Dual Modulation of Nociception and Cardiovascular Reflexes during Peripheral Ischemia through P2Y1 Receptor-Dependent Sensitization of Muscle Afferents.

Numerous musculoskeletal pain disorders are based in dysfunction of peripheral perfusion and are often comorbid with altered cardiovascular responses to muscle contraction/exercise. We have recently found in mice that 24 h peripheral ischemia induced by a surgical occlusion of the brachial artery (BAO) induces increased paw-guarding behaviors, mechanical hypersensitivity, and decreased grip strength. These behavioral changes corresponded to increased heat sensitivity as well as an increase in the numbers of chemosensitive group III/IV muscle afferents as assessed by an ex vivo forepaw muscles/median and ulnar nerves/dorsal root ganglion (DRG)/spinal cord (SC) recording preparation. Behaviors also corresponded to specific upregulation of the ADP-responsive P2Y1 receptor in the DRGs. Since group III/IV muscle afferents have separately been associated with regulating muscle nociception and exercise pressor reflexes (EPRs), and P2Y1 has been linked to heat responsiveness and phenotypic switching in cutaneous afferents, we sought to determine whether upregulation of P2Y1 was responsible for the observed alterations in muscle afferent function, leading to modulation of muscle pain-related behaviors and EPRs after BAO. Using an afferent-specific siRNA knockdown strategy, we found that inhibition of P2Y1 during BAO not only prevented the increased mean blood pressure after forced exercise, but also significantly reduced alterations in pain-related behaviors. Selective P2Y1 knockdown also prevented the increased firing to heat stimuli and the BAO-induced phenotypic switch in chemosensitive muscle afferents, potentially through regulating membrane expression of acid sensing ion channel 3. These results suggest that enhanced P2Y1 in muscle afferents during ischemic-like conditions may dually regulate muscle nociception and cardiovascular reflexes. SIGNIFICANCE STATEMENT: Our current results suggest that P2Y1 modulates heat responsiveness and chemosensation in muscle afferents to play a key role in the development of pain-related behaviors during ischemia. At the same time, under these pathological conditions, the changes in muscle sensory neurons appear to modulate an increase in mean systemic blood pressure after exercise. This is the first report of the potential peripheral mechanisms by which group III/IV muscle afferents can dually regulate muscle nociception and the exercise pressor reflex. These data provide evidence related to the potential underlying reasons for the comorbidity of muscle pain and altered sympathetic reflexes in disease states that are based in problems with peripheral perfusion and may indicate a potential target for therapeutic intervention.

Muscle IL1ß Drives Ischemic Myalgia via ASIC3-Mediated Sensory Neuron Sensitization.

UNLABELLED: Musculoskeletal pain is a significantly common clinical complaint. Although it is known that muscles are quite sensitive to alterations in blood flow/oxygenation and a number of muscle pain disorders are based in problems of peripheral perfusion, the mechanisms by which ischemic-like conditions generate myalgia remain unclear. We found, using a multidisciplinary experimental approach, that ischemia and reperfusion injury (I/R) in male Swiss Webster mice altered ongoing and evoked pain-related behaviors in addition to activity levels through enhanced muscle interleukin-1 beta (IL1ß)/IL1 receptor signaling to group III/IV muscle afferents. Peripheral sensitization depended on acid-sensing ion channels (ASICs) because treatment of sensory afferents in vitro with IL1ß-upregulated ASIC3 in single cells, and nerve-specific knock-down of ASIC3 recapitulated the results of inhibiting the enhanced IL1ß/IL1r1 signaling after I/R, which was also found to regulate afferent sensitization and pain-related behaviors. This suggests that targeting muscle IL1ß signaling may be a potential analgesic therapy for ischemic myalgia. SIGNIFICANCE STATEMENT: Here, we have described a novel pathway whereby increased inflammation within the muscle tissue during ischemia/reperfusion injury sensitizes group III and IV muscle afferents via upregulation of acid-sensing ion channel 3 (ASIC3), leading not only to alterations in mechanical and chemical responsiveness in individual afferents, but also to pain-related behavioral changes. Furthermore, these I/R-induced changes can be prevented using an afferent-specific siRNA knock-down strategy targeting either ASIC3 or the upstream mediator of its expression, interleukin 1 receptor 1. Therefore, this knowledge may contribute to the development of alternative therapeutics for muscle pain and may be especially relevant to pain caused by issues of peripheral circulation, which is commonly observed in disorders such as complex regional pain syndrome, sickle cell anemia, or fibromyalgia.

Age-dependent sensitization of cutaneous nociceptors during developmental inflammation.

