Yu Shin Kim

The vagus nerve conveys interoceptive information, yet how specific vagal sensory afferents regulate pain remains unclear. Here, we tested whether vagus nerve stimulation (VNS) modulates temporomandibular disorder (TMD)–related pain. In a mouse model of TMD, auricular VNS (aVNS) attenuated temporomandibular joint (TMJ) pain behaviors and suppressed sensitization of trigeminal nociceptors. We identified a subset of vagal sensory afferents with dopaminergic features that was sufficient to mediate these effects, as selective activation of these afferents recapitulated the analgesic actions of aVNS. These findings highlight an underappreciated peripheral interoceptive pathway and provide a mechanistic framework for targeted neuromodulation in chronic craniofacial pain.

In vivo voltage imaging is a powerful tool for monitoring action potentials and dynamic electrical events in heterogeneous sensory neurons enabling the deciphering of rapid somatosensory information processing. Virus-driven expression of genetically encoded voltage indicator (GEVI) suffers from inconsistent expression levels and offers a limited time window for optimal voltage imaging. Here, we generated and characterized a knock-in mouse line with Pirt-driven expression of Marina, a positively tuned GEVI, in primary sensory neurons. Pirt-Marina mice enable optical reporting of touch, itch, and nociceptive sensations in vivo and distinct action potential patterns in the trigeminal and dorsal root ganglion neurons. Notably, Pirt-Marina mice display robust fluorescence signals in response to mechanical, thermal, or chemical stimuli, allowing visualization of transformations in sensory coding following inflammation and injury. This Pirt-Marina mouse line provides optical access to dynamic neuronal activity and plasticity in the peripheral nervous system (PNS) with high temporal accuracy, fidelity, and reliability.

Primary somatosensory neurons, glial cells in the peripheral ganglia, and neural circuits in the spinal cord function as dynamic network circuits that transmit information to the brain. Although a variety of methods and techniques have been used to study individual neurons or tissue explants, the number of neurons that can be monitored is limited. Imaging intact primary sensory neurons, such as those in the dorsal root ganglion and trigeminal ganglia, and the spinal cord in vivo using fluorescent calcium markers helps overcome the limitations of previous methods and techniques by allowing researchers to monitor tens to thousands of cells simultaneously. This allows researchers to conduct experiments to elucidate somatosensory mechanisms and responses to axonal injury that were previously difficult or impossible to observe. Using this approach, researchers have studied dynamic neural network circuits, connectivity, responses to soft and deep touch, heat, cold, chemicals, inflammation, and injury, and they have repeatedly imaged individual neurons over long periods of time. Approaches include using calcium-sensitive fluorescent dyes and genetically encoded markers, performing terminal exposure surgeries, using chambers designed to monitor large numbers of cells or repeatedly imaging small numbers of cells, and imaging animals with or without anesthesia. This review discusses the advantages and disadvantages of in vivo calcium imaging for studying somatosensory and axonal injury in peripheral sensory ganglia and the dorsal spinal cord, as well as anticipated future directions.

Temporomandibular disorder (TMD) significantly impairs the quality of life of patients due to chronic pain and limited jaw function. Many treatment options have been used such as pharmacologic management, physical therapy, oral appliance therapy, and surgery. However, effective treatment options remain limited. In this study, we investigated the potential of botulinum toxin (BoNT) as a therapeutic approach for TMD using a forced mouth opening-induced TMD male mouse model. BoNT injection significantly alleviated mechanical hypersensitivity in the temporomandibular region over a 2 week period as demonstrated by von Frey behavioral tests. Additionally, the mouse grimace test confirmed that BoNT alleviated pain in mice. The open field test and pasta gnawing test showed that BoNT injection effectively alleviated mouth motor and food intake problems and did not cause impairments in general behavior. Moreover, direct observation of neural activity via in vivo Pirt-GCaMP3 calcium imaging of intact trigeminal ganglia (TG) revealed that BoNT suppressed both stimulus-evoked and spontaneous activity in TG neurons. Mechanistically, BoNT downregulated the expression of pain-promoting proteins (TRPV1, TRPA1, and TRPC1) and glutamate transporting protein (VGLUT2), thereby suppressing peripheral neural activity in the TG. In summary, our study identified a novel mechanism by which BoNT alleviates TMD pain. These new findings not only expand our understanding of the effects of BoNT on pain but also provide a new therapeutic approach to TMJ pain management.

