S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

Highly synchronized cortical circuit dynamics mediate spontaneous pain in mice.

Cortical neural dynamics mediate information processing for the cerebral cortex, which is implicated in fundamental biological processes such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes the cortical network into an aberrant state and, if so, whether it can be brought back to a "normal" operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with spontaneous pain-like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos-expressing neurons and selective activation of GABAergic interneurons significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More important, restoring a "normal" range of neural dynamics through attenuation of pain-induced S1 synchronization alleviated pain-like behavior. These results suggest that spontaneous pain pushed the S1 regional network into a synchronized state, whereas reversal of this synchronization alleviated pain.

Phenotypic screen identifies the natural product silymarin as a novel anti-inflammatory analgesic.

Sensory neuron hyperexcitability is a critical driver of pathological pain and can result from axon damage, inflammation, or neuronal stress. G-protein coupled receptor signaling can induce pain amplification by modulating the activation of Trp-family ionotropic receptors and voltage-gated ion channels. Here, we sought to use calcium imaging to identify novel inhibitors of the intracellular pathways that mediate sensory neuron sensitization and lead to hyperexcitability. We identified a novel stimulus cocktail, consisting of the SSTR2 agonist L-054,264 and the S1PR3 agonist CYM5541, that elicits calcium responses in mouse primary sensory neurons in vitro as well as pain and thermal hypersensitivity in mice in vivo. We screened a library of 906 bioactive compounds and identified 24 hits that reduced calcium flux elicited by L-054,264/CYM5541. Among these hits, silymarin, a natural product derived from milk thistle, strongly reduced activation by the stimulation cocktail, as well as by a distinct inflammatory cocktail containing bradykinin and prostaglandin E2. Silymarin had no effect on sensory neuron excitability at baseline, but reduced calcium flux via Orai channels and downstream mediators of phospholipase C signaling. In vivo, silymarin pretreatment blocked development of adjuvant-mediated thermal hypersensitivity, indicating potential use as an anti-inflammatory analgesic.

Heat But Not Mechanical Hypersensitivity Depends on Voltage-Gated CaV2.2 Calcium Channel Activity in Peripheral Axon Terminals Innervating Skin.

Voltage-gated CaV2.2 calcium channels are expressed in nociceptors at presynaptic terminals, soma, and axons. CaV2.2 channel inhibitors applied to the spinal cord relieve pain in humans and rodents, especially during pathologic pain, but a biological function of nociceptor CaV2.2 channels in processing of nociception, outside presynaptic terminals in the spinal cord, is underappreciated. Here, we demonstrate that functional CaV2.2 channels in peripheral axons innervating skin are required for capsaicin-induced heat hypersensitivity in male and female mice. We show that CaV2.2 channels in TRPV1-nociceptor endings are activated by capsaicin-induced depolarization and contribute to increased intracellular calcium. Capsaicin induces hypersensitivity of both thermal nociceptors and mechanoreceptors, but only heat hypersensitivity depends on peripheral CaV2.2 channel activity, and especially a cell-type-specific CaV2.2 splice isoform. CaV2.2 channels at peripheral nerve endings might be important therapeutic targets to mitigate certain forms of chronic pain.SIGNIFICANCE STATEMENT It is generally assumed that nociceptor termini in the spinal cord dorsal horn are the functionally significant sites of CaV2.2 channel in control of transmitter release and the transmission of sensory information from the periphery to central sites. We show that peripheral CaV2.2 channels are essential for the classic heat hypersensitivity response to develop in skin following capsaicin exposure. This function of CaV2.2 is highly selective for heat, but not mechanical hypersensitivity induced by capsaicin exposure, and is not a property of closely related CaV2.1 channels. Our findings suggest that interrupting CaV2.2-dependent calcium entry in skin might reduce heat hypersensitivity that develops after noxious heat exposure and may limit the degree of heat hypersensitivity associated with certain other forms of pain.

Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions.

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.

A high-content platform for physiological profiling and unbiased classification of individual neurons.

High-throughput physiological assays lose single-cell resolution, precluding subtype-specific analyses of activation mechanism and drug effects. We demonstrate APPOINT (automated physiological phenotyping of individual neuronal types), a physiological assay platform combining calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at large scale. Using unbiased techniques, we quantify responses to sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. We expand the role of neuroimmune interactions by showing that human serum directly activates sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically validate contributions of ion channel families to optical activation.

Transcriptomic signature of painful human neurofibromatosis type 2 schwannomas.

Schwannomas are benign neoplasms that can cause gain- and loss-of-function neurological phenotypes, including severe, intractable pain. To investigate the molecular mechanisms underlying schwannoma-associated pain we compared the RNA sequencing profile of painful and non-painful schwannomas from NF2 patients. Distinct segregation of painful and non-painful tumors by gene expression patterns was observed. Differential expression analysis showed the upregulation of fibroblast growth factor 7 (FGF7) in painful schwannomas. Behavioral support for this finding was observed using a xenograft human NF2-schwannoma model in nude mice. In this model, over-expression of FGF7 in intra-sciatically implanted NF2 tumor cells generated pain behavior compared with controls.

