Animal Studies

Infection-induced chronic pain is an under-studied pain condition. One example is apical periodontitis, which evokes considerable mechanical allodynia that persists after treatment in 7-12% of patients. Available analgesics often provide incomplete relief. However, a preclinical model to study pain mechanisms associated with apical periodontitis is not available. Here, we report a mouse model of AP to facilitate studies determining mechanisms mediating persistent infection-induced pain. Mice were anesthetized and the left first molar was exposed to the oral environment for 6 weeks. Bone resorption, as an indicator of apical periodontitis, was quantified using micro-computed tomography. Mechanical allodynia was determined using extraoral von-Frey filaments in both male and female mice. The expression of c-fos in the medullary dorsal horn was assessed using immunohistochemistry. Mice with apical periodontitis developed significant mechanical allodynia by day 7 that was maintained for 42 days. Mechanical thresholds were significantly lower in females compared to males. Administration of ibuprofen, morphine or MK-801 reversed mechanical allodynia. Finally, apical periodontitis triggered an up-regulation of c-fos in the medullary dorsal horn. Collectively, this model simulates signs of clinical pain experienced by patients with apical periodontitis, detects sex differences in allodynia and permits the study of peripheral and central trigeminal pain mechanisms.

Low back pain is a chronic, highly prevalent, and hard-to-treat condition in the elderly. Clinical studies indicate that AXL, which belongs to the tyrosine kinase receptor subfamily, mediates pathological pain. However, it is not clear exactly how AXL regulates pain behaviors. In the present study, we used a model of chronic compression of dorsal root ganglion (CCD)-induced neuropathic pain to recreate clinical intervertebral foramen stenosis and related lumbocrural pain to explore whether AXL in primary sensory neurons contributes to this neuropathic pain in rats. Using double-labeling immunofluorescence, we observed that both phosphorylated AXL (p-AXL) and AXL were localized primarily on isolectin B4 (IB4)-positive and calcitonin gene-related peptide (CGRP)-positive neurons, while AXL was also localized in neurofilament-200 (NF200)-positive neurons. CCD-induced pain was associated with the upregulation of AXL mRNA and protein in injured DRGs. Repeated intrathecal administration of the AXL inhibitor, TP0903, or the AXL small interfering RNA (AXL siRNA), effectively alleviated CCD-induced pain hypersensitivities. Moreover, repeated intrathecal administration of either TP0903, or AXL siRNA, reduced the expression of mTOR in injured DRGs, suggesting that mTOR may mediate AXL's actions. These results indicate that the upregulation of DRG AXL may be part of a peripheral mechanism of neuropathic pain via an intracellular mTOR-signaling pathway. Thus, while AXL inhibitors have so far primarily shown clinical efficacy in tumor treatment, AXL intervention could also serve as a potential target for the treatment of neuropathic pain.

Perioperative sleep disturbance is a risk factor for persistent pain after surgery. Clinical studies have shown that patients with insufficient sleep before and after surgery experience more intense and long-lasting postoperative pain. We hypothesize that sleep deprivation alters L-type calcium channels in the dorsal root ganglia (DRG), thus delaying the recovery from post-surgical pain. To verify this hypothesis, and to identify new predictors and therapeutic targets for persistent postoperative pain, we first established a model of postsurgical pain with perioperative sleep deprivation (SD) by administering hind paw plantar incision to sleep deprivation rats. Then we conducted behavioral tests, including tests with von Frey filaments and a laser heat test, to verify sensory pain, measured the expression of L-type calcium channels using western blotting and immunofluorescence of dorsal root ganglia (an important neural target for peripheral nociception), and examined the activity of L-type calcium channels and neuron excitability using electrophysiological measurements. We validated the findings by performing intraperitoneal injections of calcium channel blockers and microinjections of dorsal root ganglion cells with adeno-associated virus. We found that short-term sleep deprivation before and after surgery increased expression and activity of L-type calcium channels in the lumbar dorsal root ganglia, and delayed recovery from postsurgical pain. Blocking these channels reduced impact of sleep deprivation. We conclude that the increased expression and activity of L-type calcium channels is associated with the sleep deprivation-mediated prolongation of postoperative pain. L-type calcium channels are thus a potential target for management of postoperative pain.

Physical exercise has been established as a low-cost, safe, and effective way to manage chronic pain, but exact mechanisms underlying such exercise-induced hypoalgesia (EIH) are not fully understood. Since a growing body of evidence implicated the amygdala (Amyg) as a critical node in emotional affective aspects of chronic pain, we hypothesized that the Amyg may play important roles to produce EIH effects. Here, using partial sciatic nerve ligation (PSL) model mice, we investigated the effects of voluntary running (VR) on the basal amygdala (BA) and the central nuclei of amygdala (CeA). The present study indicated that VR significantly improved heat hyperalgesia which was exacerbated in PSL-Sedentary mice, and that a significant positive correlation was detected between total running distances after PSL-surgery and thermal withdrawal latency. The number of activated glutamate (Glu) neurons in the medal BA (medBA) was significantly increased in PSL-Runner mice, while those were increased in the lateral BA in sedentary mice. Furthermore, in all subdivisions of the CeA, the number of activated gamma-aminobutyric acid (GABA) neurons was dramatically increased in PSL-Sedentary mice, but these numbers were significantly decreased in PSL-Runner mice. In addition, a tracer experiment demonstrated a marked increase in activated Glu neurons in the medBA projecting into the nucleus accumbens lateral shell in runner mice. Thus, our results suggest that VR may not only produce suppression of the negative emotion such as fear and anxiety closely related with pain chronification, but also promote pleasant emotion and hypoalgesia. Therefore, we conclude that EIH effects may be produced, at least in part, via such plastic changes in the Amyg.

Chronic pain has been shown to depend on nociceptive sensitization in the spinal cord, and while multiple mechanisms involved in the initiation of plastic changes have been established, the molecular targets which maintain spinal nociceptive sensitization are still largely unknown. Building upon the established neurobiology underlying the maintenance of LTP in the hippocampus, this present study investigated the contributions of spinal atypical PKC isoforms PKCι/λ and PKM and their downstream targets (p62/GluA1 and NSF/GluA2 interactions, respectively) to the maintenance of spinal nociceptive sensitization in male and female rats. Pharmacological inhibition of atypical PKCs by ZIP reversed established allodynia produced by repeated intramuscular (i.m.) acidic saline injections in male animals only, replicating previously demonstrated sex differences. Inhibition of both PKCι/λ and downstream substrates p62/GluA1 resulted in male-specific reversals of i.m. acidic saline-induced allodynia, while female animals continued to display allodynia. Inhibition of NSF/GluA2, the downstream target to PKM, reversed allodynia induced by i.m. acidic saline in both sexes. Neither PKCι/λ, p62/GluA1 or NSF/GluA2 inhibition had any effect on formalin response for either sex.This study provides novel behavioural evidence for the male-specific role of PKCι/λ and downstream target p62/GluA1, highlighting the potential influence of ongoing afferent input. The sexually divergent pathways underlying persistent pain are shown here to converge at the interaction between NSF and the GluR2 subunit of the AMPA receptor. Though this interaction is thought to be downstream of PKM in males, these findings and previous work suggest that females may rely on a factor independent of atypical PKCs for the maintenance of spinal nociceptive sensitization.