BACKGROUND: It is well-documented that neonates can experience pain after injury. However, the contribution of individual populations of sensory neurons to neonatal pain is not clearly understood. Here we characterized the functional response properties and neurochemical phenotypes of single primary afferents after injection of carrageenan into the hairy hindpaw skin using a neonatal ex vivo recording preparation. RESULTS: During normal development, we found that individual afferent response properties are generally unaltered. However, at the time period in which some sensory neurons switch their neurotrophic factor responsiveness, we observe a functional switch in slowly conducting, broad spiking fibers ("C"-fiber nociceptors) from mechanically sensitive and thermally insensitive (CM) to polymodal (CPM). Cutaneous inflammation induced prior to this switch (postnatal day 7) specifically altered mechanical and heat responsiveness, and heat thresholds in fast conducting, broad spiking ("A"-fiber) afferents. Furthermore, hairy skin inflammation at P7 transiently delayed the functional shift from CM to CPM. Conversely, induction of cutaneous inflammation after the functional switch (at P14) caused an increase in mechanical and thermal responsiveness exclusively in the CM and CPM neurons. Immunocytochemical analysis showed that inflammation at either time point induced TRPV1 expression in normally non-TRPV1 expressing CPMs. Realtime PCR and western blotting analyses revealed that specific receptors/channels involved in sensory transduction were differentially altered in the DRGs depending on whether inflammation was induced prior to or after the functional changes in afferent prevalence. CONCLUSION: These data suggest that the mechanisms of neonatal pain development may be generated by different afferent subtypes and receptors/channels in an age-related manner.

Increasing acceptance of AI-generated digital twins through clinical trial applications.

Today's approach to medicine requires extensive trial and error to determine the proper treatment path for each patient. While many fields have benefited from technological breakthroughs in computer science, such as artificial intelligence (AI), the task of developing effective treatments is actually getting slower and more costly. With the increased availability of rich historical datasets from previous clinical trials and real-world data sources, one can leverage AI models to create holistic forecasts of future health outcomes for an individual patient in the form of an AI-generated digital twin. This could support the rapid evaluation of intervention strategies in silico and could eventually be implemented in clinical practice to make personalized medicine a reality. In this work, we focus on uses for AI-generated digital twins of clinical trial participants and contend that the regulatory outlook for this technology within drug development makes it an ideal setting for the safe application of AI-generated digital twins in healthcare. With continued research and growing regulatory acceptance, this path will serve to increase trust in this technology and provide momentum for the widespread adoption of AI-generated digital twins in clinical practice.

Interleukin 1ß inhibition contributes to the antinociceptive effects of voluntary exercise on ischemia/reperfusion-induced hypersensitivity.

Issues of peripheral circulation have been increasingly suggested as an underlying cause of musculoskeletal pain in many conditions, including sickle cell anemia and peripheral vascular disease. We have previously shown in our model of transient ischemia and reperfusion (I/R) injury of the forelimb that individual group III and IV muscle afferents display altered chemosensitivity and mechanical thresholds 1 day after injury. Functional alterations corresponded to increased evoked and spontaneous pain-related behaviors and decreased muscle strength and voluntary activity-all actions that echo clinical symptoms of ischemic myalgia. These behavioral and physiological changes appeared to originate in part from the action of increased interleukin 1ß (IL1ß) in the injured muscles at its upregulated IL1 receptor 1 within the dorsal root ganglion. Here, we describe that two days of voluntary wheel running prior to I/R blocks both injury-induced IL1ß enhancement and the subsequent development of ischemic myalgia-like behaviors. Furthermore, the protective effects of 2 days prior exercise on the I/R-evoked increases in pain-related behaviors were also paralleled with systemic injection of the IL1 receptor antagonist during I/R. Interleukin 1 receptor antagonist treatment additionally prevented the I/R-induced changes in mechanical and chemical sensitivity of individual primary muscle afferents. Altogether, these data strengthen the evidence that transient I/R injury sensitizes group III and IV muscle afferents via increased IL1ß in the muscles to stimulate ischemic myalgia development. Targeting IL1ß may, therefore, be an effective treatment strategy for this insidious type of muscle pain.

Sex differences in primary muscle afferent sensitization following ischemia and reperfusion injury.