Calcium imaging is an important tool for monitoring signaling mechanisms that either influx Ca2+ into the cytosol or release Ca2+ from intracellular Ca2+ stores. The use of Pirt-GCaMP3 mice, which have the Ca2+sensor GCaMP3 inserted downstream of the Pirt promoter region and expressed in nearly all (>95%) primary sensory neurons, allows for simultaneous monitoring of the activity of >3,000 neurons via Ca2+ imaging of trigeminal ganglion (TG) primary sensory neurons. This allows investigators to study neural network responses to noxious stimuli and somatosensory and hormonal responses in vivo, which would otherwise be very difficult to detect using other methods. Specifically, the ability to monitor the TG area innervating the face and other areas of the head separately allows for monitoring primary sensory neurons activated by stimuli of the face area before later layers of neuron processing. The number of neurons generating Ca2+ transients and the amplitude of Ca2+ transients, which indicate their sensitivity to different sensory modalities, can also be observed. Additionally, investigators can measure the diameter of neurons, which is an indicator of the type of fiber activated (non-noxious mechano-fibers vs. noxious nociceptive fibers, Aβ, Aδ, and C fibers). Therefore, Pirt-GCaMP3 calcium imaging of TG would be a powerful tool to dissect specific sensory modalities and neuronal cell types to study trigeminal origin pain (orofacial and craniofacial, dental or/tooth pain, headache, migraine, temporomandibular joint pain, trigeminal neuralgia), itch and touch and any other somatosensations, as well as hormonal responses.

Calcium imaging is an important tool for monitoring signaling mechanisms that either influx Ca2+ into the cytosol or release Ca2+ from intracellular Ca2+ stores. The use of Pirt-GCaMP3 mice, which have the Ca2+sensor GCaMP3 inserted downstream of the Pirt promoter region and expressed in nearly all (>95%) primary sensory neurons, allows for simultaneous monitoring of the activity of >3,000 neurons via Ca2+ imaging of trigeminal ganglion (TG) primary sensory neurons. This allows investigators to study neural network responses to noxious stimuli and somatosensory and hormonal responses in vivo, which would otherwise be very difficult to detect using other methods. Specifically, the ability to monitor the TG area innervating the face and other areas of the head separately allows for monitoring primary sensory neurons activated by stimuli of the face area before later layers of neuron processing. The number of neurons generating Ca2+ transients and the amplitude of Ca2+ transients, which indicate their sensitivity to different sensory modalities, can also be observed. Additionally, investigators can measure the diameter of neurons, which is an indicator of the type of fiber activated (non-noxious mechano-fibers vs. noxious nociceptive fibers, Aβ, Aδ, and C fibers). Therefore, Pirt-GCaMP3 calcium imaging of TG would be a powerful tool to dissect specific sensory modalities and neuronal cell types to study trigeminal origin pain (orofacial and craniofacial, dental or/tooth pain, headache, migraine, temporomandibular joint pain, trigeminal neuralgia), itch and touch and any other somatosensations, as well as hormonal responses.

