Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.

We developed a rat dorsal root ganglion (DRG)-derived sensory nerve organotypic model by culturing DRG explants on an organoid culture device. With this method, a large number of organotypic cultures can be produced simultaneously with high reproducibility simply by seeding DRG explants derived from rat embryos. Unlike previous DRG explant models, this organotypic model consists of a ganglion and an axon bundle with myelinated A fibers, unmyelinated C fibers, and stereo-myelin-forming nodes of Ranvier. The model also exhibits Ca2+ signaling in cell bodies in response to application of chemical stimuli to nerve terminals. Further, axonal transection increases the activating transcription factor 3 mRNA level in ganglia. Axons and myelin are shown to regenerate 14 days following transection. Our sensory organotypic model enables analysis of neuronal excitability in response to pain stimuli and tracking of morphological changes in the axon bundle over weeks.

Spinal pain affects individuals of all ages and is the most common musculoskeletal problem globally. Its clinical management remains a challenge as the underlying mechanisms leading to it are still unclear. Here, we report that significantly increased numbers of senescent osteoclasts (SnOCs) are observed in mouse models of spinal hypersensitivity, like lumbar spine instability (LSI) or aging, compared to controls. The larger population of SnOCs is associated with induced sensory nerve innervation, as well as the growth of H-type vessels, in the porous endplate. We show that deletion of senescent cells by administration of the senolytic drug Navitoclax (ABT263) results in significantly less spinal hypersensitivity, spinal degeneration, porosity of the endplate, sensory nerve innervation, and H-type vessel growth in the endplate. We also show that there is significantly increased SnOC-mediated secretion of Netrin-1 and NGF, two well-established sensory nerve growth factors, compared to non-senescent OCs. These findings suggest that pharmacological elimination of SnOCs may be a potent therapy to treat spinal pain.

Maxillofacial bone defects exhibit intricate anatomy and irregular morphology, presenting challenges for effective treatment. This study aimed to address these challenges by developing an injectable bioactive composite microsphere, termed D-P-Ak (polydopamine-PLGA-akermanite), designed to fit within the defect site while minimizing injury. The D-P-Ak microspheres biodegraded gradually, releasing calcium, magnesium, and silicon ions, which, notably, not only directly stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) but also activated sensory nerve cells to secrete calcitonin gene-related peptide (CGRP), a key factor in bone repair. Moreover, the released CGRP enhanced the osteogenic differentiation of BMSCs through epigenetic methylation modification. Specifically, inhibition of EZH2 and enhancement of KDM6A reduced the trimethylation level of histone 3 at lysine 27 (H3K27), thereby activating the transcription of osteogenic genes such as Runx2 and Osx. The efficacy of the bioactive microspheres in bone repair is validated in a rat mandibular defect model, demonstrating that peripheral nerve response facilitates bone regeneration through epigenetic modification. These findings illuminated a novel strategy for constructing neuroactive osteo-inductive biomaterials with potential for further clinical applications.

The trigeminal nerve is the sensory afferent of the orofacial regions and divided into three major branches. Cell bodies of the trigeminal nerve lie in the trigeminal ganglion and are surrounded by satellite cells. There is a close interaction between ganglion cells via satellite cells, but the function is not fully understood. In the present study, we clarified the ganglion cells\' three-dimensional (3D) localization, which is essential to understand the functions of cell-cell interactions in the trigeminal ganglion. Fast blue was injected into 12 sites of the rat orofacial regions, and ganglion cells were retrogradely labeled. The labeled trigeminal ganglia were cleared by modified 3DISCO, imaged with confocal laser-scanning microscopy, and reconstructed in 3D. Histograms of the major axes of the fast blue-positive somata revealed that the peak major axes of the cells innervating the skin/mucosa were smaller than those of cells innervating the deep structures. Ganglion cells innervating the ophthalmic, maxillary, and mandibular divisions were distributed in the anterodorsal, central, and posterolateral portions of the trigeminal ganglion, respectively, with considerable overlap in the border region. The intermingling in the distribution of ganglion cells within each division was also high, in particular, within the mandibular division. Specifically, intermingling was observed in combinations of tongue and masseter/temporal muscles, maxillary/mandibular molars and masseter/temporal muscles, and tongue and mandibular molars. Double retrograde labeling confirmed that some ganglion cells innervating these combinations were closely apposed. Our data provide essential information for understanding the function of ganglion cell-cell interactions via satellite cells.

