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Neuropathy is major source of chronic pain that can be caused by mechanically or chemically induced nerve injury. Intraplantar formalin injection produces local necrosis over a two-week period and has been used to model neuropathy in rats. To determine whether neuropathy alters dopamine (DA) receptor responsiveness in mesolimbic brain regions, we examined dopamine D-like and D-like receptor (DR) signaling and expression in male rats 14 days after bilateral intraplantar formalin injections into both rear paws. DR-mediated G-protein activation and expression of the DR long, but not short, isoform were reduced in nucleus accumbens (NAc) core, but not in NAc shell, caudate-putamen or ventral tegmental area of formalin-compared to saline-treated rats. In addition, DR-stimulated adenylyl cyclase activity was also reduced in NAc core, but not in NAc shell or prefrontal cortex, of formalin-treated rats, whereas DR expression was unaffected. Other proteins involved in dopamine neurotransmission, including dopamine uptake transporter and tyrosine hydroxylase, were unaffected by formalin treatment. In behavioral tests, the potency of a DR agonist to suppress intracranial self-stimulation (ICSS) was decreased in formalin-treated rats, whereas DR agonist effects were not altered. The combination of reduced DR expression and signaling in NAc core with reduced suppression of ICSS responding by a DR agonist suggest a reduction in D autoreceptor function. Altogether, these results indicate that intraplantar formalin produces attenuation of highly specific DA receptor signaling processes in NAc core of male rats and suggest the development of a neuropathy-induced allostatic state in both pre- and post-synaptic DA receptor function.
Learn More >Non-steroidal anti-inflammatory drugs (NSAIDs), commonly known as COX-1/COX-2 inhibitors, can be effective in treating mild to moderate migraine headache. However, the mechanism by which these drugs act in migraine is not known, nor is the specific contribution of COX-1 versus COX-2 known. We sought to investigate these unknowns using celecoxib, which selectively inhibits the enzymatic activity of COX-2, by determining its effects on several migraine-associated vascular and inflammatory events. Using in vivo two-photon microscopy, we determined intraperitoneal celecoxib effects on CSD-induced blood vessel responses, plasma protein extravasation, and immune cell activation in the dura and pia of mice and rats. Compared to vehicle (control group), celecoxib reduced significantly CSD-induced dilatation of dural arteries and activation of dural and pial macrophages but not dilatation or constriction of pial arteries and veins, or the occurrence of plasma protein extravasation. Collectively, these findings suggest that a mechanism by which celecoxib-mediated COX-2 inhibition might ease the intensity of migraine headache and potentially terminate an attack is by attenuating dural macrophages activation and arterial dilatation outside the blood brain barrier (BBB), and pial macrophages activation inside the BBB.
Learn More >Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.
Learn More >The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.
Learn More >To develop non-opioid therapies for postoperative incisional pain, we must understand its underlying molecular mechanisms. In this study, we assessed global gene expression changes in dorsal root ganglia neurons in a model of incisional pain to identify pertinent molecular pathways. Male, Sprague-Dawley rats underwent infiltration of 1% capsaicin or vehicle into the plantar hind paw (n = 6-9/group) 30 min before plantar incision. Twenty-four hours after incision or sham (control) surgery, lumbar L4-L6 dorsal root ganglias were collected from rats pretreated with vehicle or capsaicin. RNA was isolated and sequenced by next generation sequencing. The genes were then annotated to functional networks using a knowledge-based database, Ingenuity Pathway Analysis. In rats pretreated with vehicle, plantar incision caused robust hyperalgesia, up-regulated 36 genes and downregulated 90 genes in dorsal root ganglias one day after plantar incision. Capsaicin pretreatment attenuated pain behaviors, caused localized denervation of the dermis and epidermis, and prevented the incision-induced changes in 99 of 126 genes. The pathway analyses showed altered gene networks related to increased pro-inflammatory and decreased anti-inflammatory responses in dorsal root ganglias. Insulin-like growth factor signaling was identified as one of the major gene networks involved in the development of incisional pain. Expression of insulin-like growth factor -2 and IGFBP6 in dorsal root ganglia were independently validated with quantitative real-time polymerase chain reaction. We discovered a distinct subset of dorsal root ganglia genes and three key signaling pathways that are altered 24 h after plantar incision but are unchanged when incision was made after capsaicin infiltration in the skin. Further exploration of molecular mechanisms of incisional pain may yield novel therapeutic targets.
