Background & Aims
Surgeries induce post-operative pain that must be alleviated to limit suffering and promote recovery. Post-operative pain is often not effectively managed in >80% of patients in the US. Opioids are the standard of care for perioperative pain, but their dosing is limited due to adverse effects and abuse potential. Macrophages are known to play a pivotal role in pain attenuation and wound healing. Small extracellular vesicles (sEVs), including exosomes, mediate intercellular communication by carrying biomolecular cargo to recipient cells. We have previously shown that sEVs isolated from macrophage-derived RAW 264.7 cells attenuate pain in mouse models of inflammatory pain. We hypothesize that macrophage-derived sEVs can attenuate post-operative pain and inflammation and promote wound healing. Here we investigated the analgesic and wound healing properties of sEVs in a mouse model of postoperative pain.
Methods
sEVs were isolated from RAW 264.7 cell culture media by ultracentrifugation and size exclusion chromatography. A paw incisional model of postoperative pain in mice was used to assess mechanical and thermal pain thresholds after intrathecal or plantar sEV administration. F4/80, Iba1 and CD86 antibodies were used in flow cytometry and immunohistochemistry. Macrophage fas-induced apoptosis (MaFIA) mice were treated with the AP20187 (1 mg/kg, i.p.) or vehicle to transiently deplete macrophages.
Results
We generated and validated the mouse model, and our preliminary data showed that intrathecal and intraplantar injections of sEVs given immediately after incisional surgery attenuates pain in a dose-dependent manner as compared to control. Studies are ongoing to determine whether prophylactic administration of sEVs either days or hours before surgery is efficacious. Preliminary flow cytometric data in paw tissue showed macrophages traffic to the incision site in the days following surgery, peaking at day 3 and reducing around day 7 when pain thresholds return to baseline. MaFIA mice will be used to determine whether macrophages are necessary for sEV-induced attenuation of postoperative pain and preliminary characterization shows inhibition of macrophage trafficking to the paw.
Conclusions
sEVs are efficacious in attenuating postoperative pain in a paw incisional model of mice. Studies are ongoing to determine the most efficacious combination of sEV dose, route of administration, and timepoint. Additionally, studies will assess if sEVs increase macrophage numbers and promote pro-resolution phenotypes and whether they can reduce the dose and/or duration of prescribed analgesics.
References
1. Gan TJ. Poorly controlled postoperative pain: prevalence, consequences, and prevention. J Pain Res. 2017 Sep 25;10:2287-2298. doi: 10.2147/JPR.S144066. PMID: 29026331; PMCID: PMC5626380
2. Krzyszczyk P, Schloss R, Palmer A, Berthiaume F. The Role of Macrophages in Acute and Chronic Wound Healing and Interventions to Promote Pro-wound Healing Phenotypes. Front Physiol. 2018 May 1;9:419. doi: 10.3389/fphys.2018.00419. PMID: 29765329; PMCID: PMC5938667.
3. Chen O, Donnelly CR, Ji RR. Regulation of pain by neuro-immune interactions between macrophages and nociceptor sensory neurons. Curr Opin Neurobiol. 2020 Jun;62:17-25. doi: 10.1016/j.conb.2019.11.006. Epub 2019 Dec 3. PMID: 31809997; PMCID: PMC7266706.
4. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020 Feb 7;367(6478):eaau6977. doi: 10.1126/science.aau6977. PMID: 32029601; PMCID: PMC7717626.
5. Jean-Toussaint R, Lin Z, Tian Y, Gupta R, Pande R, Luo X, Hu H, Sacan A, Ajit SK. Therapeutic and prophylactic effects of macrophage-derived small extracellular vesicles in the attenuation of inflammatory pain. Brain Behav Immun. 2021 May;94:210-224. doi: 10.1016/j.bbi.2021.02.005. Epub 2021 Feb 17. PMID: 33607232; PMCID: PMC8058272
6. Wu CL, McNeill J, Goon K, Little D, Kimmerling K, Huebner J, Kraus V, Guilak F. Conditional Macrophage Depletion Increases Inflammation and Does Not Inhibit the Development of Osteoarthritis in Obese Macrophage Fas-Induced Apoptosis-Transgenic Mice. Arthritis Rheumatol. 2017 Sep;69(9):1772-1783. doi: 10.1002/art.40161. Epub 2017 Aug 8. PMID: 28544542; PMCID: PMC5611814.