Primary sensory neurons convert external stimuli into electrical signals, yet how heterogeneous neurons encode distinct sensations remains unclear. In vivo dorsal root ganglia (DRG) imaging with genetically-encoded Ca²⁺ indicators (GECIs) enables mapping of neuronal activity from over 1800 neurons per DRG in live mice, offering high spatial and populational resolution. However, GECIs’ slow Ca²⁺ response kinetics limit the temporal accuracy of neuronal electrical dynamics. Genetically-encoded voltage indicators (GEVIs) provide real-time voltage tracking but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a bright and fast positively tuned GEVI, to dissect temporal dynamics of DRG neuron responses to mechanical, thermal, or chemical stimulation in live male and female mice. ASAP4.4-Kv revealed previously unrecognized cell-to-cell electrical synchronization and robust dynamic transformations in sensory coding following tissue injury. Combining GEVI and GECI imaging empowers spatiotemporal analysis of sensory signal processing and integration mechanisms in vivo.

Calcium ions (Ca²⁺) and Ca²⁺ channels are pivotal in the regulation of pain pathways and serve as key regulators of neuronal excitability and neurotransmitter release. We review the different types of Ca²⁺ channels involved in pain processing, including voltage-gated Ca²⁺ channels (VGCCs), such as L-, N-, P/Q-, and T-type channels. Each subtype is intricately involved in different aspects of pain perception, from acute pain signaling to the development and maintenance of chronic pain states. In addition, the roles of transient receptor potential (TRP) channels, particularly TRPV1 and TRPA1, are discussed in the context of their contribution to chronic pain. Advances in Ca²⁺ imaging techniques, particularly through genetically encoded Ca²⁺ indicators (GECIs), such as GCaMPs, have provided unprecedented insight into the dynamic role of Ca²⁺ channels in pain pathways. These efforts have deepened our understanding of Ca²⁺ channels and suggest novel therapeutic targets for more effective pain management strategies within Ca²⁺ channels.

Models of IVD Degeneration, Tang et al., 2022 Rodents and humans have fundamentally different spinal anatomy. Standardization, translatability, and consistency across models in behavior and markers of IVD may improve the field.Pregnancy-related LBPP Pregnancy-related lumbopelvic pain (LBPP) is a combination of pregnancy related lower back pain and pelvic girdle pain. Study of LBPP on large populations (>70,000) reliably find statistical correlations. However, smaller, mechanistic studies give varying degrees of inconsistent results [2,3]. The efficacy of exercises to relieve pain and prevent chronification of pain, and which exercises are appropriate, are controversial [3,4]. Additionally, common problems including the difficulty of translatability of animal models, numerous changes to the body happening simultaneously during pregnancy, and differences limited applicability of mixed or all-male pain studies contribute to a making a science-based approach to management of pregnancy-related LBPP difficult. This review by Daneau et al proposes an integrated approach of studying neuromechanical, physiological (including hormonal), second or later pregnancies, and clinical changes during all trimesters of pregnancy and interactions between these factors to disentangle the mixed evidence of mechanisms of pregnancy-related LBPP [2]. They note one of the strongest predictors of LBPP is prior LBPP, suggesting (along with other evidence) that motor adaptations during pregnancy contribute to later LBPP. In addition to the value of this paper as a review of pregnancy-related LBPP, they argue studies of interactions of risk factors will be fruitful.Searching PubMed for the most recent (2022 or later) literature on interactions, motor control, and muscles with lumbopelvic pain and pregnancy found several studies about musculoskeletal adaptation during pregnancy and pain. Papers that did not consider pelvic pain or musculoskeletal structure (including exercise interventions) were not considered. There have been attempts to map changes in musculature and lumbopelvic alignment in detail during and after pregnancy [3,[5][6][7]. Interactions with age and disability and muscular strength in areas of the pelvic region are strongly correlated with pelvic pain during pregnancy, but musculature was not predictive [7]. However, Palsson et al found that deep tissue muscle sensitivity during pregnancy was independent of pain [8]. There have been limited recently published studies since the Danau paper to resolve the controversies around exercise as an intervention [9,10], and it is difficult to separate the effect of exercise from effects of participants disliking exercise [11]. Overall, there is not yet enough literature to evaluate the hypothesis of motor adaptations during pregnancy as a major contributor to LBPP. Future studies are needed to conclude the causal relationship and new contributors for LBPP.Intervertebral disc (IVD) degeneration is significantly increased after menopause [12] and after hysterectomy [13]. There is significant mechanistic evidence from human studies that alterations in estrogen could contribute to IVD [14][15][16]. Dissection of mechanisms and possible interventions may be accelerated by studying rodent models, but, in addition to normal problems correlating human and animal models, there is the obvious problem that humans are large bipeds and rodents are small quadrupeds, which leads to very different IVD stresses.Lower back pain (LBP) is a common disability, and IVD degeneration is a major cause of LBP. In an extensive review with highlights including sex differences, Tang et al discuss using mouse models to study IVD degeneration [17]. Despite large differences in mouse and human anatomy, they argue mouse IVD morphology, physiology, and behavior is more similar to humans than dissimilar. Aged and "accelerated aging" mouse models are broadly similar to humans including changes in IVD morphology and inflammatory factors. However, intervention efficacy period coincides with mice beginning to die of old age. Mechanical models (IVD puncture, forced bipedal posture, etc.) can be used on younger mice, but work best on lumbar-level IVDs. The review extensively assesses what has been learned from mouse models of IVD. The authors argue standardization in scoring phenotypes could advance assessment of mouse models by making different studies more comparable. The authors identify several common mouse pain assays that are consistent both between mouse studies and with human behavior, discuss how they determine consistency, and discuss why inconsistencies may occur.The review ends with a discussion of how to assess the best mouse model for a given clinical or mechanistic question about IVD. The authors have contributed an extensive review of mouse IVD models, their advantages and limitations, and make an argument for the most useful aspects of mouse models of IVD.Rat models have been extensively studied for anatomical and cellular changes but much less in behavior assays. Barbe et al compared a 2 lumbar intervertebral disc (IVD) puncture (DP-2) and one lumbar IVD puncture (DP-1) to sham surgery female rats as part of an effort to further develop an animal model for study acute to chronic lower back pain (LBP) [18]. The paper extensively compares their results to similar papers. They performed a battery of behavior, serum, and post-mortem histological and morphological assays. To the authors' knowledge, this study provides the most extensive set of behavioral testing of this model in the literature.Searching PubMed for recent (2022 or later) literature on mouse and rat models of IVD degeneration, excluding those which did not study behavior tests, show limited standardization of behavioral tests for mice [19][20][21][22] or rats [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. More papers used rat models than mouse models. 70% of papers used von Frey on the paws to test...

