PAIN HYPERSENSITIVITY IN SLURP1 AND SLURP2 KNOCKOUT MOUSE MODELS OF HEREDITARY PALMOPLANTAR KERATODERMA.

SLURP1 and SLURP2 are both small secreted members of the Ly6/u-PAR family of proteins and are highly expressed in keratinocytes. Loss of function mutations in SLURP1 lead to a rare autosomal recessive Palmoplantar Keratoderma (PPK), Mal de Meleda (MdM), which is characterized by diffuse, yellowish palmoplantar hyperkeratosis. Some individuals with MdM experience pain in conjunction with the hyperkeratosis that has been attributed to fissures or microbial superinfection within the affected skin. By comparison, other hereditary PPKs such as Pachyonychia congenita (PC) and Olmsted syndrome (OS) show prevalent pain in PPK lesions. Two mouse models of MdM, Slurp1 knockout and Slurp2X knockout, exhibit robust PPK in all four paws. However, whether the sensory experience of these animals including augmented pain sensitivity remains unexplored. In this study, we demonstrate that both models exhibit hypersensitivity to mechanical and thermal stimuli as well as spontaneous pain behaviors in males and females. Anatomical analysis revealed increased paw pad skin epidermal innervation and substantial alterations in palmoplantar skin immune composition in Slurp2X knockout mice. Primary sensory neurons innervating hind paw glabrous skin from Slurp2X knockout mice exhibit increased incidence of spontaneous activity and mechanical hypersensitivity both in vitro and in vivo. Thus, Slurp knockout mice exhibit polymodal PPK-associated pain that is associated with both immune alterations and neuronal hyperexcitability, and might therefore be useful for the identification of therapeutic targets to treat PPK-associated pain.Significance Statement Palmoplantar keratodermas (PPKs) are rare human skin disorders associated with thickening of the skin on the palms and soles. Pain is a common feature of some PPKs, yet the causes of PPK-associated pain are not understood. Here we show that two mouse models of one PPK, SLURP1 knockout mice and SLURP2 knockout mice, exhibit enhanced pain sensitivity and increased activity of pain-associated sensory neurons. These mouse lines will therefore be of value in defining causes of pain in PPKs and possibly developing improved therapies for that pain.

Phenotypes Associated With the Val122Ile, Leu58His, and Late-Onset Val30Met Variants in Patients With Hereditary Transthyretin Amyloidosis.

BACKGROUND AND OBJECTIVES: Hereditary transthyretin amyloidosis (hATTR) is a rare autosomal dominant systemic disease with variable penetrance and heterogeneous clinical presentation. Several effective treatments can reduce mortality and disability, though diagnosis remains challenging, especially in the United States where disease is nonendemic. Our aim is to describe the neurologic and cardiac characteristics of common US ATTR variants V122I, L58H, and late-onset V30M at presentation. METHODS: We conducted a retrospective case series of patients with a new diagnosis of ATTRv between January 2008 and January 2020 to characterize features of prominent US variants. The neurologic (examination, EMG, and skin biopsy), cardiac (echo), and laboratory assessments (pro b-type natriuretic peptide [proBNP] and reversible neuropathy screens) are described. RESULTS: A total of 56 patients with treatment-naïve ATTRv with symptoms/signs of peripheral neuropathy (PN) or cardiomyopathy and confirmatory genetic testing presenting with Val122Ile (N = 31), late-onset Val30Met (N = 12), and Leu58His ATTRv (N = 13) were included. The age at onset and sex distributions were similar (V122I: 71.5 ± 8.0, V30M: 64.8 ± 2.6, and L58H: 62.4 ± 9.8 years; 26, 25, 31% female). Only 10% of patients with V122I and 17% of patients with V30M were aware of an ATTRv family history, while 69% of patients with L58H were aware. PN was present in all 3 variants at diagnosis (90%, 100%, and 100%), though neurologic impairment scores differed: V122I: 22 ± 16, V30M: 61 ± 31, and L58H: 57 ± 25. Most points (deficits) were attributed to loss of strength. Carpal tunnel syndrome (CTS) and a positive Romberg sign were common across all groups (V122I: 97%, 39%; V30M: 58%, 58%; and L58H: 77%, 77%). ProBNP levels and interventricular septum thickness were highest among patients with V122I (5,939 ± 962 pg/mL, 1.70 ± 0.29 cm), followed by V30M (796 ± 970 pg/mL, 1.42 ± 0.38 cm) and L58H (404 ± 677 pg/mL, 1.23 ± 0.36 cm). Atrial fibrillation was present among 39% of cases with V122I and only 8% of cases with V30M and L58H. Gastrointestinal symptoms were rare (6%) among patients with V122I and common in patients with V30M (42%) and L58H (54%). DISCUSSION: Important clinical differences exist between ATTRv genotypes. While V122I is perceived to be a cardiac disease, PN is common and clinically relevant. Most patients with V30M and V122I were diagnosed de novo and therefore require clinical suspicion for diagnosis. A history of CTS and a positive Romberg sign are helpful diagnostic clues.

