patients showed early and late effects of electrical stimulation with changes noted in multiple areas, categorized by improvement status. CONCLUSION: Patients with symptoms of gastroparesis have multiple abnormalities, including systemic inflammation and disordered hormonal status. GES affects many of these abnormalities. We conclude electrical stimulation improves symptoms and physiology with (a) an early and sustained anti-emetic effect; (b) an early and durable gastric prokinetic effect in delayed emptying patients; (c) an early anti-arrhythmic effect that continues over time; (d) a late autonomic effect; (e) a late hormonal effect; (f) an early anti-inflammatory effect that persists; and (g) an early and sustained improvement in health-related quality of life. This study is registered with Clinicaltrials.gov under study # NCT03178370 (https://clinicaltrials.gov/ct2/show/NCT03178370).

Concordance between epidermal nerve fiber density and sensory examination in patients with symptoms of idiopathic small fiber neuropathy.

Quantitation of epidermal nerve fiber (ENF) density is an objective diagnostic test of small fiber neuropathy (SFN). For a diagnostic test to be clinically useful it should correspond well with clinically meaningful physical findings. We performed a retrospective analysis of the concordance between foot ENF density and clinical findings in all patients seen at our institution with possible idiopathic SFN who underwent skin biopsy for ENF density determination. We found a high concordance between reduced foot ENF density and loss of pinprick sensitivity in this patient population. Our findings indicate that ENF density determination is a clinically relevant objective test in patients undergoing evaluation for possible SFN.

Stimulated growth of human and pig epidermal nerve fibers by tape stripping.

Nerve growth studies in adults usually rely upon nerve regeneration that follows axon disruption. In this study elongation of the epidermal nerve fibers occurred in human and pig epidermis stimulated to hypertrophy by removing the stratum corneum with repetitive applications of tape (tape stripping). Epidermal thickening was accompanied by elongation of the epidermal nerve fibers. This study demonstrates that changing the cellular and chemical environment of nerves by tape stripping is a feasible method to study nerve fiber growth in a physiological manner.

Comparison of Neurovascular Characteristics of Facial Skin in Patients After Primary and Revision Rhytidectomies.

IMPORTANCE: Wound healing influences both the cosmetic and functional outcomes of facial surgery. Study of cutaneous innervation may afford insight into patients' preoperative wound healing potential and aid in their selection of appropriate surgical procedures. OBJECTIVE: To present the quantitative and qualitative differences of epidermal nerve fibers (ENFs), neurotransmitters, vasculature, and mast cells in facial skin among patients after primary and revision rhytidectomies. DESIGN, SETTING, AND PARTICIPANTS: This pilot study collected cutaneous specimens from 8 female patients aged 42 to 66 years who underwent primary rhytidectomy (n = 5) and revision rhytidectomy (n = 3) at Centennial Lakes Surgery Center, Edina, Minnesota, from July 2010 to March 2014. Tissue was processed for confocal/epifluorescence microscopy and indirect immunofluorescent localization of several neural and tissue antigens as well as basement membrane and mast cell markers. INTERVENTION: Primary rhytidectomy vs revision rhytidectomy with selection of a small area of redundant, otherwise disposed of tissue anterior to the tragus for ENF study. MAIN OUTCOMES AND MEASURES: Demographic characteristics included smoking status; 10-point rating scales for facial sensation, pain, and paresthesias; and confocal/epifluorescence microscopy to quantify ENFs, neurotransmitters, vasculature, and mast cells. RESULTS: Patients in the primary rhytidectomy group had a mean (SD) of 54.4 (31.6) ENFs/mm (range, 14.2-99.2 ENFs/mm), and those in the revision rhytidectomy group had a mean (SD) of 18.6 (5.8) ENFs/mm (range, 13.8-25.0 ENFs/mm). A patient in the primary rhytidectomy group was a 25-pack-year smoker and had 14.2 ENFs/mm, the lowest in both groups. In addition to these structural neural changes, functional neural changes in revision rhytidectomy samples included qualitative changes in normal neural antigen prevalence (substance P, calcitonin gene-related peptide, and vasoactive intestinal peptide). Capillary loops appeared less robust and were less common in dermal papilla among samples from both the primary and revision groups, and mast cells were more degranulated. No differences were found in subjective, self-reported postoperative facial sensation. CONCLUSIONS AND RELEVANCE: Previous skin elevation was associated with decreased epidermal nerve fiber density and qualitative changes in dermal nerves, capillaries, and mast cells in a clinical sample of patients undergoing rhytidectomy. Future research is needed to determine whether histological findings predict wound healing and to better understand the effects of surgery on regenerative capacity of epidermal nerve fibers. LEVEL OF EVIDENCE: NA.

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.

Re-organization of P2X3 receptor localization on epidermal nerve fibers in a murine model of cancer pain.