BACKGROUND: Chronic pain conditions are more prevalent in women, but most preclinical studies into mechanisms of pain generation are performed using male animals. Furthermore, whereas group III and IV nociceptive muscle afferents provoke central sensitization more effectively than their cutaneous counterparts, less is known about this critical population of muscle nociceptors. Here, we compare the physiology of individual muscle afferents in uninjured males and females. We then characterize the molecular, physiological, and behavioral effects of transient ischemia and reperfusion injury (I/R), a model we have extensively studied in males and in females. METHODS: Response properties and phenotypes to mechanical, thermal, and chemical stimulation were compared using an ex vivo muscle/nerve/dorsal root ganglia (DRG)/spinal cord recording preparation. Analyses of injury-related changes were also performed by assaying evoked and spontaneous pain-related behaviors, as well as mRNA expression of the affected muscle and DRGs. The appropriate analyses of variance and post hoc tests (with false discovery rate corrections when needed) were performed for each measure. RESULTS: Females have more mechanically sensitive muscle afferents and show greater mechanical and thermal responsiveness than what is found in males. With I/R, both sexes show fewer cells responsive to an innocuous metabolite solution (ATP, lactic acid, and protons), and lower mechanical thresholds in individual afferents; however, females also possess altered thermal responsiveness, which may be related to sex-dependent changes in gene expression within the affected DRGs. Regardless, both sexes show similar increases in I/R-induced pain-like behaviors. CONCLUSIONS: Here, we illustrate a unique phenomenon wherein discrete, sex-dependent mechanisms of primary muscle afferent sensitization after ischemic injury to the periphery may underlie similar behavioral changes between the sexes. Furthermore, although the group III and IV muscle afferents are fully developed functionally, the differential mechanisms of sensitization manifest prior to sexual maturity. Hence, this study illustrates the pressing need for further exploration of sex differences in afferent function throughout the lifespan for use in developing appropriately targeted pain therapies.

Peripheral Mechanisms of Ischemic Myalgia.

Musculoskeletal pain due to ischemia is present in a variety of clinical conditions including peripheral vascular disease (PVD), sickle cell disease (SCD), complex regional pain syndrome (CRPS), and even fibromyalgia (FM). The clinical features associated with deep tissue ischemia are unique because although the subjective description of pain is common to other forms of myalgia, patients with ischemic muscle pain often respond poorly to conventional analgesic therapies. Moreover, these patients also display increased cardiovascular responses to muscle contraction, which often leads to exercise intolerance or exacerbation of underlying cardiovascular conditions. This suggests that the mechanisms of myalgia development and the role of altered cardiovascular function under conditions of ischemia may be distinct compared to other injuries/diseases of the muscles. It is widely accepted that group III and IV muscle afferents play an important role in the development of pain due to ischemia. These same muscle afferents also form the sensory component of the exercise pressor reflex (EPR), which is the increase in heart rate and blood pressure (BP) experienced after muscle contraction. Studies suggest that afferent sensitization after ischemia depends on interactions between purinergic (P2X and P2Y) receptors, transient receptor potential (TRP) channels, and acid sensing ion channels (ASICs) in individual populations of peripheral sensory neurons. Specific alterations in primary afferent function through these receptor mechanisms correlate with increased pain related behaviors and altered EPRs. Recent evidence suggests that factors within the muscles during ischemic conditions including upregulation of growth factors and cytokines, and microvascular changes may be linked to the overexpression of these different receptor molecules in the dorsal root ganglia (DRG) that in turn modulate pain and sympathetic reflexes. In this review article, we will discuss the peripheral mechanisms involved in the development of ischemic myalgia and the role that primary sensory neurons play in EPR modulation.

Growth hormone regulates the sensitization of developing peripheral nociceptors during cutaneous inflammation.

Cutaneous inflammation alters the function of primary afferents and gene expression in the affected dorsal root ganglia (DRG). However, specific mechanisms of injury-induced peripheral afferent sensitization and behavioral hypersensitivity during development are not fully understood. Recent studies in children suggest a potential role for growth hormone (GH) in pain modulation. Growth hormone modulates homeostasis and tissue repair after injury, but how GH affects nociception in neonates is not known. To determine whether GH played a role in modulating sensory neuron function and hyperresponsiveness during skin inflammation in young mice, we examined behavioral hypersensitivity and the response properties of cutaneous afferents using an ex vivo hairy skin-saphenous nerve-DRG-spinal cord preparation. Results show that inflammation of the hairy hind paw skin initiated at either postnatal day 7 (P7) or P14 reduced GH levels specifically in the affected skin. Furthermore, pretreatment of inflamed mice with exogenous GH reversed mechanical and thermal hypersensitivity in addition to altering nociceptor function. These effects may be mediated through an upregulation of insulin-like growth factor 1 receptor (IGFr1) as GH modulated the transcriptional output of IGFr1 in DRG neurons in vitro and in vivo. Afferent-selective knockdown of IGFr1 during inflammation also prevented the observed injury-induced alterations in cutaneous afferents and behavioral hypersensitivity similar to that after GH pretreatment. These results suggest that GH can block inflammation-induced nociceptor sensitization during postnatal development leading to reduced pain-like behaviors, possibly by suppressing the upregulation of IGFr1 within DRG.

Sensitization of group III and IV muscle afferents in the mouse after ischemia and reperfusion injury.

