The most frequently reported use of medical marijuana is for pain relief. However, its psychoactive component Δ9-tetrahydrocannabinol (THC) causes significant side effects. Cannabidiol (CBD) and β-caryophyllene (BCP), two other cannabis constituents, possess more benign side effect profiles and are also reported to reduce neuropathic and inflammatory pain. We evaluated the analgesic potential of CBD and BCP individually and in combination in a rat spinal cord injury (SCI) clip compression chronic pain model. Individually, both phytocannabinoids produced dose-dependent reduction in tactile and cold hypersensitivity in male and female rats with SCI. When co-administered at fixed ratios based on individual A50s, CBD and BCP produced enhanced dose-dependent reduction in allodynic responses with synergistic effects observed for cold hypersensitivity in both sexes and additive effects for tactile hypersensitivity in males. Antinociceptive effects of both individual and combined treatment were generally less robust in females than males. CBD:BCP co-administration also partially reduced morphine-seeking behavior in a conditioned place preference (CPP) test. Minimal cannabinoidergic side effects were observed with high doses of the combination. The antinociceptive effects of the CBD:BCP co-administration were not altered by either CB2 or μ-opioid receptor antagonist pretreatment but, were nearly completely blocked by CB1 antagonist AM251. Since neither CBD or BCP are thought to mediate antinociception via CB1 activity, these findings suggest a novel CB1 interactive mechanism between these two phytocannabinoids in the SCI pain state. Together, these findings suggest that CBD:BCP co-administration may provide a safe and effective treatment option for the management of chronic SCI pain.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs’ miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

Neuropathic pain often accompanies the functional deficits associated with spinal cord injury (SCI) and further reduces a patient's quality of life. Clinical and pre-clinical research is beginning to highlight the beneficial role that rehabilitative therapies such as locomotor training can have not only on functional recovery but also on chronic pain management. Our group has previously developed an intensive locomotor training (ILT) treadmill protocol on rats that reduced SCI neuropathic pain symptoms for at least 3 months. We have extended these findings in the current study to evaluate the ability of regular ILT regimen over a 2 year period post-SCI to maintain neuropathic pain reduction. To assess this, the rat clip compression SCI model (T7/8) was used and treadmill training was initiated starting 4 weeks after SCI and continuing through the duration of the study. Results showed continued suppression of SCI neuropathic pain responses (reduced mechanical, heat, and cold hypersensitivity throughout the entire time course of the study). In contrast, non-exercised rats showed consistent and sustained neuropathic pain responses during this period. In addition, prolonged survival and improved locomotor outcomes were observed in rats undergoing ILT as the study longevity progressed. Potential contributory mechanisms underlying beneficial effects of ILT include reduced inflammation and restoration of anti-nociceptive inhibitory processes as indicated by neurochemical assays in spinal tissue of remaining rats at 2 years post-SCI. The benefits of chronic ILT suggest that long-term physical exercise therapy can produce powerful and prolonged management of neuropathic pain, partly through sustained reduction of spinal pathological processes.

Background: The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our ab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated the analgesic potential of these peptides when delivered via gene therapy.Results: Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanica and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of individually injected components were modest, but the combination of EMs and SHG produced robust and sustained antinociception. The onset of the analgesic effects was observed between 1–5 weeks post-injection and sustained without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in these animals using the SHG-EMs combination vectors compared to the group with early intervention.Conclusions: Findings from this study support the potential for direct gene therapy to provide a robust and sustained alleviation of chronic neuropathic pain following SCI. The combination strategy utilizing potent mu-opioid peptides with a naturally-derived NMDA antagonist may produce additive or synergistic analgesic effects without the tolerance development for long-term management of persistent pain.

Neuropathic pain induced by spinal cord injury (SCI) is clinically challenging with inadequate long-term treatment options. Partial pain relief offered by pharmacologic treatment is often counterbalanced by adverse effects after prolonged use in chronic pain patients. Cell-based therapy for neuropathic pain using GABAergic neuronal progenitor cells (NPCs) has the potential to overcome untoward effects of systemic pharmacotherapy while enhancing analgesic potency due to local activation of GABAergic signaling in the spinal cord. However, multifactorial anomalies underlying chronic pain will likely require simultaneous targeting of multiple mechanisms. Here, we explore the analgesic potential of genetically modified rat embryonic GABAergic NPCs releasing a peptidergic NMDA receptor antagonist, Serine¹-histogranin (SHG), thus targeting both spinal hyperexcitability and reduced inhibitory processes. Recombinant NPCs were designed using either lentiviral or adeno-associated viral vectors (AAV2/8) encoding single and multimeric (6 copies of SHG) cDNA. Intraspinal injection of recombinant cells elicited enhanced analgesic effects compared with nonrecombinant NPCs in SCI-induced pain in rats. Moreover, potent and sustained antinociception was achieved, even after a 5-week postinjury delay, using recombinant multimeric NPCs. Intrathecal injection of SHG antibody attenuated analgesic effects of the recombinant grafts suggesting active participation of SHG in these antinociceptive effects. Immunoblots and immunocytochemical assays indicated ongoing recombinant peptide production and secretion in the grafted host spinal cords. These results support the potential for engineered NPCs grafted into the spinal dorsal horn to alleviate chronic neuropathic pain.