OBJECTIVE: Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea.
METHODS: Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [3H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs.
RESULTS: ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [3H] acetylcholine was synthesized from [3H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist.
CONCLUSIONS: The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

Locomotor recovery after spinal cord injury (SCI) remains an unmet challenge. Nerve transfer (NT), the connection of a functional/expendable peripheral nerve to a paralyzed nerve root, has long been clinically applied, aiming to restore motor control. However, outcomes have been inconsistent, suggesting that NT-induced neurological reinstatement may require activation of mechanisms beyond motor axon reinnervation (our hypothesis). We previously reported that to enhance rat locomotion following T13-L1 hemisection, T12-L3 NT must be performed within timeframes optimal for sensory nerve regrowth. Here, T12-L3 NT was performed for adult female rats with subacute (7-9 days) or chronic (8 weeks) mild (SCImi: 10 g × 12.5 mm) or moderate (SCImo: 10 g × 25 mm) T13-L1 thoracolumbar contusion. For chronic injuries, T11-12 implantation of adult hMSCs (1-week before NT), post-NT intramuscular delivery of FGF2, and environmentally enriched/enlarged (EEE) housing were provided. NT, not control procedures, qualitatively improved locomotion in both SCImi groups and animals with subacute SCImo. However, delayed NT did not produce neurological scale upgrading conversion for SCImo rats. Ablation of the T12 ventral/motor or dorsal/sensory root determined that the T12-L3 sensory input played a key role in hindlimb reanimation. Pharmacological, electrophysiological, and trans-synaptic tracing assays revealed that NT strengthened integrity of the propriospinal network, serotonergic neuromodulation, and the neuromuscular junction. Besides key outcomes of thoracolumbar contusion modeling, the data provides the first evidence that mixed NT-induced locomotor efficacy may rely pivotally on sensory rerouting and pro-repair neuroplasticity to reactivate neurocircuits/central pattern generators. The finding describes a novel neurobiology mechanism underlying NT, which can be targeted for development of innovative neurotization therapies.

Respiratory viral infection can lead to activation of sensory afferent nerves as indicated by the consequential sore throat, sneezing, coughing, and reflex secretions. In addition to causing troubling symptoms, sensory nerve activation likely accelerates viral spreading. The mechanism how viruses activate sensory nerve terminals during infection is unknown. In this study, we investigate whether coronavirus spike protein activates sensory nerves terminating in the airways. We used isolated vagally-innervated mouse trachea-lung preparation for two-photon microscopy and extracellular electrophysiological recordings. Using two-photon Ca2+ imaging, we evaluated a total number of 786 vagal bronchopulmonary nerves in six experiments. Approximately 49% of the sensory fibers were activated by S1 protein (4 μg/mL intratracheally). Extracellular nerve recording showed the S1 protein evoked action potential discharge in sensory C-fibers; of 39 airway C-fibers (one fiber per mouse), 17 were activated. Additionally, Fura-2 Ca2+ imaging was performed on neurons dissociated from vagal sensory ganglia (n = 254 from 22 mice). The result showed that 63% of neurons responded to S1 protein. SARS-CoV-2 S1 protein can lead to direct activation of sensory C-fiber nerve terminals in the bronchopulmonary tract. Direct activation of C-fibers may contribute to coronavirus symptoms, and amplify viral spreading in a population.

BACKGROUND: The integumentary system of the skin serves as an exceptional protective barrier, with the stratum corneum situated at the forefront. This outermost layer is composed of keratinocytes that biosynthesize filaggrin (encoded by the gene Flg), a pivotal constituent in maintaining skin health. Nevertheless, the precise role of sensory nerves in restoration of the skin barrier after tape stripping-induced epidermal disruption, in contrast to the wound-healing process, remains a tantalizing enigma.
OBJECTIVE: This study aimed to elucidate the cryptic role of sensory nerves in repair of the epidermal barrier following tape stripping-induced disruption.
METHODS: Through the implementation of resiniferatoxin (RTX)-treated denervation mouse model, we investigated the kinetics of barrier repair after tape stripping and performed immunophenotyping and gene expression analysis in the skin or dorsal root ganglia (DRG) to identify potential neuropeptides. Furthermore, we assessed the functional impact of candidates on the recovery of murine keratinocytes and RTX-treated mice.
RESULTS: Ablation of TRPV1-positive sensory nerve attenuated skin barrier recovery and sustained subcutaneous inflammation, coupled with elevated IL-6 level in ear homogenates after tape stripping. Expression of the keratinocyte differentiation marker Flg in the ear skin of RTX-treated mice was decreased compared with that in control mice. Through neuropeptide screening, we found that the downregulation of Flg by IL-6 was counteracted by somatostatin or octreotide (a chemically stable somatostatin analog). Furthermore, RTX-treated mice given octreotide exhibited a partial improvement in barrier recovery after tape stripping.
CONCLUSIONS: Sensory neurons expressing TRPV1 play an indispensable role in restoring barrier function following epidermal injury. Our findings suggest the potential involvement of somatostatin in restoring epidermal repair after skin injury.

Airway nerves regulate vital airway functions including bronchoconstriction, cough, and control of respiration. Dysregulation of airway nerves underlies the development and manifestations of airway diseases such as chronic cough, where sensitization of neural pathways leads to excessive cough triggering. Nerves are heterogeneous in both expression and function. Recent advances in confocal imaging and in targeted genetic manipulation of airway nerves have expanded our ability to visualize neural organization, study neuro-immune interactions, and selectively modulate nerve activation. As a result, we have an unprecedented ability to quantitatively assess neural remodeling and its role in the development of airway disease. This review highlights our existing understanding of neural heterogeneity and how advances in methodology have illuminated airway nerve morphology and function in health and disease.