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Background and Hypothesis Cognitive deficits in schizophrenia are linked to dysfunctions of the dorsolateral prefrontal cortex (DLPFC), including alterations in parvalbumin (PV)-expressing interneurons (PVIs). Redox dysregulation and oxidative stress may represent convergence points in the pathology of schizophrenia, causing dysfunction of GABAergic interneurons and loss of PV. Here, we show that the mitochondrial matrix protein cyclophilin D (CypD), a critical initiator of the mitochondrial permeability transition pore (mPTP) and modulator of the intracellular redox state, is altered in PVIs in schizophrenia. Study Design Western blotting was used to measure CypD protein levels in postmortem DLPFC specimens of schizophrenic patients (n  = 27) and matched comparison subjects with no known history of psychiatric or neurological disorders (n  = 26). In a subset of this cohort, multilabel immunofluorescent confocal microscopy with unbiased stereological sampling methods were used to quantify (1) numbers of PVI across the cortical mantle (20 unaffected comparison, 14 schizophrenia) and (2) PV and CypD protein levels from PVIs in the cortical layers 2–4 (23 unaffected comparison, 18 schizophrenia). Study Results In schizophrenic patients, the overall number of PVIs in the DLPFC was not significantly altered, but in individual PVIs of layers 2–4 PV protein levels decreased along a superficial-to-deep gradient when compared to unaffected comparison subjects. These laminar-specific PVI alterations were reciprocally linked to significant CypD elevations both in PVIs and total DLPFC gray matter. Conclusions Our findings support previously reported PVI anomalies in schizophrenia and suggest that CypD-mediated mPTP formation could be a potential contributor to PVI dysfunction in schizophrenia. [ABSTRACT FROM AUTHOR]

Spontaneous activity in dorsal root ganglion (DRG) neurons is a key driver of neuropathic pain in patients suffering from this largely untreated disease. While many intracellular signalling mechanisms have been examined in preclinical models that drive spontaneous activity, none have been tested directly on spontaneously active human nociceptors. Using cultured DRG neurons recovered during thoracic vertebrectomy surgeries, we showed that inhibition of mitogen-activated protein kinase interacting kinase (MNK) with tomivosertib (eFT508, 25 nM) reversibly suppresses spontaneous activity in human sensory neurons that are likely nociceptors based on size and action potential characteristics associated with painful dermatomes within minutes of treatment. Tomivosertib treatment also decreased action potential amplitude and produced alterations in the magnitude of after hyperpolarizing currents, suggesting modification of Na+ and K+ channel activity as a consequence of drug treatment. Parallel to the effects on electrophysiology, eFT508 treatment led to a profound loss of eIF4E serine 209 phosphorylation in primary sensory neurons, a specific substrate of MNK, within 2 min of drug treatment. Our results create a compelling case for the future testing of MNK inhibitors in clinical trials for neuropathic pain. [ABSTRACT FROM AUTHOR]

Impairments in somatosensory function are a common and often debilitating consequence of neurological injury, with few effective interventions. Building on success in rehabilitation for motor dysfunction, the delivery of vagus nerve stimulation (VNS) combined with tactile rehabilitation has emerged as a potential approach to enhance recovery of somatosensation. In order to maximize the effectiveness of VNS therapy and promote translation to clinical implementation, we sought to optimize the stimulation paradigm and identify neural mechanisms that underlie VNS-dependent recovery. To do so, we characterized the effect of tactile rehabilitation combined with VNS across a range of stimulation intensities on recovery of somatosensory function in a rat model of chronic sensory loss in the forelimb. Consistent with previous studies in other applications, we find that moderate intensity VNS yields the most effective restoration of somatosensation, and both lower and higher VNS intensities fail to enhance recovery compared to rehabilitation without VNS. We next used the optimized, moderate intensity to evaluate the mechanisms that underlie recovery. We find that moderate intensity VNS enhances transcription of Arc, a canonical mediator of synaptic plasticity, in the cortex, and that transcript levels were correlated with the degree of somatosensory recovery. Moreover, we observe that blocking plasticity by depleting acetylcholine in the cortex prevents the VNS-dependent enhancement of somatosensory recovery. Collectively, these findings identify neural mechanisms that subserve VNS-dependent somatosensation recovery and provide a basis for selecting optimal stimulation parameters in order to facilitate translation of this potential intervention. [ABSTRACT FROM AUTHOR]

