The first point of our body’s contact with tactile stimuli (innocuous and noxious) is the epidermis, the outermost layer of skin that is largely composed of keratinocytes. Here, we sought to define the role that keratinocytes play in touch sensation in vivo and ex vivo. We show that optogenetic inhibition of keratinocytes decreases behavioral and cellular mechanosensitivity. These processes are inherently mediated by ATP signaling, as demonstrated by complementary cutaneous ATP release and degradation experiments. Specific deletion of P2X4 receptors in sensory neurons markedly decreases behavioral and primary afferent mechanical sensitivity, thus positioning keratinocyte-released ATP to sensory neuron P2X4 signaling as a critical component of baseline mammalian tactile sensation. These experiments lay a vital foundation for subsequent studies into the dysfunctional signaling that occurs in cutaneous pain and itch disorders, and ultimately, the development of novel topical therapeutics for these conditions.

Cutaneous somatosensory neurons convey innocuous and noxious mechanical, thermal, and chemical stimuli from peripheral tissues to the CNS. Among these are nociceptive neurons that express calcitonin gene-related peptide-α (CGRPα). The role of peripheral CGRPα neurons (CANs) in acute and injury-induced pain has been studied using diphtheria toxin ablation, but their functional roles remain controversial. Because ablation permanently deletes a neuronal population, compensatory changes may ensue that mask the physiological or pathophysiological roles of CANs, particularly for injuries that occur after ablation. Therefore, we sought to define the role of intact CANs under baseline and injury conditions by using noninvasive transient optogenetic inhibition. We assessed pain behavior longitudinally from acute to chronic time points. We generated adult male and female mice that selectively express the outward rectifying proton pump archaerhodopsin-3 (Arch) in CANs, and inhibited their peripheral cutaneous terminals in models of neuropathic (spared nerve injury) and inflammatory (skin-muscle incision) pain using transdermal light activation of Arch. After nerve injury, brief activation of Arch reversed the chronic mechanical, cold, and heat hypersensitivity, alleviated the spontaneous pain, and reversed the sensitized mechanical currents in primary afferent somata. In contrast, Arch inhibition of CANs did not alter incision-induced hypersensitivity. Instead, incision-induced mechanical and heat hypersensitivity was alleviated by peripheral blockade of CGRPα peptide-receptor signaling. These results reveal that CANs have distinct roles in the time course of pain during neuropathic and incisional injuries and suggest that targeting peripheral CANs or CGRPα peptide-receptor signaling could selectively treat neuropathic or postoperative pain, respectively. The contribution of sensory afferent CGRPα neurons (CANs) to neuropathic and inflammatory pain is controversial. Here, we left CANs intact during neuropathic and perioperative incision injury by using transient transdermal optogenetic inhibition of CANs. We found that peripheral CANs are required for neuropathic mechanical, cold, and heat hypersensitivity, spontaneous pain, and sensitization of mechanical currents in afferent somata. However, they are dispensable for incisional pain transmission. In contrast, peripheral pharmacological inhibition of CGRPα peptide-receptor signaling alleviated the incisional mechanical and heat hypersensitivity, but had no effect on neuropathic pain. These results show that CANs have distinct roles in neuropathic and incisional pain and suggest that their targeting via novel peripheral treatments may selectively alleviate neuropathic versus incisional pain.

Postoperative pain management continues to be suboptimal because of the lack of effective nonopioid therapies and absence of understanding of sex-driven differences. Here, we asked how the NLRP3 inflammasome contributes to postoperative pain. Inflammasomes are mediators of the innate immune system that are responsible for activation and secretion of IL-1β upon stimulation by specific molecular signals. Peripheral IL-1β is known to contribute to the mechanical sensitization induced by surgical incision. However, it is not known which inflammasome mediates the IL-1β release after surgical incision. Among the 9 known inflammasomes, the NLRP3 inflammasome is ideally positioned to drive postoperative pain through IL-1β production because NLRP3 can be activated by factors that are released by incision. Here, we show that male mice that lack NLRP3 (NLRP3KO) recover from surgery-induced behavioral and neuronal mechanical sensitization faster and display less surgical site inflammation than mice expressing NLRP3 (wild-type). By contrast, female NLRP3KO mice exhibit minimal attenuation of the postoperative mechanical hypersensitivity and no change in postoperative inflammation compared with wild-type controls. Sensory neuron-specific deletion of NLRP3 revealed that in males, NLRP3 expressed in non-neuronal cells and potentially sensory neurons drives postoperative pain. However, in females, only the NLRP3 that may be expressed in sensory neurons contributes to postoperative pain where the non-neuronal cell contribution is NLRP3 independent. This is the first evidence of a key role for NLRP3 in postoperative pain and reveals immune-mediated sex differences in postoperative pain.

The strength-duration curve, when used in conjunction with anorectal manometry, may have a role in the investigation of faecal incontinence.

Postoperative pain is highly debilitating and hinders recovery. Opioids are the main pain medication used for acute postoperative pain. Given the devastating opioid addiction and overdose epidemic across the US, non-opioid pain therapeutics are desperately needed. In order to develop novel, non-opioid therapies for the treatment of postoperative pain and identify the mechanisms underlying this pain, rodent models of incisional pain have been established. The protocol herein describes in detail how to create a mouse model of postoperative pain that was adapted from established protocols. This model of postoperative pain is frequently-used, highly reproducible, and results in peripheral and central nervous system alterations.