Animal Studies

The neuropeptide of calcitonin gene-related peptide (CGRP) plays critical roles in chronic pain, especially in migraine. Immunohistochemistry and in situ hybridization studies have shown that CGRP and its receptors are expressed in cortical areas including pain perception related prefrontal anterior cingulate cortex (ACC). However, less information is available for the functional roles of CGRP in cortical regions such as the ACC. Recent studies have consistently demonstrated that long-term potentiation (LTP) is a key cellular mechanism for chronic pain in the ACC. In the present study, we used 64-electrode array field recording system to investigate the effect of CGRP on excitatory transmission in the ACC. We found that CGRP induced potentiation of synaptic transmission in a dose-dependently manner (1, 10, 50, and 100 nM). CGRP also recruited inactive circuit in the ACC. An application of the calcitonin receptor-like receptor antagonist CGRP8-37 blocked CGRP-induced chemical LTP and the recruitment of inactive channels. CGRP-induced LTP was also blocked by NMDA receptor antagonist AP-5. Consistently, application of CGRP increased NMDA receptor mediated excitatory postsynaptic currents (EPSCs). Finally, we found that CGRP-induced LTP requires activation of calcium-stimulated adenylyl cyclase subtype 1 (AC1) and PKA. Genetic deletion of AC1 using AC1-/- mice, an AC1 inhibitor NB001 or a PKA inhibitor KT5720 all reduced or blocked CGRP induced potentiation. Our results provide direct evidence that CGRP may contribute to synaptic potentiation in important physiological and pathological conditions in the ACC, an AC1 inhibitor NB001 may be beneficial for the treatment of chronic headache.

Despite enormous investment in research and development of novel treatments, there remains a lack of predictable, effective, and safe therapeutics for human chronic neuropathic pain (NP) afflictions. NP continues to increase among the population and treatments remain a major unmet public health care need. In recent years, numerous costly (time and money) failures have occurred attempting to translate successful animal pain model results, typically using rodents, to human clinical trials. These continued failures point to the essential need for better animal models of human pain conditions. To address this challenge, we have previously developed a peripheral neuritis trauma (PNT) model of chronic pain induced by a proximal sciatic nerve irritation in pigs, which have a body size, metabolism, skin structure, and cutaneous innervation more similar to humans. Here, we set out to determine the extent that the PNT model presents with cutaneous neuropathologies consistent with those associated with human chronic NP afflictions. Exactly as is performed in human skin biopsies, extensive quantitative multi-molecular immunofluorescence analyses of porcine skin biopsies were performed to assess cutaneous innervation and skin structure. ChemoMorphometric Analysis (CMA) results demonstrated a significant reduction in small caliber intraepidermal nerve fiber (IENF) innervation, altered dermal vascular innervation, and aberrant analgesic/algesic neurochemical properties among epidermal keratinocytes, which are implicated in modulating sensory innervation. These comprehensive pathologic changes very closely resemble those observed from CMA of human skin biopsies collected from NP afflictions. The results indicate that the porcine PNT model is more appropriate for translational NP research compared with commonly utilized rodent models. Because the PNT model creates cutaneous innervation and keratinocyte immunolabeling alterations consistent with human NP conditions, use of this animal model for NP testing and treatment response characteristics will likely provide more realistic results to direct successful translation to humans.

Neurons of the peripheral nervous system are able to regenerate injured axons, a process requiring significant cellular resources to establish and maintain long-distance growth. Genetic activation of mTORC1, a potent regulator of cellular metabolism and protein translation, improves axon regeneration of peripheral neurons by an unresolved mechanism. To gain insight into this process, we activated mTORC1 signaling in mouse nociceptors via genetic deletion of its negative regulator Tsc2. Perinatal deletion of Tsc2 in nociceptors enhanced initial axon growth after sciatic nerve crush, however by three days post-injury axon elongation rate became similar to controls. mTORC1 inhibition prior to nerve injury was required to suppress the enhanced axon growth. Gene expression analysis in purified nociceptors revealed that Tsc2-deficient nociceptors had increased activity of regeneration-associated transcription factors (RATFs), including cJun and Atf3, in the absence of injury. Additionally, nociceptor deletion of Tsc2 activated satellite glial cells and macrophages in the dorsal root ganglia (DRG) in a similar manner to nerve injury. Surprisingly, these changes improved axon length but not percentage of initiating axons in dissociated cultures. The pro-regenerative environment in naïve DRG was recapitulated by AAV8-mediated deletion of Tsc2 in adult mice, suggesting that this phenotype does not result from a developmental effect. Consistently, AAV8-mediated Tsc2 deletion did not improve behavioral recovery after a sciatic nerve crush injury despite initially enhanced axon growth. Together, these data show that neuronal mTORC1 activation induces an incomplete pro-regenerative environment in the DRG that improves initial but not later axon growth after nerve injury. Long distance axon regeneration poses a significant hurdle to recovery following nervous system injury. Increased mTORC1 signaling improves axon regeneration, however the underlying mechanisms are incompletely understood. We activated neuronal mTORC1 signaling by genetically deleting Tsc2 in Nav1.8-positive neurons perinatally or by AAV8-mediated viral infection in adult mice and observed improved short- but not long-term axon regeneration after sciatic nerve injury. We suggest that Tsc2 deletion promotes initial but not later peripheral axon regeneration by upregulating expression of neuronal pro-regenerative genes and activating non-neuronal responses in the surrounding environment. Activating mTORC1 signaling in peripheral neurons may provide therapeutic benefit in circumstances with poor initial growth such as after spinal cord injury to the dorsal column or peripheral nerve repair.

