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Luminal distention and abdominal pain are major clinical hallmarks of obstructive bowel disorders and functional bowel disorders linked to gut dysbiosis. Our recent studies found that chronic lumen distention increased visceral sensitivity and decreased abundance of gut commensal Lactobacillus reuteri in a rodent model of partial colon obstruction (OB). To establish causation, we performed precision microbial therapy to assess whether recolonization of L. reuteri prevents visceral hypersensitivity in lumen distention, and if so, to identify the gut-microbiota mechanism. Lumen distention was induced in Sprague-Dawley rats by implanting an obstruction band in the distal colon for up to 7 days. L. reuteri strains or vehicle were gavage ingested 1X10 CFU/g daily starting 2 days prior to obstruction. L. reuteri rat strains that were able to recolonize obstructed colon significantly improved food intake and body weight in OB rats, and attenuated referred visceral hyperalgesia measured by the withdrawal response to von Frey filament applications to the abdomen. Mechanistically, L. reuteri treatment attenuated hyper-excitability of the dorsal root ganglia neurons projecting to the distended colon by promoting opioid receptor function in affected tissues. The expression of µ, δ, and κ opioid receptors was significantly downregulated in colonic muscularis externae and sensory neurons in OB rats. However, L. reuteri treatment prevented the loss of opioid receptors. Further, administration of peripheral opioid receptor antagonist naloxone methiodide abolished the analgesic effect of L. reuteri in OB. In conclusion, precision L. reuteri therapy prevents lumen distention-associated visceral hypersensitivity by local bacterial induction of opioid receptors.
Learn More >Acute pain that persists for a few days is associated with a reduction in patients' quality of life. Orofacial persistent pain promotes psychological disorders such as anxiety, impairs daily essential activities such as eating, and results in decreased social interaction. Here, we investigated whether rats subjected to orofacial formalin injection or intraoral incision surgery display persistent facial heat hyperalgesia, ongoing pain, anxiety-like behavior and changes in ultrasonic vocalization. Orofacial formalin injection or intraoral incision caused facial heat hyperalgesia for 3 days compared to saline-injected and sham animals. Additionally, both experimental groups showed a reduction in the number of entries and in the time spent in the open arms in the elevated plus maze test on day 3, suggesting that anxiety-like behavior developed as a consequence of persistent pain. At this time point, both groups also displayed a reduction in the number of 50-kHz calls, specifically in the flat subtype, which suggests a decrease in social communication. Moreover, on day 3 after surgery, systemic morphine produced robust conditioned place preference in rats subjected to intraoral incision compared to sham, and the former group also presented increased spontaneous facial grooming, revealing the presence of ongoing pain. Finally, western blot and immunohistochemistry analysis showed a reduction in tyrosine hydroxylase expression in the nucleus accumbens (NAc), which may reflect a decrease in mesolimbic dopaminergic activity. Altogether, the results demonstrate that acute orofacial pain causes prolonged changes in behavioral and affective pain components, which may be related to dopaminergic changes in the NAc.
Learn More >While dipyrone is a widely used analgesic, its mechanism of action is not completely understood. Recently we have reported that the dipyrone metabolite 4-aminoantipirine (4-AA) reduces prostaglandin E (PGE )-induced pain-related behaviour through the cannabinoid type 1 (CB1) receptor. Here, we ascertained, in naive and PGE -induced "inflamed" conditions, both in vivo and in vitro, the molecular mechanisms involved in the 4-AA-induced analgesic effects.
Learn More >PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.
Learn More >Activation of kappa opioid receptor (KOR) produces analgesia, antipruritic effect, sedation and dysphoria. To characterize neuroanatomy of KOR at high resolutions and circumvent issues of specificity of KOR antibodies, we generated a knock-in mouse line expressing KOR fused at the C-terminus with the fluorescent protein tdTomato (KtdT). The selective KOR agonist U50,488H caused anti-scratch effect and hypolocomotion, indicating intact KOR neuronal circuitries. Clearing of brains with CLARITY revealed 3-dimensional (3-D) images of distribution of KOR, and any G protein-coupled receptors, for the first time. 3-D brain images of KtdT and immunohistochemistry (IHC) on brain sections with antibodies against tdTomato show similar distribution to that of autoradiography of [H]U69,593 binding to KOR in wildtype mice. KtdT was observed in regions involved in reward and aversion, pain modulation and neuroendocrine regulation. KOR is present in several areas with unknown roles, including the claustrum, dorsal endopiriform nucleus, paraventricular nucleus of the thalamus, lateral habenula and substantia nigra pars reticulata (SNr), which are discussed. Prominent KtdT-containing fibers were observed to project from caudate putamen (CP) and nucleus accumbens (ACB) to substantia innominata (SI) and SNr. Double IHC revealed co-localization of KtdT with tyrosine hydroxylase (TH) in brain regions, including CP, ACB and ventral tegmental area (VTA). KOR was visualized at the cellular level, such as co-localization with TH and agonist-induced KOR translocation into intracellular space in some VTA neurons. These mice thus represent a powerful and heretofore unparalleled tool for neuroanatomy of KOR at both the 3-D and cellular levels. A combination of tagging KOR with tdTomato and tissue clearing with CLARITY enables 3-D mouse brain imaging of KOR, or any G protein-coupled receptors, for the first time. This approach reveals prominent KOR-expressing fiber bundles from caudate putamen and nucleus accumbens to substantia nigra pars reticulata and allows visualization of the KOR at the cellular level, including co-localization with TH and agonist-induced KOR translocation in some neurons. Regions expressing moderate to high KOR, but with no known functions, are highlighted and discussed, including claustrum, dorsal endopiriform nucleus, paraventricular nucleus of the thalamus and lateral habenula. The mouse line will be a valuable tool for investigation of KOR neurobiology. This approach paves ways for future similar studies.