UNLABELLED: Ischemic myalgia is a unique type of muscle pain in the patient population. The role that discrete muscle afferent subpopulations play in the generation of pain during ischemic events, however, has yet to be determined. Using 2 brachial artery occlusion models to compare prolonged ischemia or transient ischemia with reperfusion of the muscles, we found that both injuries caused behavioral decrements in grip strength, as well as increased spontaneous pain behaviors. Using our ex vivo forepaw muscles, median and ulnar nerves, dorsal root ganglion, and spinal cord recording preparation, we found after both prolonged and transient ischemia that there was a significant increase in the number of afferents that responded to both noxious and non-noxious chemical (lactate, adenosine triphosphate, varying pH) stimulation of the muscles compared to uninjured controls. However, we found an increase in firing to heat stimuli specifically in muscle afferents during prolonged ischemia, but a distinct increase in afferent firing to non-noxious chemicals and decreased mechanical thresholds after transient ischemia. The unique changes in afferent function observed also corresponded with distinct patterns of gene expression in the dorsal root ganglia. Thus, the development of ischemic myalgia may be generated by unique afferent-based mechanisms during prolonged and transient ischemia. PERSPECTIVE: This study analyzed the response properties of thinly myelinated group III and unmyelinated group IV muscle afferents during prolonged and transient ischemia in addition to pain behaviors and alterations in DRG gene expression in the mouse. Results suggest that mechanisms of pain generation during prolonged ischemia may be different from ischemia/reperfusion.

Ketogenic diets and thermal pain: dissociation of hypoalgesia, elevated ketones, and lowered glucose in rats.

UNLABELLED: Ketogenic diets (KDs) are high-fat, low-carbohydrate formulations effective in treating medically refractory epilepsy, and recently we demonstrated lowered sensitivity to thermal pain in rats fed a KD for 3 to 4 weeks. Regarding anticonvulsant and hypoalgesic mechanisms, theories are divided as to direct effects of increased ketones and/or decreased glucose, metabolic hallmarks of these diets. To address this point, we characterized the time course of KD-induced thermal hypoalgesia, ketosis, and lowered glucose in young male rats fed ad libitum on normal chow or KDs. A strict 6.6:1 (fat:[carbohydrates + protein], by weight) KD increased blood ketones and reduced blood glucose by 2 days of feeding, but thermal hypoalgesia did not appear until 10 days. Thus, ketosis and decreased glucose are not sufficient for hypoalgesia. After feeding a 6.6:1 KD for 19 days, decreased thermal pain sensitivity and changes in blood chemistry reversed 1 day after return to normal chow. Effects were consistent between 2 different diet formulations: a more moderate and clinically relevant KD formula (3.0:1) produced hypoalgesia and similar changes in blood chemistry as the 6.6:1 diet, thus increasing translational potential. Furthermore, feeding the 3.0:1 diet throughout an extended protocol (10-11 weeks) revealed that significant hypoalgesia and increased ketones persisted whereas low glucose did not, demonstrating that KD-induced hypoalgesia does not depend on reduced glucose. In separate experiments we determined that effects on thermal pain responses were not secondary to motor or cognitive changes. Together, these findings dissociate diet-related changes in nociception from direct actions of elevated ketones or decreased glucose, and suggest mechanisms with a slower onset in this paradigm. Overall, our data indicate that metabolic approaches can relieve pain. PERSPECTIVE: Chronic pain is a common and debilitating condition. We show that a KD, a high-fat, very low carbohydrate diet well known for treating epilepsy, lowers sensitivity to thermal pain in rats. This reduced pain is not temporally correlated with hallmark diet-induced changes in blood glucose and ketones.

A ketogenic diet delays weight loss and does not impair working memory or motor function in the R6/2 1J mouse model of Huntington's disease.

Ketogenic diets are high in fat and low in carbohydrates, and have long been used as an anticonvulsant therapy for drug-intractable and pediatric epilepsy. Additionally, ketogenic diets have been shown to provide neuroprotective effects against acute and chronic brain injury, including beneficial effects in various rodent models of neurodegeneration. Huntington's disease is a progressive neurodegenerative disease characterized by neurological, behavioral and metabolic dysfunction, and ketogenic diets have been shown to increase energy molecules and mitochondrial function. We tested the effects of a ketogenic diet in a transgenic mouse model of Huntington's disease (R6/2 1J), with a focus on life-long behavioral and physiological effects. Matched male and female wild-type and transgenic mice were maintained on a control diet or were switched to a ketogenic diet fed ad libitum starting at six weeks of age. We found no negative effects of the ketogenic diet on any behavioral parameter tested (locomotor activity and coordination, working memory) and no significant change in lifespan. Progressive weight loss is a hallmark feature of Huntington's disease, yet we found that the ketogenic diet-which generally causes weight loss in normal animals-delayed the reduction in body weight of the transgenic mice. These results suggest that metabolic therapies could offer important benefits for Huntington's disease without negative behavioral or physiological consequences.