Learn More >Activation of glial cells has been shown to play an important role in chronic itch. However, whether glial cells play an important role in the development of psoriasis-induced chronic itch has not been fully elucidated. This study investigated the role of spinal glial cells in psoriasis-induced chronic itch. To develop a mouse model of psoriasis-induce chronic itch, we used 5% imiquimod cream to receive a daily topical application on the shaved back skin for seven consecutive days. The results showed that the expression of microglial marker ionized calcium binding adaptor molecule-1 was significantly increased after 5% imiquimod treatment in cervical spinal cord dorsal horn (C3-C4), and the intrathecal microglial inhibitor minocycline or PLX5622 diet suppressed both spontaneous itch and microglial activation. Furthermore, we found that the number of scratches and alloknesis score in female mice was significantly greater than in male mice after 5% imiquimod treatment. Our results indicate that microglia mediate chronic psoriatic itch induced by imiquimod.
Learn More >Morphine is the most commonly used drug for treating physical and psychological suffering caused by advanced cancer. Although morphine is known to elicit multiple supraspinal analgesic effects, its behavioral correlates with respect to the whole brain metabolic activity during cancer-induced bone pain have not been elucidated. We injected 4T1 mouse breast cancer cells into the left femur bone marrow cavity of BALB/c mice. All mice developed limb use deficits, mechanical allodynia, and hypersensitivity to cold, which were effectively suppressed with morphine. Serial 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) was performed for each mouse before cancer induction (0 d), after cancer-induced bone pain was established (14 d), and during effective morphine treatment (16 d). The longitudinal FDG-PET imaging analysis demonstrated that cancer-induced bone pain increased glucose uptake in the insular cortex and hypothalamus and decreased the activity of the retrosplenial cortex. Morphine reversed the activation of the insular cortex and hypothalamus. Furthermore, morphine activated the amygdala and rostral ventromedial medulla and suppressed the activity of anterior cingulate cortex. Our findings of hypothalamic and insular cortical activation support the hypothesis that cancer-induced bone pain has strong inflammatory and affective components in freely moving animals. Morphine may provide descending inhibitory and facilitatory actions in the treatment of cancer-induced bone pain in a clinical setting.
Learn More >The glutamate type 1 transporter (GLT1) plays a major role in glutamate homeostasis in the brain. Although alterations of GLT1 activity have been linked to persistent pain, the significance of these changes is poorly understood. Focusing on the rostral ventromedial medulla, a key site in pain modulation, we examined the expression and function of GLT1 and related transcription factor kappa B-motif binding phosphoprotein (KBBP) in rats after adjuvant-induced hind paw inflammation.
Learn More >Transient receptor potential melastatin member 8 (TRPM8) is a Ca-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo-electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP, and Ca and in complex with the menthol analog WS-12 and PIP Our structures reveal the binding sites for cooling agonists and PIP in TRPM8. Notably, PIP binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.
Learn More >Human and animal studies suggest that both traumatic nerve injury and toxic challenge with chemotherapeutic agents involves the reorganization of neural circuits in the brain. However, there have been no prospective studies, human or animal, using magnetic resonance imaging (MRI) to identify changes in brain neural circuitry that accompany the development of chemotherapy-induced neuropathic pain (i.e. within days following cessation of chemotherapy treatment and without the confound cancer). To this end, different MRI protocols were used to ascertain whether a reorganization of brain neural circuits is observed in otherwise normal rats exposed to the taxane chemotherapeutic agent paclitaxel. We conducted an imaging study to evaluate the impact of a well-established paclitaxel dosing regimen, validated to induce allodynia in control rats within eight days of treatment, on brain neural circuitry. Rats received either paclitaxel (2 mg/kg/day i.p; cumulative dose of 8 mg/kg) or its vehicle four times on alternate days (i.e. day 0, 2, 4, 6). Following the cessation of treatments (i.e. on day 8), all rats were tested for responsiveness to cold followed by diffusion weighted magnetic resonance imaging and assessment of resting state functional connectivity. Imaging data were analyzed using a 3D MRI rat with 173 segmented and annotated brain areas. Paclitaxel-treated rats were more sensitive to a cold stimulus compared to controls. Diffusion weighted imaging identified brain areas involved in the emotional and motivational response to chronic pain that were impacted by paclitaxel treatment. Affected brain regions included the prefrontal cortex, amygdala, hippocampus, hypothalamus and the striatum/nucleus accumbens. This putative reorganization of gray matter microarchitecture formed a continuum of brain areas stretching from the basal medial/lateral forebrain to the midbrain. Resting state functional connectivity showed reorganization between the periaqueductal gray, a key node in nociceptive neural circuitry, and connections to the brainstem. Our results, employing different imaging modalities to assess the central nervous system effects of chemotherapy, fit the theory that chronic pain is regulated by emotion and motivation and influences activity in the periaqueductal gray and brainstem to modulate pain perception.
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