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.

Combining In Vivo Corneal Confocal Microscopy With Deep Learning-Based Analysis Reveals Sensory Nerve Fiber Loss in Acute Simian Immunodeficiency Virus Infection.

PURPOSE: To characterize corneal subbasal nerve plexus features of normal and simian immunodeficiency virus (SIV)-infected macaques by combining in vivo corneal confocal microscopy (IVCM) with automated assessments using deep learning-based methods customized for macaques. METHODS: IVCM images were collected from both male and female age-matched rhesus and pigtailed macaques housed at the Johns Hopkins University breeding colony using the Heidelberg HRTIII with Rostock Corneal Module. We also obtained repeat IVCM images of 12 SIV-infected animals including preinfection and 10-day post-SIV infection time points. All IVCM images were analyzed using a deep convolutional neural network architecture developed specifically for macaque studies. RESULTS: Deep learning-based segmentation of subbasal nerves in IVCM images from macaques demonstrated that corneal nerve fiber length and fractal dimension measurements did not differ between species, but pigtailed macaques had significantly higher baseline corneal nerve fiber tortuosity than rhesus macaques (P = 0.005). Neither sex nor age of macaques was associated with differences in any of the assessed corneal subbasal nerve parameters. In the SIV/macaque model of human immunodeficiency virus, acute SIV infection induced significant decreases in both corneal nerve fiber length and fractal dimension (P = 0.01 and P = 0.008, respectively). CONCLUSIONS: The combination of IVCM and robust objective deep learning analysis is a powerful tool to track sensory nerve damage, enabling early detection of neuropathy. Adapting deep learning analyses to clinical corneal nerve assessments will improve monitoring of small sensory nerve fiber damage in numerous clinical settings including human immunodeficiency virus.

Deep learning-based analysis of macaque corneal sub-basal nerve fibers in confocal microscopy images.

BACKGROUND: To develop and validate a deep learning-based approach to the fully-automated analysis of macaque corneal sub-basal nerves using in vivo confocal microscopy (IVCM). METHODS: IVCM was used to collect 108 images from 35 macaques. 58 of the images from 22 macaques were used to evaluate different deep convolutional neural network (CNN) architectures for the automatic analysis of sub-basal nerves relative to manual tracings. The remaining images were used to independently assess correlations and inter-observer performance relative to three readers. RESULTS: Correlation scores using the coefficient of determination between readers and the best CNN averaged 0.80. For inter-observer comparison, inter-correlation coefficients (ICCs) between the three expert readers and the automated approach were 0.75, 0.85 and 0.92. The ICC between all four observers was 0.84, the same as the average between the CNN and individual readers. CONCLUSIONS: Deep learning-based segmentation of sub-basal nerves in IVCM images shows high to very high correlation to manual segmentations in macaque data and is indistinguishable across readers. As quantitative measurements of corneal sub-basal nerves are important biomarkers for disease screening and management, the reported work offers utility to a variety of research and clinical studies using IVCM.

Comparative Anatomy of the Mammalian Corneal Subbasal Nerve Plexus.

Purpose: The subbasal nerve plexus (SNP) is the densest and most recognizable component of the mammalian corneal innervation; however, the anatomical configuration of the SNP in most animal models remains incompletely described. The purpose of the current study is to describe in detail the SNP architecture in eight different mammals, including several popular animal models used in cornea research. Methods: Corneal nerves in mouse, rat, guinea pig, rabbit, dog, macaque, domestic pig, and cow eyes were stained immunohistochemically with antiserum directed against neurotubulin. SNP architecture was documented by digital photomicrography and large-scale reconstructions, that is, corneal nerve maps, using a drawing tube attached to a light microscope. Results: Subbasal nerve fibers (SNFs) in mice, rats, guinea pigs, dogs, and macaques radiated centrally from the corneoscleral limbus toward the corneal apex in a whorl-like or spiraling pattern. SNFs in rabbit and bovine corneas swept horizontally across the ocular surface in a temporal-to-nasal direction and converged on the inferonasal limbus without forming a spiral. SNFs in the pig cornea radiated centrifugally in all directions, like a starburst, from a focal point located equidistant between the corneal apex and the superior pole. Conclusions: The results of the present study have demonstrated for the first time substantial interspecies differences in the architectural organization of the mammalian SNP. The physiological significance of these different patterns and the mechanisms that regulate SNP pattern formation in the mammalian cornea remain incompletely understood and warrant additional investigation.

SIV-Induced Immune Activation and Metabolic Alterations in the Dorsal Root Ganglia During Acute Infection.

Human immunodeficiency virus-associated sensory neuropathy (HIV-SN) remains a frequent neurologic complication of HIV infection. Little is known about alterations in the peripheral nervous system during the early stages of HIV, a time when neuroprotective interventions may be most beneficial. We performed Nanostring gene expression analysis on lumbar dorsal root ganglia (DRG) from 6 simian immunodeficiency virus (SIV)-infected pigtailed macaques killed at 7 days post-inoculation and 8 uninfected controls. We found significant upregulation of many genes involved in immune signaling and activation in the DRG. Among genes related to glutamate metabolism, there was significant upregulation of glutamine synthetase (GS), while glutaminase (GLS) was downregulated. Several genes involved in the oxidative stress response also showed significant differential regulation in the DRG of 7d SIV-infected animals, with superoxide dismutase-2 (SOD2) showing the greatest median fold change compared to controls. Novel findings in the DRG were compared to corresponding brain data and further investigated at the protein level by Western blotting and immunohistochemistry. Together with our previous finding of significant epidermal nerve fiber loss at 14 days post-SIV infection, results of this study demonstrate that immune activation and altered cellular metabolism at in the DRG precede and likely contribute to early sensory nerve injury in HIV-SN.

Tracking Epidermal Nerve Fiber Changes in Asian Macaques: Tools and Techniques for Quantitative Assessment.

Quantitative assessment of epidermal nerve fibers (ENFs) has become a widely used clinical tool for the diagnosis of small fiber neuropathies such as diabetic neuropathy and human immunodeficiency virus-associated sensory neuropathy (HIV-SN). To model and investigate the pathogenesis of HIV-SN using simian immunodeficiency virus (SIV)-infected Asian macaques, we adapted the skin biopsy and immunostaining techniques currently employed in human patients and then developed two unbiased image analysis techniques for quantifying ENF in macaque footpad skin. This report provides detailed descriptions of these tools and techniques for ENF assessment in macaques and outlines important experimental considerations that we have identified in the course of our long-term studies. Although initially developed for studies of HIV-SN in the SIV-infected macaque model, these methods could be readily translated to a range of studies involving peripheral nerve degeneration and neurotoxicity in nonhuman primates as well as preclinical investigations of agents aimed at neuroprotection and regeneration.