Background: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. Methods: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. Results: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. Conclusions: We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders. [ABSTRACT FROM AUTHOR]

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., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The nasal, oropharyngeal, and bronchial mucosa are primary contact points for airborne pathogens like Mycobacterium tuberculosis (Mtb), SARS-CoV-2, and influenza virus. While mucosal surfaces can function as both entry points and barriers to infection, mucosa-associated lymphoid tissues (MALT) facilitate early immune responses to mucosal antigens. MALT contains a variety of specialized epithelial cells, including a rare cell type called a microfold cell (M cell) that functions to transport apical antigens to basolateral antigen-presenting cells, a crucial step in the initiation of mucosal immunity. M cells have been extensively characterized in the gastrointestinal (GI) tract in murine and human models. However, the precise development and functions of human airway M cells is unknown. Here, using single-nucleus RNA sequencing (snRNA-seq), we generated an atlas of cells from the human adenoid and identified 16 unique cell types representing basal, club, hillock, and hematopoietic lineages, defined their developmental trajectories, and determined cell-cell relationships. Using trajectory analysis, we found that human airway M cells develop from progenitor club cells and express a gene signature distinct from intestinal M cells. Surprisingly, we also identified a heretofore unknown epithelial cell type demonstrating a robust interferon-stimulated gene signature. Our analysis of human adenoid cells enhances our understanding of mucosal immune responses and the role of M cells in airway immunity. This work also provides a resource for understanding early interactions of pathogens with airway mucosa and a platform for development of mucosal vaccines.

Abstract: Neuropathic pain remains difficult to treat, with drug development hampered by an incomplete understanding of the pathogenesis of the condition, as well as a lack of biomarkers. The problem is compounded by the scarcity of relevant human peripheral tissues, including skin, nerves, and dorsal root ganglia. Efforts to obtain such samples are accelerating, increasing the need for standardisation across laboratories. In this white paper, we report on a consensus meeting attended by neuropathic pain experts, designed to accelerate protocol alignment and harmonization of studies involving relevant peripheral tissues. The meeting was held in London in March 2024 and attended by 28 networking partners, including industry and patient representatives. We achieved consensus on minimal recommended phenotyping, harmonised wet laboratory protocols, statistical design, reporting, and data sharing. Here, we also share a variety of relevant standard operating procedures as supplementary protocols. We envision that our recommendations will help unify human tissue research in the field and accelerate our understanding of how abnormal interactions between sensory neurons and their local peripheral environment contribute towards neuropathic pain., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

Telocytes are interstitial cells with long processes that cover distances in tissues and likely coordinate interacts with other cell types. Though present in central and peripheral neuronal tissues, their role remains unclear. Dorsal root ganglia (DRG) house pseudounipolar afferent neurons responsible for signals such as temperature, proprioception and nociception. This study aimed to investigate the presence and function of telocytes in human DRG by investigating their transcriptional profile, location and ultrastructure. Sequencing data revealed CD34 and PDGFRA expressing cells comprise roughly 1.5-3% of DRG cells. Combined expression of CD34 and PDGFRA is a putative marker gene set for telocytes. Further analysis identified nine subclusters with enriched cluster-specific genes. KEGG and GO pathway analysis suggested vascular, immune and connective tissue associated putative telocyte subtypes. Over 3000 potential receptor-ligand interactions between sensory neurons and these CD34 and PDGFRA expressing putative telocytes were identified using a ligand-receptors interactome platform. Immunohisto-chemistry showed CD34+ telocytes in the endoneural space of DRGs, next to neuron-satellite complexes, in perivascular spaces and in the endoneural space between nerve fibre bundles, consistent with pathway analysis. Transmission electron microscopy (TEM) confirmed their location identifying characteristic elongated nucleus, long and thin telopods containing vesicles, surrounded by a basal lamina. This is the first study that provides gene expression analysis of telocytes in complex human tissue such as the DRG, highlighting functional differences based on tissue location with no significant ultrastructural variation., Competing Interests: Conflict of interest disclosure: The authors declare no conflict of interest.