There is a need to increase the performance and longevity of dental composites and accelerate the translation of novel composites to the market. This study explores the use of artificial intelligence (AI), specifically machine learning (ML) models, to predict the performance outcomes (POs) of dental composites from their composite attributes. A comprehensive dataset was carefully curated and refined from 200+ publications. Nine ML models were trained to predict discrete POs, and their performance was evaluated. Five models were used for regression analysis of continuous POs. Different ML models performed better on different POs. The k-nearest neighbors (KNN) model excelled in predicting flexural modulus (FlexMod), Decision Tree model in flexural strength (FlexStr) and volumetric shrinkage (ShrinkV), and Logistic Regression and Support Vector Machine models in shrinkage stress (ShrinkStr). Receiver-operating characteristic area under the curve analysis confirmed these results but found that Random Forest was more effective for FlexStr and ShrinkV, suggesting the possibility of Decision Tree overfitting the data. Regression analysis revealed that the voting regressor was superior for FlexMod and ShrinkV predictions, while Decision Tree Regression was optimal for FlexStr and ShrinkStr. Feature importance analysis indicated triethylene glycol dimethacrylate is a key contributor to FlexMod and ShrinkV, bisphenol A glycidyl dimethacrylate and urethane dimethacrylate to FlexStr, and depth of cure, degree of monomer-to-polymer conversion, and filler loading to ShrinkStr. There is a need to conduct a full analysis using multiple ML models because different models predict different POs better and for a large, comprehensive dataset to train robust AI models to facilitate the prediction and optimization of composite properties and support the development of new dental materials.