Animal Studies

Increased afferent input resulting from painful injury augments the activity of central nociceptive circuits both neuron-neuron and neuron-glia interactions. Microglia, resident immune cells of the central nervous system (CNS), play a crucial role in the pathogenesis of chronic pain. This study provides a framework for understanding how peripheral joint injury signals the CNS to engage spinal microglial responses. During the first week of monosodium iodoacetate (MIA)-induced knee joint injury in male rats, inflammatory and neuropathic pain were characterized by increased firing of peripheral joint afferents. This increased peripheral afferent activity was accompanied by increased Iba1 immunoreactivity within the spinal dorsal horn indicating microglial activation. Pharmacological silencing of C and A afferents with co-injections of QX-314 and bupivacaine, capsaicin, or flagellin prevented the development of mechanical allodynia and spinal microglial activity after MIA injection. Elevated levels of ATP in the cerebrospinal fluid (CSF) and increased expression of the ATP transporter vesicular nucleotide transporter (VNUT) in the ipsilateral spinal dorsal horn were also observed after MIA injections. Selective silencing of primary joint afferents subsequently inhibited ATP release into the CSF. Furthermore, increased spinal microglial reactivity, and alleviation of MIA-induced arthralgia with co-administration of QX-314 with bupivacaine were recapitulated in female rats. Our results demonstrate that early peripheral joint injury activates joint nociceptors, which triggers a central spinal microglial response. Elevation of ATP in the CSF, and spinal expression of VNUT suggest ATP signaling may modulate communication between sensory neurons and spinal microglia at 2 weeks of joint degeneration.

Preclinical evidence suggests the potential of Ph1, a toxin obtained from the venom of spider , as a new analgesic drug. Molecular brain imaging techniques have afforded exciting opportunities to examine brain processes in clinical pain conditions. This paper aims to study the brain regions involved in the analgesic effects of Ph1 compared with Morphine, in a model of acute pain induced by formalin in Sprague Dawley rats. We used F-fluorodeoxyglucose as a metabolic radiotracer to perform brain imaging of rats pretreated with Ph1 or Morphine in a model of acute inflammatory pain caused by intraplantar injection of formalin. The rats' hind paw's formalin stimulation resulted in a brain metabolic increase at the bilateral motor cortex, visual cortex, somatosensory cortex, thalamus, and cingulate cortex.In rats treated with Ph1, selective inhibition of unilateral motor cortex and cingulate cortex was observed. Morphine treatment leads to small and selective inhibition at the bilateral amygdala striatum and accumbens. Our results indicate that the analgesic effect of Ph1 and Morphine possesses a differential profile of central processing in the pain state.

Rheumatoid arthritis (RA) causes inflammation, pain, edema, and articular degradation and its treatment can be based on anti-inflammatory drugs, photobiomodulation (PBM) and/or platelet-rich plasma (PRP) that can decrease cell flow and promote local healing. In the present study, we evaluate the effects of PBM and PRP on acute arthritis in Wistar rats through inflammatory and oxidative stress parameters. Thirty female Wistar rats were assigned to five groups (n=6, each group): Control, Sham, PRP, Laser, and PRP+Laser. For arthritis induction, all animals of groups Sham, PRP, Laser and PRP+Laser received an intraarticular injection of Zymosan® (200µg) in the right knee. Twenty-four hours post-arthritis induction, PRP was prepared and injected (8 × 10 of platelets) in animals of PRP and PRP+Laser groups. PBM was performed in Laser and PRP+Laser groups by single-dose therapy with the GaAlAs laser (λ=808 nm, P=25 mW, fluence=30 J/cm, beam area=0.02 mm, t=33 seconds, E=0.825 J, punctual application). After seven days of induction, serum samples were collected and thiobarbituric acid reactive substances (TBARS), nitric oxide (NO) and catalase activity were analysed. Morphological parameters were measured for inflammation areas, cartilage thickness, and C3 protein expression in knee samples. Statistical analysis was performed with an ANOVA test and Tukey's post-hoc test with a significance level of 5% (<0.05). NO was lower in the treated groups compared to the Sham group, and TBARS did not show any differences, while catalase showed greater activity between PRP+Laser versus PRP (<0.05). Inflammatory areas and cartilage thickness were lower in the treated groups compared to Sham (<0.05), while no differences in C3 protein expression was observed. PBM associated with PRP is better for anti-inflammatory and joint preservation by morphological aspects and NO levels that concern a potential clinical application.