Neuropathic symptoms with SARS-CoV-2 vaccination.

Background and Objectives: Various peripheral neuropathies, particularly those with sensory and autonomic dysfunction may occur during or shortly after acute COVID-19 illnesses. These appear most likely to reflect immune dysregulation. If similar manifestations can occur with the vaccination remains unknown. Results: In an observational study, we studied 23 patients (92% female; median age 40years) reporting new neuropathic symptoms beginning within 1 month after SARS-CoV-2 vaccination. 100% reported sensory symptoms comprising severe face and/or limb paresthesias, and 61% had orthostasis, heat intolerance and palpitations. Autonomic testing in 12 identified seven with reduced distal sweat production and six with positional orthostatic tachycardia syndrome. Among 16 with lower-leg skin biopsies, 31% had diagnostic/subthreshold epidermal neurite densities (≤5%), 13% were borderline (5.01-10%) and 19% showed abnormal axonal swelling. Biopsies from randomly selected five patients that were evaluated for immune complexes showed deposition of complement C4d in endothelial cells. Electrodiagnostic test results were normal in 94% (16/17). Together, 52% (12/23) of patients had objective evidence of small-fiber peripheral neuropathy. 58% patients (7/12) treated with oral corticosteroids had complete or near-complete improvement after two weeks as compared to 9% (1/11) of patients who did not receive immunotherapy having full recovery at 12 weeks. At 5-9 months post-symptom onset, 3 non-recovering patients received intravenous immunoglobulin with symptom resolution within two weeks. Conclusions: This observational study suggests that a variety of neuropathic symptoms may manifest after SARS-CoV-2 vaccinations and in some patients might be an immune-mediated process.

Major Improvement in Wound Healing Through Pharmacologic Mobilization of Stem Cells in Severely Diabetic Rats.

Current therapeutic strategies for diabetic foot ulcer (DFU) have focused on developing topical healing agents, but few agents have controlled prospective data to support their effectiveness in promoting wound healing. We tested a stem cell mobilizing therapy for DFU using a combination of AMD3100 and low-dose FK506 (tacrolimus) (AF) in streptozocin-induced type 1 diabetic (T1DM) rats and type 2 diabetic Goto-Kakizaki (GK) rats that had developed peripheral artery disease and neuropathy. Here, we show that the time for healing back wounds in T1DM rats was reduced from 27 to 19 days, and the foot wound healing time was reduced from 25 to 20 days by treatment with AF (subcutaneously, every other day). Similarly, in GK rats treated with AF, the healing time on back wounds was reduced from 26 to 21 days. Further, this shortened healing time was accompanied by reduced scar and by regeneration of hair follicles. We found that AF therapy mobilized and recruited bone marrow-derived CD133+ and CD34+ endothelial progenitor cells and Ym1/2+ M2 macrophages into the wound sites, associated with enhanced capillary and hair follicle neogenesis. Moreover, AF therapy improved microcirculation in diabetic and neuropathic feet in GK rats. This study provides a novel systemic therapy for healing DFU.

Spinal cord organogenesis model reveals role of Flk1+ cells in self-organization of neural progenitor cells into complex spinal cord tissue.

A platform for studying spinal cord organogenesis in vivo where embryonic stem cell (ESC)-derived neural progenitor cells (NPC) self-organize into spinal cord-like tissue after transplantation in subarachnoid space of the spinal cord has been described. We advance the applicability of this platform by imaging in vivo the formed graft through T2w magnetic resonance imaging (MRI). Furthermore, we used diffusion tensor imaging (DTI) to verify the stereotypical organization of the graft showing that it mimics the host spinal cord. Within the graft white matter (WM) we identified astrocytes that form glial limitans, myelinating oligodendrocytes, and myelinated axons with paranodes. Within the graft grey matter (GM) we identified cholinergic, glutamatergic, serotonergic and dopaminergic neurons. Furthermore, we demonstrate the presence of ESC-derived complex vasculature that includes the presence of blood brain barrier. In addition to the formation of mature spinal cord tissue, we describe factors that drive this process. Specifically, we identify Flk1+ cells as necessary for spinal cord formation, and synaptic connectivity with the host spinal cord and formation of host-graft chimeric vasculature as contributing factors. This model can be used to study spinal cord organogenesis, and as an in vivo drug discovery platform for screening potential therapeutic compounds and their toxicity.

