Animal Studies

Brain-derived neurotrophic factor (BDNF) signaling through its cognate receptor, TrkB, is a well-known promoter of synaptic plasticity at nociceptive synapses in the dorsal horn of the spinal cord. Existing evidence suggests that BDNF/TrkB signaling in neuropathic pain is sex dependent. We tested the hypothesis that the effects of BDNF/TrkB signaling in hyperalgesic priming might also be sexually dimorphic. Using the incision postsurgical pain model in male mice, we show that BDNF sequestration with TrkB-Fc administered at the time of surgery blocks the initiation and maintenance of hyperalgesic priming. However, when BDNF signaling was blocked prior to the precipitation of hyperalgesic priming with prostaglandin E (PGE), priming was not reversed. This result is in contrast to our findings in male mice with interleukin-6 (IL6) as the priming stimulus where TrkB-Fc was effective in reversing the maintenance of hyperalgesic priming. Furthermore, in IL6-induced hyperalgesic priming, the BDNF sequestering agent, TrkB-fc, was effective in reversing the maintenance of hyperalgesic priming in male mice; however, when this experiment was conducted in female mice, we did not observe any effect of TrkB-fc. This markedly sexual dimorphic effect in mice is consistent with recent studies showing a similar effect in neuropathic pain models. We tested whether the sexual dimorphic role for BDNF was consistent across species. Importantly, we find that this sexual dimorphism does not occur in rats where TrkB-fc reverses hyperalgesic priming fully in both sexes. Finally, to determine the source of BDNF in hyperalgesic priming in mice, we used transgenic mice (  ×  mice) with BDNF eliminated from microglia. From these experiments we conclude that BDNF from microglia does not contribute to hyperalgesic priming and that the key source of BDNF for hyperalgesic priming is likely nociceptors in the dorsal root ganglion. These experiments demonstrate the importance of testing mechanistic hypotheses in both sexes in multiple species to gain insight into complex biology underlying chronic pain.

Pain is one of the most common and distressing symptoms suffered by patients with progression of bone cancer; however, the mechanisms responsible for hyperalgesia are not well understood. The purpose of our current study was to determine contributions of the sensory signaling pathways of inflammatory tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and downstream transient receptor potential ankyrin 1 (TRPA1) to neuropathic pain induced by bone cancer. We further determined if influencing these pathways can improve bone cancer pain. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats to induce mechanical and thermal hyperalgesia. ELISA and western blot analysis were used to examine 1) the levels of TNF-α and IL-6 in dorsal root ganglion (DRG); and 2) protein expression of TNF-α and IL-6 receptors (TNFR1 and IL-6R) and TRPA1 as well as intracellular signals (p38-MAPK and JNK). TNF-α and IL-6 were elevated in the DRG of bone cancer rats and expression of TNFR1, IL-6R and TRPA1 was upregulated. In addition, inhibition of TNFR1 and IL-6R alleviated mechanical and thermal hyperalgesia in bone cancer rats, accompanied with downregulated TRPA1 and p38-MAPK and JNK. We revealed specific signaling pathways leading to neuropathic pain during the development of bone cancer, including TNF-α-TRPA1 and IL-6-TRPA1 signal pathways. Overall, our data suggest that blocking these signals is beneficial to alleviate bone cancer pain.

Peripheral nerve injury can lead to remodeling of brain circuits, and this can cause chronification of pain. We have recently reported that male mice subjected to spared injury of the sciatic nerve undergo changes in the function of the medial prefrontal cortex (mPFC) that culminate in reduced output of layer 5 pyramidal cells. More recently, we have shown that this is mediated by alterations in synaptic inputs from the basolateral amygdala (BLA) into GABAergic interneurons in the mPFC. Optogenetic inhibition of these inputs reversed mechanical allodynia and thermal hyperalgesia in male mice. It is known that the processing of pain signals can exhibit marked sex differences. We therefore tested whether the dysregulation of BLA to mPFC signaling is equally altered in female mice. Injection of AAV-Arch3.0 constructs into the BLA followed by implantation of a fiberoptic cannula into the mPFC in sham and SNI operated female mice was carried out, and pain behavioral responses were measured in response to yellow light mediated activation of this inhibitory opsin. Our data reveal that Arch3.0 activation leads to a marked increase in paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively. However, we did not observe nerve injury-induced changes in mPFC layer 5 pyramidal cell output in female mice. Hence, the observed light-induced analgesic effects may be due to compensation for dysregulated neuronal circuits downstream of the mPFC.

Neuropathic pain is difficult to treat and remains a major clinical challenge worldwide. While the mechanisms which underlie the development of neuropathic pain are incompletely understood, interferon signaling by the immune system is known to play a role. Here, we demonstrate a role for IFNβ in attenuating mechanical allodynia induced by the spared nerve injury in mice. The results show that intrathecal administration of IFNβ (dosages up to 5000U) produces significant, transient, and dose-dependent attenuation of mechanical allodynia without observable effects on motor activity or feeding behavior, as is common with IFN administration. This analgesic effect is mediated by the ubiquitin-like protein ISG15, which is potently induced within the spinal cord following intrathecal delivery of IFNβ. Both free and conjugated ISG15 are elevated following IFNβ treatment, and this effect is increased in UBP43 mice lacking a key deconjugating enzyme. The IFNβ-mediated analgesia reduces MAPK signaling activation following nerve injury, and this effect requires induction of ISG15. These findings highlight a new role for IFNβ, ISG15 and MAPK signaling in immunomodulation of neuropathic pain and may lead to new therapeutic possibilities. Perspective: Neuropathic pain is frequently intractable in a clinical setting, and new treatment options are needed. Characterizing the anti-nociceptive potential of IFNβ and the associated downstream signaling pathways in preclinical models may lead to the development of new therapeutic options for debilitating neuropathies.