To determine whether ATP and P2X3 receptors contribute to bone-cancer pain in a mouse model, immunohistochemical techniques were used to identify whether changes in the labeling of P2X3 receptors on epidermal nerve fibers (ENFs) occurred during tumor development. C3H mice were injected with osteolytic fibrosarcoma cells in and around the calcaneus bone. These mice exhibited mechanical hyperalgesia by day 10 post-implantation as assessed using von Frey monofilaments. Biopsies of the plantar skin overlying the tumor were obtained at days 10, 14, and 18 post-implantation. Confocal images were analyzed for the number of PGP 9.5, P2X3, and CGRP immunoreactive (ir) ENFs. The overall ENF population (PGP-ir) decreased progressively over time, whereas the subsets of P2X3-ir fibers demonstrated a modest increase and CGRP-ir nerve fibers remained fairly constant. Importantly, the proportion of CGRP-ir fibers that labeled for P2X3 increased from approximately 6% in control animals to nearly 30% at day 14 following tumor cell implantation. These studies demonstrate increased expression of P2X3 receptors on CGRP-ir ENFs during tumor growth and suggest a role for ATP in cancer-related pain.

Assessing function and pathology in familial dysautonomia: assessment of temperature perception, sweating and cutaneous innervation.

This study was performed to assess cutaneous nerve fibre loss in conjunction with temperature and sweating dysfunction in familial dysautonomia (FD). In ten FD patients, we determined warm and cold thresholds at the calf and shoulder, and sweating in response to acetylcholine iontophoresis over the calf and forearm. Punch skin biopsies from calf and back were immunostained and imaged to assess nerve fibre density and neuropeptide content. Mean temperature thresholds and baseline sweat rate were elevated in the patients, while total sweat volume and response time did not differ from controls. The average density of epidermal nerve fibres was greatly diminished in the calf and back. There was also severe nerve loss from the subepidermal neural plexus (SNP) and deep dermis. The few sweat glands present within the biopsies had had reduced innervation density. Substance P immunoreactive (-ir) and calcitonin gene related peptide-ir (CGRP-ir) were virtually absent, but vasoactive intestinal peptide-ir (VIP-ir) nerves were present in the SNP. Empty Schwann cell sheaths were observed. Temperature perception was more impaired than sweating. Epidermal nerve fibre density was found to be profoundly reduced in FD. Decreased SP and CGRP-ir nerves suggest that the FD gene mutation causes secondary neurotransmitter depletions. Empty Schwann cell sheaths and VIP-ir nerves suggest active denervation and regeneration.

Peripheral Innervation in Children With Global Developmental Delay: Biomarker for Risk for Self-Injurious Behavior?

The relation between somatosensory mechanisms and self-injury among children with neurologic impairments associated with developmental delay is not well understood. We evaluated the feasibility of procuring skin biopsies to examine epidermal nerve fiber density and reported self-injury. Following informed parental consent, epidermal skin biopsies were obtained from a distal leg site with no pre-existing skin damage from 11 children with global developmental delay (55% male; mean age = 36.8 months, 17-63 months). Visual microscopic examination and quantitative analyses showed extremely high epidermal nerve fiber density values for some children. Children with reported self-injury (5/11) had significantly (P < .02) greater density values (138.8, standard deviation = 45.5) than children without self-injury (80.5, standard deviation = 17.5). Results from this novel immunohistologic analysis of skin in very young children with neurodevelopmental delays suggest it may be a useful tool to study peripheral innervation as a possible sensory risk factor for self-injury.

Influence of thermode size for detecting heat pain dysfunction in a capsaicin model of epidermal nerve fiber loss.

Quantitative sensory testing of heat pain sensation has become an important tool to evaluate small caliber afferent nerve function in peripheral neuropathy. In earlier studies, we found that topical application of capsaicin in humans results in the loss of epidermal nerve fibers (ENFs) with a corresponding decrease in detection of heat pain sensation. Capsaicin may therefore be a useful model for developing optimal psychophysical testing procedures for detection of neuropathy in its early stages. Here we determined the influence of thermal probe (thermode) size in detecting the diminished heat pain sensation following capsaicin application. Twelve healthy volunteers applied 0.075% capsaicin topically to the volar forearm four times daily for 7 days. Psychophysical measures of heat pain, mechanical (sharp) pain, and tactile threshold were obtained daily from untreated control skin and from capsaicin-treated skin during capsaicin application, and once weekly for 5 weeks following discontinuation of capsaicin. Heat pain sensation was assessed using a large (30 x 30 mm) and small (3 x 3 mm) thermode and different algorithms to assess pain threshold and suprathreshold heat pain. Skin biopsies were obtained and were processed for immunohistochemical localization of (ENFs) using the pan neuronal marker protein gene product 9.5. Capsaicin produced a rapid decrease in the number of ENFs, with nearly complete disappearance after 3 days of treatment. Heat pain evoked by the small, but not the large, thermode decreased dramatically after capsaicin treatment. The sensation of heat pain returned toward normal after 2--3 weeks following discontinuation of capsaicin treatment concordant with gradual reinnervation of the epidermis. Regression analysis indicated that the sensation of heat pain evoked by the small thermode correlated much better with the number of ENFs than heat pain evoked by the large thermode. The detection of sharp pain decreased moderately after capsaicin treatment. Assessment of heat pain sensation using small thermodes has potential for detecting sensory deficits in early stages of small fiber neuropathy.