Nociceptin/orphanin FQ opioid peptide-receptor expression in pachyonychia congenita.

Nociceptin/orphanin FQ opioid peptide (NOP)-receptor (NOP-R) is a member of the opioid receptor family. NOP-R activation has demonstrated analgesic effects in preclinical pain models without the addiction risks associated with other opiate targets. Pachyonychia congenita (PC) is a palmoplantar keratoderma characterized by neuropathic pain in affected skin. A cohort of KRT6A gene mutation PC patients with no other explanation for their neuropathic pain offered a unique opportunity to assess potential of NOP-R as a therapeutic target. Plantar biopsies from 10 PC patients and 10 age/gender matched controls were performed at the ball (PC-affected) and the arch (PC-unaffected) of the foot. NOP-R expression was assessed by immunohistochemistry. Localization of NOP-R in subsets of epidermal nerve fibers was investigated using the pan-neuronal marker PGP9.5, markers for unmyelinated peptidergic fibers (calcitonin gene-related peptide [CGRP] and substance P [SP]), as well as for myelinated Aδ and Aß fibers (neurofilament H [NFH]). Robust NOP-R expression was detected in epidermal keratinocytes and in a subset of PGP9.5+ fibers in both epidermis and dermis, confirmed by western blot and absorption experiments with NOP-R peptide. NOP-R expression in keratinocytes was significantly reduced in PC-affected plantar skin compared with PC-unaffected skin. In addition, NOP-R expression occurred in dermal NFH+ myelinated fibers in all groups, although few CGRP+ fibers co-expressed NOP-R. Furthermore, most SP+ fibers also co-expressed NOP-R. These findings indicate that NOP-R is expressed on epidermal keratinocytes, as well as on epidermal and dermal nerve fibers and has potential as a promising target to treat neuropathic pain in PC.

Sensory and autonomic function and structure in footpads of a diabetic mouse model.

Sensory and autonomic neuropathy affects the majority of type II diabetic patients. Clinically, autonomic evaluation often focuses on sudomotor function yet this is rarely assessed in animal models. We undertook morphological and functional studies to assess large myelinated and small unmyelinated axons in the db/db type II diabetes mouse model. We observed that autonomic innervation of sweat glands in the footpads was significantly reduced in db/db mice compared to control db/+ mice and this deficit was greater compared to reductions in intraepidermal sensory innervation of adjacent epidermis. Additionally, db/db mice formed significantly fewer sweat droplets compared to controls as early as 6 weeks of age, a time when no statistical differences were observed electrophysiologically between db/db and db/+ mice studies of large myelinated sensory and motor nerves. The rate of sweat droplet formation was significantly slower and the sweat droplet size larger and more variable in db/db mice compared to controls. Whereas pilocarpine and glycopyrrolate increased and decreased sweating, respectively, in 6 month-old controls, db/db mice did not respond to pharmacologic manipulations. Our findings indicate autonomic neuropathy is an early and prominent deficit in the db/db model and have implications for the development of therapies for peripheral diabetic neuropathy.

Peripheral neuropathic changes in pachyonychia congenita.

We compared patterns of intraepidermal nerve fibers and mechanoreceptors from affected and unaffected plantar skin from patients with pachyonychia congenita (PC) and control subjects. Plantar biopsies from 10 genetically confirmed patients with PC (with a mutation in KRT6A) were performed at the ball of the foot (affected skin) and the arch (unaffected) and were compared to biopsies from corresponding locations in 10 control subjects. Tissue was processed to visualize intraepidermal nerve fibers (IENF) (PGP9.5), subsets of IENF (CGRP, substance P, tyrosine hydroxylase), myelinated nerve fiber (neurofilament H, NFH), blood vessels (CD31), Meissner corpuscles, and Merkel cells (MCs). Structures were quantified using stereology or validated quantification methods. We observed that PC-affected plantar skin had significantly lower sweat gland innervation (sweat gland nerve fiber density) and reduced numbers of Meissner corpuscles compared to PC-unaffected or anatomically matched control skin. In contrast, Merkel cell densities and blood vessel counts were higher in PC-affected skin compared to either control or PC-unaffected skin. There were no differences in myelinated nerve fiber densities, SP, or CGRP between the groups. Pressure pain thresholds in PC-affected skin were lower compared to PC-unaffected and anatomically matched control skin. Additionally, MC densities in callused plantar skin from healthy runners with callus and one subject with a nonpainful palmoplantar keratoderma (AQP5 mutation) were similar to PC-unaffected and control skin consistent with callus alone not being sufficient to increase MC number. These findings suggest that alterations in PC extend beyond keratinocytes and may provide strategies to study neuropathic pain in PC.

