Animal Studies

Preoperative sleep loss can amplify post-operative mechanical hyperalgesia. However, the underlying mechanisms are still largely unknown. In the current study, rats were randomly allocated to a control group and an acute sleep deprivation (ASD) group which experienced 6 h ASD before surgery. Then the variations in cerebral function and activity were investigated with multi-modal techniques, such as nuclear magnetic resonance, functional magnetic resonance imaging, c-Fos immunofluorescence, and electrophysiology. The results indicated that ASD induced hyperalgesia, and the metabolic kinetics were remarkably decreased in the striatum and midbrain. The functional connectivity (FC) between the nucleus accumbens (NAc, a subregion of the ventral striatum) and the ventrolateral periaqueductal gray (vLPAG) was significantly reduced, and the c-Fos expression in the NAc and the vLPAG was suppressed. Furthermore, the electrophysiological recordings demonstrated that both the neuronal activity in the NAc and the vLPAG, and the coherence of the NAc-vLPAG were suppressed in both resting and task states. This study showed that neuronal activity in the NAc and the vLPAG were weakened and the FC between the NAc and the vLPAG was also suppressed in rats with ASD-induced hyperalgesia. This study highlights the importance of preoperative sleep management for surgical patients.

Calcitonin gene related peptide (CGRP) is a neuropeptide produced by sensory nerves and functions as a pain sensor. It acts by binding to calcitonin like receptor (CLR, protein; Calcrl, gene). CGRP inhibition has been recently introduced as therapeutic treatment of migraine-associated pain. Previous studies have shown that CGRP stimulates bone formation. The aim of our study was to determine whether the inhibition of CGRP signaling negatively impacted fracture healing. Using α-smooth muscle actin (αSMA) Cre animals crossed with Ai9 reporter mice, we showed that CGRP expressing nerves are near αSMA+ cells in the periosteum. In vitro experiments revealed that periosteal cells express Calcrl and Receptor activity modifying protein 1 (Ramp1); and CGRP stimulation increased periosteal cell proliferation. Using a tamoxifen-inducible model αSMACre/CLR we targeted deletion of CLR to periosteal progenitor cells and examined fracture healing. Micro-computed tomography of fractured femurs showed a reduction in bone mass in αSMACre+/CLR female mice relative to controls and callus volume in males. Pharmacological CGRP-CLR inhibition was achieved by subcutaneous delivery of customized pellets with small molecule inhibitor Olcegepant (BIBN-4096) at a dose of 10 μg/day. BIBN-4096-treated C57BL/6J mice had a higher latency toward thermal nociception than placebo treated mice, indicating impaired sensory function through CGRP inhibition. CGRP inhibition also resulted in reduced callus volume, bone mass and bone strength compared to placebo controls. These results indicate that inhibiting CGRP by deleting CLR or by using BIBN-4096, contributes to delayed bone-healing. This article is protected by copyright. All rights reserved.

TRPA1 and TRPM8 are transient receptor potential channels expressed in trigeminal neurons that are related to pathophysiology in migraine models. Here we use a mouse model of nitroglycerine-induced chronic migraine that displays a sexually dimorphic phenotype, characterized by mechanical hypersensitivity that develops in males and females, and is persistent up to day 20 in female mice, but disappears by day 18 in male mice. TRPA1 is required for development of hypersensitivity in males and females, whereas TRPM8 contributes to the faster recovery from hypersensitivity in males. TRPM8-mediated antinociception effects required the presence of endogenous testosterone in males. Administration of exogenous testosterone to females and orchidectomized males led to recovery from hypersensitivity. Calcium imaging and electrophysiological recordings in in vitro systems confirmed testosterone activity on murine and human TRPM8, independent of androgen receptor expression. Our findings suggest a protective function of TRPM8 in shortening the time frame of hypersensitivity in a mouse model of migraine.

The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here we investigated the role of low-voltage activated T-type calcium (CaV3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence we revealed ubiquitous expression of CaV3.2, but not CaV3.1 or CaV3.3 in individual bladder-innervating dorsal root ganglia (DRG) neurons. In an ex vivo bladder-nerve recording preparation pharmacological inhibition of CaV3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed CaV3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delaying activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in-vivo were also significantly reduced by inhibition of CaV3 with TTA-A2. Together these data provide evidence of a major role for CaV3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

Inflammatory pain is the most important clinical symptom of inflammatory diseases. Despite intensive research into inflammatory pain mechanisms, the majority of analgesics available are based on mechanistic classes of compounds that have been known for many years, as a result, inflammatory pain control remains a challenge for drug design in the context of clinically unmet needs in terms of safety and efficacy. A growing literature supports that pro-inflammatory cytokine signaling plays a crucial role in the development of inflammatory pain. Modulation of the pro-inflammatory cytokine may hold the key to improved pain management. Previous studies have reported that dorsomorphin played key roles in inflammation. But the role of dorsomorphin in the formalin-induced inflammatory nociception in mice has never been reported. Here, we report a new function of dorsomorphin which can inhibit formalin-induced inflammatory nociception in mice. The antinociceptive effect of dorsomorphin mainly depended on inhibiting the p38 MAPK/c-fos signaling and regulating inflammatory mediators.

Stroke can be followed by immediate severe headaches. As headaches are initiated by the activation of trigeminal meningeal afferents, we assessed changes in the activity of meningeal afferents in mice subjected to cortical photothrombosis. Cortical photothrombosis induced ipsilateral lesions of variable sizes that were associated with contralateral sensorimotor impairment. Nociceptive firing of mechanosensitive Piezo1 channels, activated by the agonist Yoda1, was increased in meningeal afferents in the ischemic hemispheres. These meningeal afferents also had a higher maximal spike frequency at baseline and during activation of the mechanosensitive Piezo1 channel by Yoda1. Moreover, in these meningeal afferents, nociceptive firing was active during the entire induction of transient receptor potential vanilloid 1 (TRPV1) channels by capsaicin. No such activation was observed on the contralateral hemi-skulls of the same group of mice or in control mice. Our data suggest the involvement of mechanosensitive Piezo1 channels capable of maintaining high-frequency spiking activity and of nociceptive TRPV1 channels in trigeminal headache pain responses after experimental ischemic stroke in mice.

Mounting evidence indicates that the neuropeptide FF (NPFF) system is involved in the side effects of opioid usage, including antinociceptive tolerance, hyperalgesia, abuse, constipation, and respiratory depression. Our group recently discovered that the multitarget opioid/NPFF receptor agonist DN-9 exhibits peripheral antinociceptive activity. To improve its metabolic stability, antinociceptive potency, and duration, in this study, we designed and synthesized a novel cyclic disulfide analogue of DN-9, OFP011, and examined its bioactivity through cyclic adenosine monophosphate (cAMP) functional assays and behavioral experiments. OFP011 exhibited multifunctional agonistic effects at the μ-opioid and the NPFF and NPFF receptors and partial agonistic effects at the δ- and κ-opioid , as determined the cAMP functional assays. Pharmacokinetic and pharmacological experiments revealed improvement in its blood-brain barrier permeability after systemic administration. In addition, subcutaneous OFP011 exhibited potent and long-lasting antinociceptive activity the central μ- and κ-opioid receptors, as observed in different physiological and pathological pain models. At the highest antinociceptive doses, subcutaneous OFP011 exhibited limited tolerance, gastrointestinal transit, motor coordination, addiction, reward, and respiration depression. Notably, OFP011 exhibited potent oral antinociceptive activities in mouse models of acute, inflammatory, and neuropathic pain. These results suggest that the multifunctional opioid/NPFF receptor agonists with improved blood-brain barrier penetration are a promising strategy for long-term treatment of moderate to severe nociceptive and pathological pain with fewer side effects.

Age is known to be the major risk factor for both pain sensation and sporadic Alzheimer's disease (sAD). Pain management in AD is a critical health condition. However, assessing pain in sAD patients is challenging. The intracerebroventricularly injected streptozotocin (icv-STZ) rat model of sAD has been brought to the fore as a hopefully suitable model that could mimic some features of sAD. However, the exact mechanism by which this agent may induce AD-like pathology is largely unknown. In some studies, analgesic drugs have been suggested as possible prevention of AD and icv-STZ-induced AD-like pathology. Therefore, this study used formalin and tail-flick tests to investigate whether different doses of icv-STZ injections could affect acute and inflammatory pain sensation and edema volume over time. Behavioral responses were observed at four testing time points (1, 2.5, 3.5, and 6 months postinjection). The results indicate that icv-STZ was able to significantly decrease the animals' formalin pain threshold in both a time- and dose-dependent manner. Formalin-induced acute and chronic pain scores of animals treated with streptozotocin 3 mg/kg (STZ3) increased dramatically 2.5 months after injection and persisted thereafter. The augmentation in pain score induced by streptozotocin 1 mg/kg (STZ1) was observed from 3.5 months after STZ injection. However, the effect of streptozotocin 0.5 mg/kg (STZ0.5) was NS until 6 months after injection. However, formalin-induced paw edema occurred with a longer delay and was not detectable in STZ0.5-treated animals. In addition, only STZ3-treated animals significantly reduced the thermal pain threshold of animals 6 months after injection. These observations indicate that icv-STZ can sensitize central and/or peripheral receptors to pain. The effect of STZ is dose- and time-dependent. AD-like pathology induced by icv-STZ could be partially activated via pain processing pathways. Therefore, anti-inflammatory agents could alleviate AD-like symptoms via pain treatments.