Abstract: As the incidence and survival rates of patients with cancer continues to grow, an increasing number of people are living with comorbidities, which often manifests as cancer-induced bone pain (CIBP). The majority of patients with CIBP report poor pain control from currently available analgesics. A conotoxin, Contulakin-G (CGX), has been demonstrated to be an antinociceptive agent in postsurgical and neuropathic pain states via a neurotensin receptor 2 (NTSR2)-mediated pathway. However, the efficacy and side effect profile of CGX have never been assessed in CIBP. Here, we evaluated CGX's antinociceptive potential in a rodent model of CIBP. We hypothesized that CGX engages the NTSR2 pathway, providing pain relief with minimal tolerance and motor side effects. Our results demonstrated that CGX intrathecal injection in mice with CIBP attenuated both spontaneous pain behaviors and evoked mechanical hypersensitivity, regardless of their sex. Furthermore, the antinociceptive effect of CGX was dependent upon expression of NTSR2 and the R-type voltage-gated calcium channel (Cav2.3); gene editing of these targets abolished CGX antinociception without affecting morphine antinociception. Examination of the side effect profile of CGX demonstrated that, unlike morphine, chronic intrathecal infusion maintained antinociception with reduced tolerance in rats with CIBP. Moreover, at antinociceptive doses, CGX had no impact on motor behavior in rodents with CIBP. Finally, RNAScope and immunoblotting analysis revealed expression of NTSR2 in both dorsal and ventral horns, while Cav2.3 was minimally expressed in the ventral horn, possibly explaining the sensory selectivity of CGX. Together, these findings support advancing CGX as a potential therapeutic for cancer pain., (Copyright © 2024 International Association for the Study of Pain.)

Spontaneous activity in dorsal root ganglion (DRG) neurons is a key driver of neuropathic pain in patients suffering from this largely untreated disease. While many intracellular signalling mechanisms have been examined in preclinical models that drive spontaneous activity, none have been tested directly on spontaneously active human nociceptors. Using cultured DRG neurons recovered during thoracic vertebrectomy surgeries, we showed that inhibition of mitogen-activated protein kinase interacting kinase (MNK) with tomivosertib (eFT508, 25 nM) reversibly suppresses spontaneous activity in human sensory neurons that are likely nociceptors based on size and action potential characteristics associated with painful dermatomes within minutes of treatment. Tomivosertib treatment also decreased action potential amplitude and produced alterations in the magnitude of after hyperpolarizing currents, suggesting modification of Na+ and K+ channel activity as a consequence of drug treatment. Parallel to the effects on electrophysiology, eFT508 treatment led to a profound loss of eIF4E serine 209 phosphorylation in primary sensory neurons, a specific substrate of MNK, within 2 min of drug treatment. Our results create a compelling case for the future testing of MNK inhibitors in clinical trials for neuropathic pain., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Diabetic neuropathy is frequently accompanied by pain and loss of sensation attributed to axonal dieback. We recovered dorsal root ganglia (DRGs) from 90 organ donors, 19 of whom had medical indices for diabetic painful neuropathy (DPN). Nageotte nodules, dead sensory neurons engulfed by non-neuronal cells, were abundant in DPN DRGs and accounted for 25% of all neurons. Peripherin-and Nav1.7-positive dystrophic axons invaded Nageotte nodules, forming small neuroma-like structures. Using histology and spatial sequencing, we demonstrate that Nageotte nodules are mainly composed of satellite glia and non-myelinating Schwann cells that express SPP1 and are intertwined with sprouting sensory axons originating from neighboring neurons. Our findings solve a 100-year mystery of the nature of Nageotte nodules linking these pathological structures to pain and sensory loss in DPN., Competing Interests: Competing interests: TJP is a co-founder of 4E Therapeutics. The authors declare no other competing interests related to this work.