Factors influencing sweat gland innervation in diabetes.

BACKGROUND: Using a stereologic approach, the density of nerve fibers innervating sweat gland (SG) fragments in patients with diabetes mellitus (DM) and healthy controls using protein gene product (PGP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) was measured to determine which marker best detected differences between the groups. Factors associated with SG nerve fiber (SGNF) innervation were assessed and the change in SG innervation over a 1-year time period was determined. METHODS: Ninety-two control subjects and 2 groups of subjects with DM totaling 97 were assessed in this cross-sectional study. Intraepidermal nerve fiber density and SG innervation were determined from leg skin biopsies that were immunohistochemically stained for ubiquitin hydrolase, VIP, and TH. Factors associated with SG innervation were assessed and 15 subjects were longitudinally followed for 1 year. RESULTS: SGNF innervation was reduced in subjects with DM compared with controls. Lower SG innervation values were associated with increasing glycated hemoglobin A1c, body mass index (BMI), men compared with women, and tobacco use, but not diabetes type or age. Sex, A1c, and BMI remained significant in multivariate modeling. SG innervation measured by VIP+ fibers is a more sensitive marker for neuropathy than either PGP or TH. Fifteen subjects with DM followed for 1 year showed a significant decrease in SGNF innervation but not intraepidermal nerve fiber density. CONCLUSIONS: Stereologic measurement of SG innervation is feasible to assess postganglionic autonomic nerve fiber densities. SG innervation was reduced in subjects with DM compared with control subjects and was associated with sex, A1c, and BMI in multivariate modeling. VIP+ SGNF is more severely reduced in DM than TH+ or PGP9.5+-based assessments. Progression of diabetic polyneuropathy was detected by SGNF over a 1-year time period.

The lateral thoracic nerve and the cutaneous maximus muscle--a novel in vivo model system for nerve degeneration and regeneration studies.

We report a novel in vivo mouse model system to study regeneration of injured motor nerve and spatiotemporal pattern of denervation in experimental nerve diseases. The lateral thoracic nerve (LTN), as a pure motor nerve, innervates the cutaneous maximus muscle (CMM) by some of the shortest and the longest motor nerve fibers in the mouse body. Its branches and nerve terminals can be imaged in whole mount preparations. Here we describe the branching pattern of the LTN and its innervation of the CMM, and characterize degeneration and regeneration over time after a LTN crush by morphological and electrophysiological analyses. We demonstrate the utility of this model in a well-established neurotoxicity paradigm and in a genetic disease model of the peripheral neuropathy. Furthermore, this system enables punch biopsies that allow repeated and multi-location examinations for LTN regeneration and CMM reinnervation over time. The presence of the LTN and the CMM in a variety of species and its easy accessibility suggests that this in vivo model system offers considerable promise for future nerve degeneration and regeneration research.

Measuring nerve regeneration in the mouse.

Genetic engineering of mice has become a major tool in understanding the roles of individual molecules in regeneration of nerves, and will play an increasing role in the future. Mice are in many ways well suited to assessment both of nerve regeneration after axotomy and of collateral sprouting of intact fibers into areas of denervation. However, mouse models present special challenges because of their small size, their inherent capacity for regeneration, and the potential strain effects. The most widely used model of regeneration, sciatic nerve injury, has its inherent limitations, and there is a need for other models of injury to long nerves. Measures of regeneration in the mouse can be divided into those that assess the latency to initiate growth, those sensitive to the rate of growth and the proportion of fibers growing at fast rates, those that assess the time to reinnervation of specific targets and the completeness of reinnervation, and those that assess specificity of reinnervation and functional recovery. The short length of nerve available in the mouse limits measures of the rates of outgrowth, and thus introduces a greater potential for "noise" of measurement than is seen in larger animals such as the rat. For both regeneration of interrupted fibers and collateral regeneration from intact fibers histological and physiological measures of "time to target" have the advantages of direct correlation with restoration of function, the ability to assess regeneration of different fiber types efficiently, and the fact that most of these measures are easier in the mouse than in the rat.

Effects of corticospinal tract stimulation on renal sympathetic nerve activity in rats with intact and chronically lesioned spinal cords.

