Animal Studies

Until recently, the majority of preclinical neuroinflammatory studies were performed on males. Because of this, less is known about the female neuroinflammatory response to injury, formation of pain, or response to pain-relieving therapies. A major focus has been on the macrophage and its contribution to the development of neuropathic pain. Under a typical response, the macrophage upregulates expression of cyclooxygenase-2 (COX-2), which in turn leads to the production of proinflammatory prostaglandin E (PGE ), which is linked to neuronal sensitization and pain. Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to attenuate PGE by the inhibition of COX-2. However, a problem with NSAIDs is that due to the systemic dosage needed to achieve neuropathic pain relief there are risks of off-target toxicity. This study evaluated a COX-2 inhibiting nanotherapeutic in its ability to relieve neuropathic inflammation and pain-like behavior of a chronic constriction injury (CCI) of the right sciatic nerve. We find that under the same injury, both males and females exhibit the same degree of hypersensitivity represented as pain-like behavior. We also find that the nanotherapeutic was able to relieve pain-like behavior in both sexes, but females experienced less relief. This behavioral response was reflected in the number of infiltrating macrophages at the site of injury; where again, both sexes had decreased inflammation but the females had significantly more macrophages present than the males.

Diagnoses of maternal opioid use disorder (MOUD) at delivery increased more than 500% between 1999 and 2017 in the United States. Today, cases of in utero opioid exposure due to MOUD exceed cases of the most common birth defects, including cleft lip, cleft palate, clubfoot, and Down syndrome, combined. Children exposed to opioids in utero are at increased risk of adverse childhood experiences (ACEs) due to parental care being compromised by SUDs. These ACEs include abuse, neglect, loss of a parent from death or incarceration, parental divorce, and household domestic violence. Altered sensitivity to opioid analgesia later in life is one potential outcome shared by in utero opioid exposure and ACEs that may contribute to poorly controlled pain and increased risk of opioid addiction. Studies using rat models of prenatal opioid exposure (POE) or early life adversity (ELA) have shown that each decreases opioid-induced antinociception long after exposure has ceased. However, the contributions of POE and ELA, alone and in combination, to opioid analgesic response in humans remains unknown. In this study, we used a rodent model of combined POE and ELA to disambiguate their long-term effects more quickly and with greater experimental control than human studies. We hypothesized that combined POE and ELA will decrease morphine-induced antinociception in adolescence relative to POE or ELA alone. We used a two-by-two between-subjects factorial design in which timed-pregnant Long-Evans rats were exposed to morphine (15 mg/kg/day; "POE" group) or saline ("vehicle" group) via subcutaneous osmotic minipump from gestation day 9 until delivery. Litters were fostered to untreated dams then randomly assigned to normal housing or ELA conditions using a limited bedding and nesting procedure from postnatal days 3-11. Morphine-induced antinociception was measured between postnatal days 30 and 54 using a warm-water tail withdrawal (WWTW) assay. Cumulative dose-response of morphine-induced antinociception was determined on test day 0 (1.0-30 mg/kg morphine, s.c.) and test day 13 (3.0-100 mg/kg, s.c.). On test days 1-12, rats were dosed with 18 mg/kg, s.c., every 12 hours, and WWTW was conducted on odd test days. Interesting, preliminary results of this ongoing study indicate that morphine-induced antinociception was enhanced on test day 0 in POE + normal-housed males (n = 2). These rats exhibited 100% maximum possible effect (MPE) and significantly higher potency (mean ED50=5.96 mg/kg; 95% CI = -12.12, 24.05), while mean MPE for the other groups (n=2-3) was less than 70% and mean ED50>17 mg/kg. Males exhibited partial tolerance throughout the 12-day chronic morphine treatment period. In the POE + normal-housed males, MPE decreased from 100% on test day 1 to 58% on test day 11. For the remaining groups, MPE decreased from 44-57% on test day 1 to 12-23% on test day 11. Morphine was more potent in the POE + normal-housed males on test day 13 (mean ED50=66.21 mg/kg; 95% CI = -281.6, 414) than in other males (mean ED50>104.4 mg/kg). No clear group effect was observed in females. These unexpected results suggest a complex sex-dependent interaction between POE and ELA on opioid antinociception.

Local anesthetics are commonly used for the management of intraoperative and postoperative acute and chronic pain caused by small invasive procedures. However, their short half-life and duration of action limit their clinical benefits. In this study, we proposed the incorporation of graphene oxide (GO) nanosheets to chitosan (CS)/β-glycerophosphate (GP) thermosensitive hydrogel system to form an injectable nanocomposite hydrogel (NCH) with improved mechanical properties and better control over the release of bupivacaine hydrochloride (BH). The prepared nanocomposite hydrogels were characterized for their gelation time, porosity, swelling ratio, injectability, mechanical strength and in vitro drug release. In vivo, the efficacy of the prepared NCH containing 0.5 % w/v BH was evaluated using a thermal nociceptive assay in a rat model. The incorporation of GO significantly enhanced the physicochemical and mechanical properties of the hydrogel scaffolds in a concentration-dependent manner. Inclusion of 0.1% w/v GO resulted in 84% reduction in gelation time and 16% and 40% decrease in the porosity and swelling ratio of the nanocomposite hydrogels, respectively. The mechanical strength of the CS/GP hydrogel scaffolds was also significantly improved in presence of GO. BH was slowly released from the NCHs containing 0.1% w/v GO and resulted in a 55% and 86.43% drug release after 6 and 24 h, respectively. In vivo studies showed that BH-loaded NCH significantly prolonged the local anesthetic effect and resulted in a 6.5-fold increase in blocking the pain sensory reflex compared to BH solution. These results indicate that the incorporation of GO significantly improved the physical and mechanical properties of CS/GP thermosensitive hydrogels and successfully sustained the effect of local anesthesia for more effective pain management.