Hyperalgesic priming is a model system that has been widely used to understand plasticity in painful stimulus-detecting sensory neurons, called nociceptors. A key feature of this model system is that following priming, stimuli that do not normally cause hyperalgesia now readily provoke this state. We hypothesized that hyperalgesic priming occurs due to reorganization of translation of mRNA in nociceptors. To test this hypothesis, we used paclitaxel treatment as the priming stimulus and translating ribosome affinity purification (TRAP) to measure persistent changes in mRNA translation in Nav1.8+ nociceptors. TRAP sequencing revealed 161 genes with persistently altered mRNA translation in the primed state. We identified Gpr88 as upregulated and Metrn as downregulated. We confirmed a functional role for these genes, wherein a GPR88 agonist causes pain only in primed mice and established hyperalgesic priming is reversed by Meteorin. Our work demonstrates that altered nociceptor translatomes are causative in producing hyperalgesic priming., Competing Interests: Conflict of Interest Statement: G Munro and KA Petersen are employees of Hoba Therapeutics. The authors declare no other conflicts of interest.

This study investigated the time course of gene expression changes during the progression of persistent painful neuropathy caused by paclitaxel (PTX) in male and female mouse hindpaws and dorsal root ganglia (DRG). Bulk RNA-seq was used to examine these gene expression changes at 1, 16, and 31 days post-last PTX. At these time points, differentially expressed genes (DEGs) were predominantly related to the reduction or increase in epithelial, skin, bone, and muscle development and to angiogenesis, myelination, axonogenesis, and neurogenesis. These processes are accompanied by the regulation of DEGs related to the cytoskeleton, extracellular matrix organization, and cellular energy production. This gene plasticity during the progression of persistent painful neuropathy could be interpreted as a biological process linked to tissue regeneration/degeneration. In contrast, gene plasticity related to immune processes was minimal at 1-31 days after PTX. It was also noted that despite similarities in biological processes and pain chronicity between males and females, specific DEGs differed dramatically according to sex. The main conclusions of this study are that gene expression plasticity in hindpaw and DRG during PTX neuropathy progression similar to tissue regeneration and degeneration, minimally affects immune system processes and is heavily sex-dependent at the individual gene level., (© 2024. The Author(s).)

Gene expression is influenced by chromatin architecture via controlled access of regulatory factors to DNA. To better understand gene regulation in the human dorsal root ganglion (hDRG) we used bulk and spatial transposase-accessible chromatin technology followed by sequencing (ATAC-seq). Using bulk ATAC-seq, we detected that in females diverse differentially accessible chromatin regions (DARs) mapped to the X chromosome and in males to autosomal genes. EGR1/3 and SP1/4 transcription factor binding motifs were abundant within DARs in females, and JUN, FOS and other AP-1 factors in males. To dissect the open chromatin profile in hDRG neurons, we used spatial ATAC-seq. The neuron cluster showed higher chromatin accessibility in GABAergic, glutamatergic, and interferon-related genes in females, and in Ca 2+ - signaling-related genes in males. Sex differences in transcription factor binding sites in neuron-proximal barcodes were consistent with the trends observed in bulk ATAC-seq data. We validated that EGR1 expression is biased to female hDRG compared to male. Strikingly, XIST , the long-noncoding RNA responsible for X inactivation, hybridization signal was found to be highly dispersed in the female neuronal but not non-neuronal nuclei suggesting weak X inactivation in female hDRG neurons. Our findings point to baseline epigenomic sex differences in the hDRG that likely underlie divergent transcriptional responses that determine mechanistic sex differences in pain., Competing Interests: Conflict of Interest Statement The authors declare no financial conflicts of interest related to this work.