Learn More >Individuals with spinal cord injury (SCI) often develop debilitating neuropathic pain, which may be driven by neuronal damage and neuroinflammation. We have previously demonstrated that treatment using 670 nm (red) light irradiation alters microglia/macrophage responses and alleviates mechanical hypersensitivity at 7-days post-injury. Here, we investigated the effect of red-light on the development of mechanical hypersensitivity, neuronal markers, and glial response in the subacute stage (days 1-7) following SCI. Wistar rats were subjected to a mild T10 hemi-contusion SCI or sham surgery followed by daily red-light treatment (30 min/day; 670 nm LED; 35 mW/cm2) or sham treatment. Mechanical sensitivity of the rat dorsum was assessed from 1-day post-injury and repeated every second day. Spinal cords were collected at 1, 3, 5 and 7-days post injury for analysis of myelination, neurofilament protein NF200 expression, neuronal cell death, reactive astrocytes (GFAP+ cells), interleukin1β (IL1β) expression, and inducible nitric oxide synthase (iNOS) production in IBA1+ microglia/macrophages. Red-light treatment significantly reduced the cumulative mechanical sensitivity and the hypersensitivity incidence following SCI. This effect was accompanied by significantly reduced neuronal cell death, reduced astrocyte activation and reduced iNOS expression in IBA1+ cells at the level of the injury. However, myelin and NF200 immunoreactivity and IL1β expression in GFAP+ and IBA1+ cells were not altered by red-light treatment. Thus, red-light therapy may represent a useful non-pharmacological approach for treating pain during the subacute period after SCI by decreasing neuronal loss and modulating the inflammatory glial response.
Learn More >Tissue injury and inflammation may result in chronic pain, a severe debilitating disease that is associated with great impairment of quality of life. An increasing body of evidence indicates that members of the Rab family of small GTPases contribute to pain processing; however, their specific functions remain poorly understood. Here, we found using immunofluorescence staining and in situ hybridization that the small GTPase Rab27a is highly expressed in sensory neurons and in the superficial dorsal horn of the spinal cord of mice. Rab27a mutant mice, which carry a single-nucleotide missense mutation of Rab27a leading to the expression of a nonfunctional protein, show reduced mechanical hyperalgesia and spontaneous pain behavior in inflammatory pain models, while their responses to acute noxious mechanical and thermal stimuli is not affected. Our study uncovers a previously unrecognized function of Rab27a in the processing of persistent inflammatory pain in mice.
Learn More >Painful peripheral neuropathy is a common adverse effect of paclitaxel (PTX) treatment. To analyze the contribution of transient receptor potential vanilloid 1 (TRPV1) in the development of PTX-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia, TRPV1 expression in the rat spinal cord was analyzed after intraperitoneal administration of 2 and 4 mg/kg PTX. PTX treatment increased the expression of TRPV1 protein in the spinal cord. Immunohistochemistry showed that PTX (4 mg/kg) treatment increased TRPV1 protein expression in the superficial layers of the spinal dorsal horn 14 days after treatment. Behavioral assessment using the paw withdrawal response showed that PTX-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia after 14 days was significantly inhibited by oral or intrathecal administration of the TRPV1 antagonist AMG9810. We found that intrathecal administration of small interfering RNA (siRNA) to knock down TRPV1 protein expression in the spinal cord significantly decreased PTX-induced mechanical allodynia/hyperalgesia and thermal hyperalgesia. Together, these results demonstrate that TRPV1 receptor expression in spinal cord contributes, at least in part, to the development of PTX-induced painful peripheral neuropathy. TRPV1 receptor antagonists may be useful in the prevention and treatment of PTX-induced peripheral neuropathic pain.
Learn More >Recently, oxytocin (OT) has been studied as a potential modulator of endogenous analgesia by acting upon pain circuits at the spinal cord and supraspinal levels. Yet the detailed action mechanisms of OT are still undetermined. The present study aimed to evaluate the action of OT in the spinal cord dorsal horn network under nociceptive-like conditions induced by the activation of the N-methyl-d-aspartate (NMDA) receptor and formalin injection, using calcium imaging techniques. Results demonstrate that the spontaneous Ca-dependent activity of the dorsal horn cells was scarce, and the coactivity of cells was mainly absent. When NMDA was applied, high rates of activity and coactivity occurred in the dorsal horn cells; these rates of high activity mimicked the activity dynamics evoked by a neuropathic pain condition. In addition, although OT treatment increased activity rates, it was also capable of disrupting the conformation of coordinated activity previously consolidated by NMDA treatment, without showing any effect by itself. Altogether, our results suggest that OT globally prevents the formation of coordinated patterns previously generated by nociceptive-like conditions on dorsal horn cells by NMDA application, which supports previous evidence showing that OT represents a potential therapeutic alternative for the treatment of chronic neuropathic pain.
Learn More >Increased descending pain facilitation accounts for opioid-induced hyperalgesia, but the underlying mechanisms remain elusive. Given the role of µ-opioid receptors in opioid-induced hyperalgesia in animals, the authors hypothesized that the dorsal reticular nucleus, a medullary pain facilitatory area, is involved in opioid-induced hyperalgesia through altered µ-opioid receptor signaling.
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