Translational control in the spinal cord regulates gene expression and pain hypersensitivity in the chronic phase of neuropathic pain.

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV + ) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV + neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV + neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Early life adversity promotes gastrointestinal dysfunction through a sex-dependent phenotypic switch in enteric glia.

Irritable bowel syndrome and related disorders of gut-brain interaction (DGBI) are common and exhibit a complex, poorly understood etiology that manifests as abnormal gut motility and pain. Risk factors such as biological sex, stressors during critical periods, and inflammation are thought to influence DGBI vulnerability by reprogramming gut-brain circuits, but the specific cells affected are unclear. Here, we used a model of early life stress to understand cellular mechanisms in the gut that produce DGBIs. Our findings identify enteric glia as a key cellular substrate in which stress and biological sex converge to dictate DGBI susceptibility. Enteric glia exhibit sexual dimorphism in genes and functions related to cellular communication, inflammation, and disease susceptibility. Experiencing early life stress has sex-specific effects on enteric glia that cause a phenotypic switch in male glia toward a phenotype normally observed in females. This phenotypic transformation is followed by physiological changes in the gut, mirroring those observed in DGBI in humans. These effects are mediated, in part, by alterations to glial prostaglandin and endocannabinoid signaling. Together, these data identify enteric glia as a cellular integration site through which DGBI risk factors produce changes in gut physiology and suggest that manipulating glial signaling may represent an attractive target for sex-specific therapeutic strategies in DGBIs.

Deciphering the molecular landscape of human peripheral nerves: implications for diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is a prevalent complication of diabetes mellitus that is caused by metabolic toxicity to peripheral axons. We aimed to gain deep mechanistic insight into the disease process using bulk and spatial RNA sequencing on tibial and sural nerves recovered from lower leg amputations in a mostly diabetic population. First, our approach comparing mixed sensory and motor tibial and purely sensory sural nerves shows key pathway differences in affected nerves, with distinct immunological features observed in sural nerves. Second, spatial transcriptomics analysis of sural nerves reveals substantial shifts in endothelial and immune cell types associated with severe axonal loss. We also find clear evidence of neuronal gene transcript changes, like PRPH, in nerves with axonal loss suggesting perturbed RNA transport into distal sensory axons. This motivated further investigation into neuronal mRNA localization in peripheral nerve axons generating clear evidence of robust localization of mRNAs such as SCN9A and TRPV1 in human sensory axons. Our work gives new insight into the altered cellular and transcriptomic profiles in human nerves in DPN and highlights the importance of sensory axon mRNA transport as an unappreciated potential contributor to peripheral nerve degeneration.

Transcriptome Analysis of Rheumatoid Arthritis Uncovers Genes Linked to Inflammation-Induced Pain.

Autoimmune diseases such as rheumatoid arthritis (RA) can promote states of chronic Inflammation with accompanying tissue destruction and pain. RA can cause inflammatory synovitis in peripheral joints, particularly within the hands and feet, but can also sometimes trigger temporomandibular joint (TMJ) arthralgia. To better understand the effects of ongoing Inflammation-induced pain signaling, dorsal root ganglia (DRGs) were acquired from individuals with RA for transcriptomic study. We conducted RNA sequencing from the L5 DRGs because it contains the soma of the sensory neurons that innervate the affected joints in the foot. DRGs from 5 RA patients were compared with 9 non-arthritic controls. RNA-seq of L5 DRGs identified 128 differentially expressed genes (DEGs) that were dysregulated in the RA subjects as compared to the non-arthritic controls. The DRG resides outside the blood brain barrier and, as such, our initial transcriptome analysis detected signs of an autoimmune disorder including the upregulated expression of immunoglobulins and other immunologically related genes within the DRGs of the RA donors. Additionally, we saw the upregulation in genes implicated in neurogenesis that could promote pain hypersensitivity. overall, our DRG analysis suggests that there are upregulated inflammatory and pain signaling pathways that can contribute to chronic pain in RA.

Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Neuroimmune Mechanisms Underlying Post-acute Sequelae of SARS-CoV-2 (PASC) Pain, Predictions from a Ligand-Receptor Interactome.

PURPOSE OF REVIEW: Individuals with post-acute sequelae of SARS-CoV-2 (PASC) complain of persistent musculoskeletal pain. Determining how COVID-19 infection produces persistent pain would be valuable for the development of therapeutics aimed at alleviating these symptoms. RECENT FINDINGS: To generate hypotheses regarding neuroimmune interactions in PASC, we used a ligand-receptor interactome to make predictions about how ligands from PBMCs in individuals with COVID-19 communicate with dorsal root ganglia (DRG) neurons to induce persistent pain. In a structured literature review of -omics COVID-19 studies, we identified ligands capable of binding to receptors on DRG neurons, which stimulate signaling pathways including immune cell activation and chemotaxis, the complement system, and type I interferon signaling. The most consistent finding across immune cell types was an upregulation of genes encoding the alarmins S100A8/9 and MHC-I. This ligand-receptor interactome, from our hypothesis-generating literature review, can be used to guide future research surrounding mechanisms of PASC-induced pain.

Relationship Between Automated Pupillometry Measurements and Ventricular Volume in Patients With Aneurysmal Subarachnoid Hemorrhage.

ABSTRACT: INTRODUCTION: Pupillometry allows for a standardized assessment of the pupillary light reflex. Acute hydrocephalus (HCP) is a common complication in patients with aneurysmal subarachnoid hemorrhage (aSAH). HCP may affect the pupillary light reflex because of increased intracranial pressure or dilation of the rostral aqueduct. The association between the pupillary light reflex and HCP in aSAH patients has not been clearly established. The objective of this study is to analyze the correlation between the Neurological Pupil index (NPi) and the degree of HCP in aSAH patients. METHODS: The Establishing Normative Data for Pupillometer Assessments in Neuroscience Intensive Care Registry is a prospectively collected database of pupillometry readings in patients admitted to 4 different neurological intensive care units. Patients in the registry with aSAH who had pupillometry assessments within 6 hours of a brain computed tomography were studied. The degree of HCP was quantified using the HCP score, and the relationship between the NPi and HCP was analyzed after controlling for confounders. RESULTS: A total of 43 patients were analyzed (mean age, 54 ± 15 years; 53.2% male; mean HCP score, 5.3 ± 3.8). Thirty-eight patients had HCP. Mean NPi for the right eye was 4.02 (±1.2), and that for the left eye was 3.7 (±1.5). After adjusting for age, sex, race, and sedation, there was no significant correlation between HCP and NPi (right eye: r = 0.12, P = .44; left eye: r = 0.04, P = .8). CONCLUSION: In patients with aSAH, NPi was not correlated with HCP score. A small sample size could be a limitation of this study. Additional studies are needed to characterize the clinical significance of pupillometry in the evaluation of patients with aSAH and HCP.

Supratentorial intracerebral hemorrhage volume and other CT variables predict the neurological pupil index.

OBJECTIVES: There is growing interest in the ability of automated infrared pupillometry to assess severity of neurological illness. We studied the correlation between computed tomography (CT) indicators of intracerebral hemorrhage (ICH) severity with objective measures of the pupillary light reflex (PLR), and hypothesized that hemorrhage volume would predict the Neurological Pupil index™ (NPi™), an indicator of pupillary reactivity. METHODS: This study examined data from patients with supratentorial ICH who underwent serial pupillometer evaluations. CT images were examined to determine the location and laterality of the hemorrhage, along with hematoma volume (using the simplified ABC/2 method), midline shift, hydrocephalus score, and modified Graeb score (indicating interventricular hemorrhage). Demographics were examined with standard measures of central tendency, hypotheses with logistic regression, categorical data with Fisher's Exact X2, and multivariate modeling with constructed MAX-R models. RESULTS: Data were gathered from 44 subjects. ICH volume exhibited the strongest correlation with NPi (ipsilateral [r2 = 0.48, p < 0.0001, contralateral [(r2 = 0.39, p < 0.0001]). Horizontal midline shift of the septum pellucidum also correlated with NPi (ipsilateral [r2 = 0.25, p = 0.0006], contralateral [r2 = 0.15, p = 0.0106]), as did shift of the pineal gland (ipsilateral [r2 = 0.21, p = 0.0017], contralateral[r2 = 0.11, p = 0.0328]). ICH volume was the most predictive of abnormal NPi (AUC = 0.85 for ipsilateral and 0.88 for contralateral NPi), and multivariate modeling identified additional independent predictors of NPi. CONCLUSION: ICH volume and shift of midline structures correlate with NPi, and abnormalities in NPi can be predicted by hematoma volume and other CT indicators of ICH severity. Future studies should explore the role of NPi in detecting early hematoma expansion and worsening midline shift.