Sympathetic preganglionic neurons and interneurons are closely apposed (presumably synapsed upon) by corticospinal tract (CST) axons. Sprouting of the thoracic CST rostral to lumbar spinal cord injuries (SCI) substantially increases the incidence of these appositions. To test our hypothesis that these additional synapses would increase CST control of sympathetic activity after SCI, we measured the effects of electrical stimulation of the CST on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) in alpha-chloralose-anesthetized rats with either chronically intact or chronically lesioned spinal cords. Stimuli were delivered to the CST at intensities between 25-150 muA and frequencies between 25 and 75 Hz. Stimulation of the CST at the midcervical level decreased RSNA and AP. These decreases were not mediated by direct projections of the CST to the thoracic spinal cord because we could still elicit them by midcervical stimulation after acute lesions of the CST at caudal cervical levels. In contrast, caudal thoracic CST stimulation increased RSNA and AP. Neither the responses to cervical nor thoracic stimulation were affected by chronic lumbar SCI. These data show that the CST mediates decreases in RSNA via a cervical spinal system but excites spinal sympathetic neurons at caudal thoracic levels. Because chronic lumber spinal cord injury affected responses evoked from neither the cervical nor thoracic CST, we conclude that lesion-induced or regeneration-induced formation of new synapses between the CST and sympathetic neurons may not affect cardiovascular regulation.

Increased close appositions between corticospinal tract axons and spinal sympathetic neurons after spinal cord injury in rats.

Treatments for spinal cord injury may promote new spinal cord synapses. However, the potential for new synapses between descending somatomotor and spinal sympathetic neurons has not been investigated. We studied rats with intact spinal cords and rats after a chronic, bilateral, dorsal spinal hemisection. We identified sympathetically related spinal neurons by transynaptic, retrograde transport of renally injected pseudorabies virus. We counted retrogradely labeled sympathetic preganglionic neurons (SPN) and putative sympathetic interneurons (IN) that, under light microscopy, appeared closely apposed by anterogradely labeled axons of the corticospinal tract (CST) and by axons descending from the well-established sympathetic regulatory region in the rostral ventrolateral medulla (RVLM). Spinal sympathetic neurons that were closely apposed by CST axons were significantly more numerous in lesioned rats than in unlesioned rats. CST axons closely apposed 5.4% of SPN and 10.3% of IN in rats with intact spinal cords, and 38.0% of SPN and 37.3% of IN in rats with chronically lesioned spinal cords. Further, CST appositions in SCI rats consisted of many more varicosities than those in uninjured rats. SPN and IN closely apposed by axons from the RVLM were not more numerous in lesioned rats. However, RVLM axons apposed many more SPN than IN in both control and lesioned rats. Therefore, RVLM sympathoexcitation may be mediated largely by direct synapses on SPN. Although we have not determined the functional significance of close appositions between the CST and spinal sympathetic neurons, we suggest that future studies of spinal cord repair and regeneration include an evaluation of potential, new, somatic-autonomic interactions.

Myelin-associated glycoprotein and complementary axonal ligands, gangliosides, mediate axon stability in the CNS and PNS: neuropathology and behavioral deficits in single- and double-null mice.

Complementary interacting molecules on myelin and axons are required for long-term axon-myelin stability. Their disruption results in axon degeneration, contributing to the pathogenesis of demyelinating diseases. Myelin-associated glycoprotein (MAG), a minor constituent of central and peripheral nervous system myelin, is a member of the Siglec family of sialic acid-binding lectins and binds to gangliosides GD1a and GT1b, prominent molecules on the axon surface. Mice lacking the ganglioside biosynthetic gene Galgt1 fail to express complex gangliosides, including GD1a and GT1b. In the current studies, CNS and PNS histopathology and behavior of Mag-null, Galgt1-null, and double-null mice were compared on the same mouse strain background. When back-crossed to >99% C57BL/6 strain purity, Mag-null mice demonstrated marked CNS, as well as PNS, axon degeneration, in contrast to prior findings using mice of mixed strain background. On the same background, Mag- and Galgt1-null mice exhibited quantitatively and qualitatively similar CNS and PNS axon degeneration and nearly identical decreases in axon diameter and neurofilament spacing. Double-null mice had qualitatively similar changes. Consistent with these findings, Mag- and Galgt1-null mice had similar motor behavioral deficits, with double-null mice only modestly more impaired. Despite their motor deficits, Mag- and Galgt1-null mice demonstrated hyperactivity, with spontaneous locomotor activity significantly above that of wild type mice. These data demonstrate that MAG and complex gangliosides contribute to axon stability in both the CNS and PNS. Similar neuropathological and behavioral deficits in Galgt1-, Mag-, and double-null mice support the hypothesis that MAG binding to gangliosides contributes to long-term axon-myelin stability.