Animal Studies

Recapitulating human disease pathophysiology using genetic animal models is a powerful approach to enable mechanistic understanding of genotype-phenotype relationships for drug development. NaV1.7 is a sodium channel expressed in the peripheral nervous system with strong human genetic validation as a pain target. Efforts to identify novel analgesics that are non-addictive, resulted in industry exploration of a class of sulfonamide compounds that bind to the fourth voltage-sensor domain of NaV1.7. Due to sequence differences in this region, sulfonamide blockers generally are potent on human but not rat NaV1.7 channels. To test sulfonamide-based chemical matter in rat models of pain, we generated a humanized NaV1.7 rat expressing a chimeric NaV1.7 protein containing the sulfonamide-binding site of the human gene sequence as a replacement for the equivalent rat sequence. Unexpectedly, upon transcription the human insert was spliced out, resulting in a premature stop codon. Using a validated antibody, NaV1.7 protein was confirmed to be lost in the brainstem, dorsal root ganglia (DRG), sciatic nerve and gastrointestinal tissue but not in nasal turbinates or olfactory bulb in rats homozygous for the knock-in allele (HOM-KI). HOM-KI rats exhibited normal intraepidermal nerve fiber density with reduced tetrodotoxin-sensitive current density and action potential firing in small diameter DRG neurons. HOM-KI rats did not exhibit nociceptive pain responses in hot plate or capsaicin-induced flinching assays and did not exhibit neuropathic pain responses following spinal nerve ligation. Consistent with expression of chimeric NaV1.7 in olfactory tissue, HOM-KI rats retained olfactory function. This new genetic model highlights the necessity of NaV1.7 for pain behavior in rats and indicates that sufficient inhibition of NaV1.7 in humans may reduce pain in neuropathic conditions. Due to preserved olfactory function, this rat model represents an alternative to global NaV1.7 knockout mice that require time-intensive hand feeding during early postnatal development.

Glutamate is a neurotransmitter present in most excitatory synapses in the nervous system. It also plays a key role in the spinal cord's physiological excitatory circuit and is involved in pathological neurotransmissions such as those observed in inflammatory and neuropathic pain conditions. The actions of glutamate are mediated by different types of ionotropic (iGluRs) and metabotropic (mGluRs) receptors. Although expressions of iGluRs are well studied, those of mGluRs are not fully elucidated in the spinal cord. In this study, we examined the expressions of mGluRs (mGluR1-8) and investigated which mGluR subtypes can modulate pain transmission in the dorsal horn of the spinal cord using an inflammatory pain model. Reverse transcription-polymerase chain reaction revealed that mGluR mRNAs, except for mGluR2 and 6 mRNAs, were detected in the spinal cord. Double labeling analysis, in situ hybridization histochemistry with immunohistochemistry, was used to examine the distribution of each mGluR in neurons or glial cells in the lamina I-II of the spinal dorsal horn. mGluR1, 5, and 7 were generally, and 4 and 8 were frequently, expressed in neurons. mGluR3 was expressed not only in neurons but also in oligodendrocytes. We next examined the distribution of mGluR4 and 8 were expressed in excitatory or inhibitory neurons. Both mGluR4 and 8 were preferentially expressed in inhibitory neurons rather than in excitatory neurons. Futhermore, intrathecal delivery of CPPG, an antagonist for mGluR 4 and 8, attenuated nocifensive behaviors and the increase in fos positive-excitatory neurons of the dorsal horn induced by intraplantar injection of formalin. These findings suggest that mGluR4 and 8, which are preferentially expressed in inhibitory neurons, may play roles in the modulation of pain transmission through mGluRs in the spinal dorsal horn.

Chronic pain is often linked to comorbidities such as anxiety and cognitive dysfunction, alterations that are reflected in brain plasticity in regions such as the prefrontal cortex and the limbic area. Despite the growing interest in pain-related cognitive deficits, little is known about the relationship between the emotional valence of the stimulus and the salience of its memory following painful injuries. We used the tibia fracture model of chronic pain in mice to determine whether pleasant and unpleasant odor location memories differ in their salience 7 weeks following the onset of the painful injury. Our results indicate that injured mice show a bias towards recalling unpleasant memories, thereby propagating the vicious cycle of chronic pain and negative affect. Next, we linked these behavioral differences to mechanisms of molecular plasticity by measuring the levels of global methylation and hydroxymethylation in the olfactory bulb. Compared to controls, global methylation levels were shown to be increased while hydroxymethylation levels were decreased in the olfactory bulb of injured mice, indicative of overall changes in DNA regulation machinery and the subsequent alterations in sensory systems.

Microglia are specialized brain-resident macrophages with important functions in health and disease. To improve our understanding of these cells, the research community needs genetic tools to identify and control them in a manner that distinguishes them from closely related cell-types. We have targeted the recently discovered microglia-specific gene to generate knock-in mice expressing EGFP (JAX#031823) or CreERT2 (JAX#031820) for the identification and manipulation of microglia, respectively. Genetic characterization of the locus and qPCR-based analysis demonstrate correct positioning of the transgenes and intact expression of endogenous in the knock-in mouse models. Immunofluorescence analysis further shows that parenchymal microglia, but not other brain macrophages, are completely and faithfully labeled in the EGFP-line at different time points of development. Flow cytometry indicates highly selective expression of EGFP in CD11bCD45lo microglia. Similarly, immunofluorescence and flow cytometry analyses using a Cre-dependent reporter mouse line demonstrate activity of CreERT2 primarily in microglia upon tamoxifen administration with the caveat of activity in leptomeningeal cells. Finally, flow cytometric analyses reveal absence of EGFP expression and minimal activity of CreERT2 in blood monocytes of the and lines, respectively. These new transgenic lines extend the microglia toolbox by providing the currently most specific genetic labeling and control over these cells in the myeloid compartment of mice. Tools that specifically label and manipulate only microglia are currently unavailable, but are critically needed to further our understanding of this cell type. Complementing and significantly extending recently introduced microglia-specific immunostaining methods that have quickly become a new standard in the field, we generated two mouse lines that label and control gene expression in microglia with high specificity and made them publicly available. Using these readily accessible mice, the research community will be able to study microglia biology with improved specificity.

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.