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.

Transcutaneous electrical nerve stimulation (TENS) promotes antinociception by activating the descending pain modulation pathway and consequently releasing endogenous analgesic substances. In addition, recent studies have shown that the endocannabinoid system controls pain. Thus, the present study investigated the involvement of the endocannabinoid system in TENS-induced antinociception of cancer pain using a cancer pain model induced by intraplantar (i.pl.) injections of Ehrlich tumor cells in male Swiss mice. Low- and high-frequency TENS was applied for 20 min to the mice's paws, and to investigate the involvement of the endocannabinoid system were used the N-(peperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pitazole-3-carboixamide (AM251), a cannabinoid CB receptor antagonist and (5Z,8Z,11Z,14Z)-5,8,11,14-eicosatetraenyl-methylester phosphonofluoridic acid (MAFP), an inhibitor of the endocannabinoid metabolizing enzyme fatty acid amide hydrolase, injected by via i.pl., intrathecal (i.t.), and intra-dorsolateral periaqueductal gray matter (i.dl.PAG). Furthermore, liquid chromatography-tandem mass spectrometry, western blot, and immunofluorescence assays were used to evaluate the endocannabinoid anandamide (AEA) levels, cannabinoid CB receptor protein levels, and cannabinoid CB receptor immunoreactivity, respectively. Low- and high-frequency TENS reduced the mechanical allodynia induced by Ehrlich tumor cells and this effect was reversed by AM251 and potentiated by MAFP at the peripheral and central levels. In addition, TENS increased the AEA levels and the cannabinoid CB receptor protein levels and immunoreactivity in the paw, spinal cord, and dorsolateral PAG. These results suggest that low- and high-frequency TENS is effective in controlling cancer pain, and the endocannabinoid system is involved in this effect at both the peripheral and central levels. Perspective: TENS is a non-pharmacological strategy that may be used to control cancer pain. Identification of a new mechanism involved in its analgesic effect could lead to the development of clinical studies as well as an increase in its application, lessening the need for pharmacological treatments.

Visceral sensory neurons encode distinct sensations from healthy organs and initiate pain states that are resistant to common analgesics. Transcriptome analysis is transforming our understanding of sensory neuron subtypes but has generally focused on somatic sensory neurons or the total population of neurons in which visceral neurons form the minority. Our aim was to define transcripts specifically expressed by sacral visceral sensory neurons, as a step towards understanding the unique biology of these neurons and potentially leading to identification of new analgesic targets for pelvic visceral pain. Our strategy was to identify genes differentially expressed between sacral dorsal root ganglia (DRG) that include somatic neurons and sacral visceral neurons, and adjacent lumbar DRG that comprise exclusively of somatic sensory neurons. This was performed in adult and E18.5 male and female mice. By developing a method to restrict analyses to nociceptive Trpv1 neurons, a larger group of genes were detected as differentially expressed between spinal levels. We identified many novel genes that had not previously been associated with pelvic visceral sensation or nociception. Limited sex differences were detected across the transcriptome of sensory ganglia, but more were revealed in sacral levels and especially in Trpv1 nociceptive neurons. These data will facilitate development of new tools to modify mature and developing sensory neurons and nociceptive pathways. In this study of mouse dorsal root ganglia, we have identified numerous features of sensory neurons that vary between lumbar and sacral spinal levels and that are potentially involved in unique physiology and pathophysiology of visceral sensation and pain. We further identify maturational components of this sacral visceral transcriptome by comparing data from embryonic and adult mice. There are limited sex differences across the transcriptome of embryonic or adult sensory ganglia, but in adults these can be revealed in sacral levels and especially in Trpv1 nociceptive neurons. These data sets will encourage identification of new tools to modify mature or developing sensory neurons and adult nociceptive pathways.