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., Competing Interests: Declaration of Interests: Hien T. Zhao and Bethany Fitzsimmons are full-time employees and shareholders of Ionis Pharmaceutical, Inc. Other authors declare no competing interests.

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.

Sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli to the central nervous system. Single-cell RNA sequencing has provided insights into the diversity of sensory ganglia cell types in rodents, nonhuman primates, and humans, but it remains difficult to compare cell types across studies and species. We thus constructed harmonized atlases of the DRG and TG that describe and facilitate comparison of 18 neuronal and 11 non-neuronal cell types across six species and 31 datasets. We then performed single-cell/nucleus RNA sequencing of DRG from both human and the highly regenerative axolotl and found that the harmonized atlas also improves cell type annotation, particularly of sparse neuronal subtypes. We observed that the transcriptomes of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The resources presented here can guide future studies in comparative transcriptomics, simplify cell-type nomenclature differences across studies, and help prioritize targets for future analgesic development.

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., Competing Interests: Conflict-of-interest statement: The authors declare no conflicts of interest.

The profound pain accompanying bone fracture is mediated by somatosensory neurons, which also appear to be required to initiate bone regeneration following fracture. Surprisingly, the precise neuroanatomical circuitry mediating skeletal nociception and regeneration remains incompletely understood. Here, we characterized somatosensory dorsal root ganglia (DRG) afferent neurons innervating murine long bones before and after experimental long bone fracture in mice. Retrograde labeling of DRG neurons by an adeno-associated virus with peripheral nerve tropism showed AAV-tdT signal. Single cell transcriptomic profiling of 6,648 DRG neurons showed highest labeling across CGRP+ neuron clusters (6.9-17.2%) belonging to unmyelinated C fibers, thinly myelinated Aδ fibers and Aβ-Field LTMR (9.2%). Gene expression profiles of retrograde labeled DRG neurons over multiple timepoints following experimental stress fracture revealed dynamic changes in gene expression corresponding to the acute inflammatory ( S100a8 , S100a9 ) and mechanical force ( Piezo2 ). Reparative phase after fracture included morphogens such as Tgfb1, Fgf9 and Fgf18 . Two methods to surgically or genetically denervate fractured bones were used in combination with scRNA-seq to implicate defective mesenchymal cell proliferation and osteodifferentiation as underlying the poor bone repair capacity in the presence of attenuated innervation. Finally, multi-tissue scRNA-seq and interactome analyses implicated neuron-derived FGF9 as a potent regulator of fracture repair, a finding compatible with in vitro assessments of neuron-to-skeletal mesenchyme interactions.

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.

During surgery of delicate regions, differentiation between nerve and surrounding tissue is crucial. Hyperspectral imaging (HSI) techniques can enhance the contrast between types of tissue beyond what the human eye can differentiate. Whereas an RGB image captures 3 bands within the visible light range ( e.g., 400 nm to 700 nm), HSI can acquire many bands in wavelength increments that highlight regions of an image across a wavelength spectrum. We developed a workflow to identify nerve tissues from other similar tissues such as fat, bone, and muscle. Our workflow uses spectral angle mapper (SAM) and endmember selection. The method is robust for different types of environment and lighting conditions. We validated our workflow on two samples of human tissues. We used a compact HSI system that can image from 400 to 1700 nm to produce HSI of the samples. On these two samples, we achieved an intersection-over-union (IoU) segmentation score of 84.15% and 76.73%, respectively. We showed that our workflow identifies nerve segments that are not easily seen in RGB images. This method is fast, does not rely on special hardware, and can be applied in real time. The hyperspectral imaging and nerve detection approach may provide a powerful tool for image-guided surgery.