Ultrafast Near-Infrared Light-triggered Intracellular Uncaging to Probe Cell Signaling.

The possibility of regulating cell signaling with high spatial and temporal resolution within individual cells and complex cellular networks has important implications in biomedicine. In this report, we demonstrate a general strategy that uses near-infrared tissue-penetrating laser pulses to uncage biomolecules from plasmonic gold-coated liposomes, i.e. plasmonic liposomes, to activate cell signaling in a non-thermal, ultrafast and highly controllable fashion. Near-infrared picosecond laser pulse induces transient nanobubbles around plasmonic liposomes. The mechanical force generated from the collapse of nanobubbles rapidly ejects encapsulated compound within 0.1 ms. We showed that single pulse irradiation triggers the rapid intracellular uncaging of calcein from plasmonic liposomes inside endo-lysosomes. The uncaged calcein then evenly distributes over the entire cytosol and nucleus. Furthermore, we demonstrated the ability to trigger calcium signaling in both an immortalized cell line and primary dorsal root ganglion (DRG) neurons by intracellular uncaging of inositol triphosphate (IP3), an endogenous cell calcium signaling second messenger. Compared with other uncaging techniques, this ultrafast near-infrared light-driven molecular uncaging method is easily adaptable to deliver a wide range of bioactive molecules with an ultrafast optical switch, enabling new possibilities to investigate signaling pathways within individual cells and cellular networks.

Pharmacological activation of AMPK inhibits incision-evoked mechanical hypersensitivity and the development of hyperalgesic priming in mice.

New therapeutics to manage post-surgical pain are needed to mitigate the liabilities of opioid and other analgesics. Our previous work shows that key modulators of excitability in peripheral nociceptors, such as extracellular signal-regulated kinases (ERK) are inhibited by activation of adenosine monophosphate activated protein kinase (AMPK). We hypothesized that AMPK activation would attenuate acute incision-evoked mechanical hypersensitivity and the development of hyperalgesic priming caused by surgery in mice. Here we have used a variety of administration routes and combinations of AMPK activators to test this hypothesis. Topical administration of a resveratrol-based cream inhibited acute mechanical hypersensitivity evoked by incision and blocked the development of hyperalgesic priming. We also observed that systemic administration of metformin dose-dependently inhibited incision-evoked mechanical hypersensitivity and hyperalgesic priming. Interestingly, low doses of systemic metformin and local resveratrol that had no acute effect were able to mitigate development of hyperalgesic priming. Combined treatment with doses of systemic metformin and local resveratrol that were not effective on their own enhanced the acute efficacy of the individual AMPK activators for post-surgical mechanical pain alleviation and blocked the development of hyperalgesic priming. Finally, we used dorsal root ganglion (DRG) neurons in culture to show that resveratrol and metformin given in combination shift the concentration-response curve for AMPK activation to the left and increase the magnitude of AMPK activation. Therefore, we find that topical administration is an effective treatment route of administration and combining systemic and local treatments led to anti-nociceptive efficacy in acute mechanical hypersensitivity at doses that were not effective alone. Collectively our work demonstrates a specific effect of AMPK activators on post-surgical pain and points to novel therapeutic opportunities with potential immediate impact in the clinical setting.