Pharmacological Profiling of Purified Human Stem Cell-Derived and Primary Mouse Motor Neurons.

Directed differentiation of human pluripotent stem cells (hPSCs) has enabled the generation of specific neuronal subtypes that approximate the intended primary mammalian cells on both the RNA and protein levels. These cells offer unique opportunities, including insights into mechanistic understanding of the early driving events in neurodegenerative disease, replacement of degenerating cell populations, and compound identification and evaluation in the context of precision medicine. However, whether the derived neurons indeed recapitulate the physiological features of the desired bona fide neuronal subgroups remains an unanswered question and one important for validating stem cell models as accurate functional representations of the primary cell types. Here, we purified both hPSC-derived and primary mouse spinal motor neurons in parallel and used extracellular multi-electrode array (MEA) recording to compare the pharmacological sensitivity of neuronal excitability and network function. We observed similar effects for most receptor and channel agonists and antagonists, supporting the consistency between human PSC-derived and mouse primary spinal motor neuron models from a physiological perspective.

Contained rupture of left ventricular false aneurysm after acute myocardial infarction secondary to left anterior descending artery embolism.

Left ventricular free wall rupture is a rare complication of acute myocardial infarction and accounts for a significant number of fatalities. Pseudoaneurysm is a variety of left ventricular rupture whereby the pericardium seals the defect, forming the wall of the pseudoaneurysm. The diagnosis is usually confirmed with echocardiography, and emergent surgical repair is required in suspected impending rupture. The present report describes the case of a 58-year-old woman who presented with a myocardial infarction due to distal left anterior descending artery occlusion, complicated by ventricular pseudoaneurysm with impending rupture. The patient had an otherwise normal coronary tree.

Optical and electrical properties between 0.4 and 12 microm for Sn-doped In2O3 films by pulsed laser deposition and cathode sputtering.

Optical properties of Sn-doped In(2)O(3) (ITO) have been studied in the optical range of 0.4-12 microm. A deposition has been made on BK7 glass, magnesium fluoride, sapphire, and zinc sulfide substrates. The layers have been characterized by their optical properties, DC electrical sheet resistivity, and Hall mobility. Sheet resistivity lies in the range of 6.8-318 Omega/sq for thicknesses between 16 and 280 nm. The best carrier mobility is obtained on BK7 and sapphire substrates, up to approximately 50 cm(2)/V s. The material shows good infrared transparency in the 3-5 microm range on magnesium fluoride and 0.4-4 microm on sapphire, and it is usable for practical applications up to 12 microm on zinc sulfide. Simulations have been carried out for optical indices and spectra calculations. The Drude model has been used to exploit the results in either direction: from electrical measured data to the simulation of optical spectra and indices, and from measured optical spectra to simulated optical indices and electrical parameters (mobility, carrier density). Hall mobility is considered a worthy and convenient material quality criteria for materials aimed at optics.

Lateral internal sphincterotomy is superior to topical nitroglycerin for healing chronic anal fissure and does not compromise long-term fecal continence: six-year follow-up of a multicenter, randomized, controlled trial.

PURPOSE: Although there is enthusiasm for nonoperative management of anal fissures, most trials have been of short duration (6-8 weeks) and long-term outcome is unknown. The purpose of this study was to assess long-term outcome in two cohorts of patients who had participated in a randomized, controlled trial to compare the effectiveness of topical nitroglycerin with internal sphincterotomy in the treatment of chronic anal fissure. METHODS: Between February 1997 and October 1998, 82 patients with chronic anal fissure were accrued and randomized to 0.25 percent nitroglycerin ointment t.i.d. or lateral internal sphincterotomy. In 2004, a telephone survey of trial participants was conducted to determine symptom recurrence, the need for further medical and/or surgical treatment, and patient satisfaction. Furthermore, patients were assessed for symptoms of fecal incontinence using the Jorge and Wexner Fecal Incontinence Score and the Fecal Incontinence Quality of Life questionnaire. RESULTS: Overall, 51 of the original 82 patients (62 percent, 27 nitroglycerin, 24 lateral internal sphincterotomy) completed our survey. Mean follow-up was 79 (+/-1) months. Sphincterotomy patients were less likely to have experienced fissure symptoms within the past year (0 vs. 41 percent; P = 0.0004) and were less likely to require subsequent surgical treatment (0 vs. 59 percent; P < 0.0001) than patients treated with nitroglycerin. Patients in the lateral internal sphincterotomy group were more likely to say that they were "very" or "moderately" satisfied with their treatment (100 vs. 56 percent; P = 0.04) and that they would choose the same treatment again (92 vs. 63 percent; P = 0.02) than patients in the nitroglycerin group. Finally, the fecal incontinence and fecal incontinence quality of life scores at six-year follow-up were similar in both groups. CONCLUSIONS: After six years of follow-up, it seems that lateral internal sphincterotomy is a more durable treatment for chronic anal fissure compared with topical nitroglycerin therapy and does not compromise long-term fecal continence. Thus, sphincterotomy continues to be a good treatment for patients with chronic anal fissure.