Inflammatory bowel disease (IBD), mainly comprising Crohn's disease and ulcerative colitis, is characterized by chronic inflammation in the digestive tract. Approximately 60% of the patients experience abdominal pain during acute IBD episodes, which severely impairs their quality of life. Both peripheral and central mechanisms are thought to be involved in such abdominal pain in IBD. Although much attention has been paid to peripheral mechanisms of abdominal pain in IBD pathophysiology, the involvement of supraspinal mechanisms remains poorly understood. To address this issue, we investigated regional brain activity in response to colorectal distension (CRD) in normal and IBD model rats using voxel-based statistical analysis of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) PET imaging. The rat IBD model was generated by colorectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS), a chemical compound widely used to generate colitis. Tissue damage and inflammation were induced and dynamically changed with time after TNBS injection, while CRD-induced visceromotor response showed corresponding temporal changes. We found that characteristic brain activations were observed in response to visceral innocuous and noxious CRD and supraspinal nociception shared some physiological sensory pathway. Moreover, widespread brain regions were activated, and the functional coupling between the central medial thalamic nucleus (CMT) and anterior cingulate cortex (ACC) was enhanced after noxious CRD in IBD model of rats. Increased brain activity in the anterior insular cortex (aINS) and ACC positively correlated with noxious CRD-induced pain severity in normal and IBD rats respectively. These findings suggest that the pain matrix was shifted following persistent colonic inflammation, and thalamocortical sensitization in the pathway from CMT to ACC might be a central mechanism of the visceral hyperalgesia in IBD pathophysiology.

Endogenous Pain Modulation (EPM) impairment is a significant contributor to chronic pain. Conditioned pain modulation (CPM) testing assesses EPM function. Osteoarthritic (OA) dogs are good translational models, but CPM has not been explored. Our aim was to assess EPM impairment in OA dogs compared to controls using CPM. We hypothesized that CPM testing would demonstrate EPM impairment in OA dogs compared to controls. Dogs with stifle/hip OA and demographically-matched controls were recruited. The pre-conditioning test stimulus, using mechanical/thermal quantitative sensory testing (MQST or TQST), were performed at the metatarsus. A 22N blunt probe (conditioning stimulus) was applied to the contralateral antebrachium for 2 minutes, followed by MQST or TQST (post-conditioning test stimulus). The threshold changes from pre to post-conditioning (∆MQST and ∆TQST) were compared between OA and control dogs. Twenty-four client-owned dogs (OA, n = 11; controls, n = 13) were recruited. The ∆MQST(p < 0.001) and ∆TQST(p < 0.001) increased in control dogs but not OA dogs (∆MQST p = 0.65; ∆TQST p = 0.76). Both ∆MQST(p < 0.001) and ∆TQST(p < 0.001) were different between the OA and control groups. These are the first data showing that EPM impairment is associated with canine OA pain. The spontaneous OA dog model may be used to test drugs that normalize EPM function.

Descending control from supraspinal neuronal networks onto spinal cord neurons can modulate nociceptionEndogenous opioids in these brain circuits participate in pain modulationA differential opioidergic role for brain nuclei involved in supraspinal pain modulation has not been previously reported WHAT THIS ARTICLE TELLS US THAT IS NEW: In vivo electrophysiologic recordings from the dorsal horn of the spinal cord in male rats reveal differential effects of morphine at the anterior cingulate cortex, right amygdala, and the ventromedial medulla on evoked pain responsesThese data differentiate supraspinal opioid circuit regulation of spinal nociceptive processing and suggest that the regulation of sensory and affective components of pain are likely separate BACKGROUND:: The anterior cingulate cortex and central nucleus of the amygdala connect widely with brainstem nuclei involved in descending modulation, including the rostral ventromedial medulla. Endogenous opioids in these circuits participate in pain modulation. The hypothesis was that a differential opioidergic role for the brain nuclei listed in regulation of spinal neuronal responses because separable effects on pain behaviors in awake animals were previously observed.

C-LTMRs are known to convey affective aspects of touch and to modulate injury-induced pain in humans and mice. However, a role for these neurons in temperature sensation has been suggested, but not fully demonstrated. Here, we report that deletion of C-low-threshold mechanoreceptor (C-LTMR)-expressed bhlha9 causes impaired thermotaxis behavior and exacerbated formalin-evoked pain in male, but not female, mice. Positive modulators of GABA receptors failed to relieve inflammatory formalin pain and failed to decrease the frequency of spontaneous excitatory post-synaptic currents (sEPSCs) selectively in bhlha9 knockout (KO) males. This could be explained by a drastic change in the GABA content of lamina II inner inhibitory interneurons contacting C-LTMR central terminals. Finally, C-LTMR-specific deep RNA sequencing revealed more genes differentially expressed in male than in female bhlha9 KO C-LTMRs. Our data consolidate the role of C-LTMRs in modulation of formalin pain and provide in vivo evidence of their role in the discriminative aspects of temperature sensation.