Morphine abuse is a devastating disorder that affects millions of people worldwide, and literature evidence indicates a relationship between opioid abuse and the circadian clock.

The impact of rheumatoid arthritis (RA) on the shaping of the oral and gut microbiome raises the question of whether and how RA treatment modifies microbial communities. We examined changes in the oral and gut microbiota in a mouse model of antigen-induced arthritis (AIA) treated or not with methotrexate (MTX).

Soft polymer nanoparticles designed to disassemble and release an antagonist of the neurokinin 1 receptor (NKR) in endosomes provide efficacious yet transient relief from chronic pain. These micellar nanoparticles are unstable and rapidly release cargo, which may limit the duration of analgesia. We examined the efficacy of stable star polymer nanostars containing the NKR antagonist aprepitant-amine for the treatment of chronic pain in mice. Nanostars continually released cargo for 24 h, trafficked through the endosomal system, and disrupted NKR endosomal signaling. After intrathecal injection, nanostars accumulated in endosomes of spinal neurons. Nanostar-aprepitant reversed mechanical, thermal and cold allodynia and normalized nociceptive behavior more efficaciously than free aprepitant in preclinical models of neuropathic and inflammatory pain. Analgesia was maintained for >10 h. The sustained endosomal delivery of antagonists from slow-release nanostars provides effective and long-lasting reversal of chronic pain.

The current study tries to discuss the functional role of microRNA-497 (miR-497) in diabetic neuropathic pain (DNP) and the related downstream mechanism. Bioinformatics analysis was implemented for the identification of differentially expressed miRNAs and genes. DNP was simulated in rats through intraperitoneal injection of streptozotocin. The expression patterns of miR-497, USP15, NRF2, and G6PD were then determined. The binding of miR-497 and USP15 was confirmed. Using gain- and loss-of-function assays, we analyzed the critical role of miR-497-mediated USP15 in DNP through the NRF2/G6PD axis. Downregulated miR-497 and elevated USP15 were observed in the dorsal root ganglion neurons isolated from spinal cord tissues of STZ-induced DNP rats. miR-497 could alleviate DNP, which was associated with suppression of USP15, a confirmed target of miR-497. USP15 enhanced the degradation and ubiquitination of NRF2 and induced G6PD expression, leading to the progression of DNP. We highlighted the crucial role of miR-497-mediated USP15 in DNP through the NRF2/G6PD axis. 1. miR-497 is downregulated in DRG neurons from spinal cord tissues of STZ-induced DNP rats. 2. miR-497 inhibits the expression of USP15, thereby alleviating STZ-induced DNP in rats. 3. USP15 promotes ubiquitination and degradation of NRF2, reducing the expression of G6PD. 4. miR-497 alleviates STZ-induced DNP in rats by regulating the USP15/NRF2/G6PD axis.

Current pain management is largely limited to opioids and non-steroidal anti-inflammatory drugs. Developing new analgesic drugs remains important to address the unmet medical needs of chronic pain patients. Calcium-activated chloride channel anoctamin-1 (ANO1) is a potential analgesic target. ANO1 is activated by noxious stimuli in peripheral sensory neurons and further induced neural depolarization. Downregulation of ANO1 reduced hyperalgesia and allodynia caused by inflammation and nerve injury. Here we developed a series of 4-arylthiophene-3-carboxylic acid derivatives for proof-of-concept studies of ANO1-targeted analgesia. These efforts led to the identification of the compound DFBTA, 4-(4-chlorophenyl)-2-(2,5-difluorobenzamido)thiophene-3-carboxylic acid, which displays dramatic ANO1 inhibition with IC of 24 nM. DFBTA displays very weak cytotoxicity, cardiotoxicity, and acute toxicity (HEK293 proliferation IC > 30 μM, hERG IC > 30 μM, mouse minimum lethal dosage, MLD>1000 mg/kg), as well as excellent pharmacokinetics properties with oral bioavailability >75% and little brain penetration (<1.5% brain/plasma). Finally, the analgesic efficacy of ANO1 inhibitor was evaluated in animal models. DFBTA shown comparable efficacy to clinical drugs in all inflammatory pain models induced by complete Freund's adjuvant, formalin, and capsaicin. These works provide a useful tool compound and promising results for ANO1-targenting analgesic development.