Inflammation-associated chronic pain is a global clinical problem, affecting millions of people worldwide. However, the underlying mechanisms that mediate inflammation-associated chronic pain remain unclear. A rat model of cutaneous inflammation induced by Complete Freund's Adjuvant (CFA) has been widely used as an inflammation-induced pain hypersensitivity model. We present the transcriptomics profile of CFA-induced inflammation in the rat dorsal root ganglion (DRG) an approach that targets gene expression, DNA methylation, and post-transcriptional regulation. We identified 418 differentially expressed mRNAs, 120 differentially expressed microRNAs (miRNAs), and 2,670 differentially methylated regions (DMRs), which were all highly associated with multiple inflammation-related pathways, including nuclear factor kappa B (NF-κB) and interferon (IFN) signaling pathways. An integrated analysis further demonstrated that the activator protein 1 (AP-1) network, which may act as a regulator of the inflammatory response, is regulated at both the transcriptomic and epigenetic levels. We believe our data will not only provide drug screening targets for the treatment of chronic pain and inflammation but will also shed light on the molecular network associated with inflammation-induced hyperalgesia.

Neonatal pain such as that experienced by infants in the neonatal intensive care unit is known to produce later-life dysfunction including heightened pain sensitivity and anxiety, although the mechanisms remain unclear. Both chronic pain and stress in adult organisms are known to influence the corticotropin-releasing factor (CRF) system in the Central Nucleus of the Amygdala, making this system a likely candidate for changes following neonatal trauma. To examine this, neonatal rats were subjected to daily pain, non-painful handling or left undisturbed for the first week of life. Beginning on postnatal day, 24 male and female rats were subjected to a 4-day fear conditioning and sensory testing protocol. Some subjects received intra-amygdalar administration of either Vehicle, the CRF receptor 1 (CRF) receptor antagonist Antalarmin, or the CRF receptor 2 (CRF) receptor antagonist Astressin 2B prior to fear conditioning and somatosensory testing, while others had tissue collected following fear conditioning and CRF expression in the CeA and BLA was assessed using fluorescent hybridization. CRF antagonism attenuated fear-induced hypersensitivity in neonatal pain and handled rats, while CRF antagonism produced a general antinociception. In addition, neonatal pain and handling produced a lateralized sex-dependent decrease in CRF expression, with males showing a diminished number of CRF-expressing cells in the right CeA and females showing a similar reduction in the number of CRF-expressing cells in the left BLA compared to undisturbed controls. These data show that the amygdalar CRF system is a likely target for alleviating dysfunction produced by early life trauma and that this system continues to play a major role in the lasting effects of such trauma into the juvenile stage of development.

To elucidate the relationship between chronic pain conditions with cast immobilization and autonomic function, we investigated the functional changes of the autonomic nervous system in conscious rats with chronic post-cast pain (CPCP) induced by a two-week cast immobilization of one hind limb. We telemetrically examined the time courses of systolic arterial blood pressure (SBP), heart rate (HR), and the middle-frequency (MF) component obtained from the power spectral analysis of SBP variability as a vasomotor sympathetic index. We also investigated the baroreflex sensitivity to phentolamine, an α-adrenoceptor antagonist, and the SBP and HR responses to a low ambient temperature (LT; 9.0 ± 0.2°C) exposure, a sympathetic stimulant. Rats exposed to cast immobilization exhibited mechanical allodynia lasting for at least 10 weeks after cast removal in the calf area (skin and muscle) of the bilateral hind limbs. Under resting conditions, the SBP, HR, and MF components were significantly increased during cast immobilization (all p < 0.001). Following cast removal, these parameters gradually decreased and within 1 week reached lower than baseline levels, lasting for over 10 weeks. Phentolamine administration (10 mg/kg, intraperitoneally) significantly decreased the SBP before and during cast immobilization (before, p < 0.001; during, p = 0.001) but did not lower the SBP after cast removal. The baroreflex gain after phentolamine administration, calculated as the HR increase divided by the SBP reduction, was significantly increased after cast removal (p = 0.002). The SBP increase on LT exposure was significantly greater after cast removal than that before cast immobilization, suggesting hypersensitivity to sympathetic neurotransmitters. These results revealed that, in the CPCP model, sympathetic activation was augmented during cast immobilization, which then decreased after cast removal and remained below normal levels with persisting pain behaviors. Additionally, the responsiveness of the autonomic nervous system was impaired in the CPCP model.

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain .