Animal Studies

As an emerging dietary essential fatty acid, pentadecanoic acid (C15:0) is expected to have bioactive metabolites with broad health benefits. Here, we evaluated pentadecanoylcarnitine, an endogenous C15:0 metabolite, for dose dependent cell-based activities, including measurement of its effects on 148 clinically relevant biomarkers across twelve primary human cell systems mimicking various disease states. Mechanisms of action for pentadecanoylcarnitine were also assessed across 78 cell-based target assays. Pentadecanoylcarnitine had dose-dependent anti-inflammatory activities, including lower IL-1α, ITAC, MCP-1, and IP-10, across five cell systems relevant to treating cardiovascular, immune, neoplastic, pulmonary, and skin diseases. Targeted assays showed pentadecanoylcarnitine as a full-acting cannabinoid 1 and 2 receptor agonist (EC50 3.7 and 3.2 µM, 111% and 106% maximum activity compared to the positive control, respectively). Pentadecanoylcarnitine also had 5-HT1A and 5-HT1B receptor agonist and histamine H1 and H2 receptor antagonist activities. In summary, pentadecanoylcarnitine, a second discovered full-acting endocannabinoid, had broad pleiotropic activities relevant to regulating inflammation, pain, mood, and sleep. This study's findings further the need to evaluate the potential health impacts of C15:0 nutritional deficiencies caused by population-wide avoidance of all dietary saturated fats, including C15:0.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and social interaction and the presence of restricted, repetitive behaviors. Additionally, difficulties in sensory processing commonly occur in ASD. Sensory abnormalities include heightened or reduced sensitivity to pain, but the mechanism underlying sensory phenotypes in ASD remain unknown. Emerging evidence suggests that microglia play an important role in forming and refining neuronal circuitry, and thus contribute to neuronal plasticity and nociceptive signaling. In the present study, we investigated the age-dependent tactile sensitivity in an animal model of ASD induced by prenatal exposure to valproic acid (VPA) and subsequently assessed the involvement of microglia in the spinal cord in pain processing. Pregnant ICR (CD1) mice were intraperitoneally injected with either saline or VPA (500 mg/kg) on embryonic day 12.5. Male offspring of VPA-treated mothers showed mechanical allodynia at both 4 and 8 weeks of age. In the spinal cord dorsal horn in prenatally VPA-treated mice, the numbers and staining intensities of ionized calcium-binding adapter molecule 1-positive cells were increased and the cell bodies became enlarged, indicating microglial activation. Treatment with PLX3397, a colony-stimulating factor 1 receptor inhibitor, for 10 days resulted in a decreased number of spinal microglia and attenuated mechanical allodynia in adult mice prenatally exposed to VPA. Additionally, intrathecal injection of Mac-1-saporin, a saporin-conjugated anti-CD11b antibody to deplete microglia, abolished mechanical allodynia. These findings suggest that prenatal VPA treatment causes allodynia and that spinal microglia contribute to the increased nociceptive responses.

Pain and itch are recognized as antagonistic sensations; pain suppresses itch and inhibition of pain generates itch. There is still a lack of evidence about the neural mechanism of the interaction between pain and itch in the central nervous system. In this study, we focused on the orexin (ORX) neurons in the lateral hypothalamus (LH), which mediate various "defense responses" when animals confront stressors. We found that the scratching behaviors induced by the pruritogen were significantly suppressed in ORX-neuron-ablated (ORX-abl) mice. The exaggerated pain behavior and attenuated itch behavior observed in ORX-abl mice indicated that ORX neurons modulate pain and itch in an opposite way, i.e., pain relief and itch exacerbation. In addition, most of the ORX neurons responded to both pain and itch input. Our results suggest that ORX neurons inversely regulate pain- and itch-related behaviors, which could be understood as a defense response to cope with stress environment.

Alpha1-adrenoceptors are over-expressed in the epidermis of a subgroup of patients with complex regional pain syndrome (CRPS). Activating α1-adrenoceptors in epidermal cells increases production of the pro-inflammatory cytokine interleukin-6 (IL-6), a mediator of inflammation. To investigate whether this might exacerbate inflammation in CRPS, primary keratinocytes and/or dermal fibroblasts were cultured from skin biopsies obtained from the affected limb of 25 patients and a similar site in 28 controls. The fundamental pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα), was administered for 24 hours to initiate inflammation. Following this, cells were incubated for 6 hours with the α1-adrenoceptor agonist phenylephrine. Exposure to TNFα induced pro-inflammatory cytokine mRNA production and protein secretion in keratinocytes and fibroblasts, and enhanced α1B-adrenoceptor mRNA expression in keratinocytes. Additional stimulation of α1-adrenoceptors with phenylephrine increased the production of interleukin-6 (IL-6) mRNA and protein secretion in both cell types. Under all conditions, gene and protein α1-adrenoceptor levels and cytokine gene expression and protein secretion were similar, overall, in patients and controls, except for abnormally high α1-adrenoceptor protein levels in the keratinocytes of three of 17 patients. These findings suggest that persistent inflammation in CRPS is not due to dysfunction of skin cells but is a normal response to extrinsic signals. After α1-adrenoceptor stimulation of keratinocytes, increases in IL-6 mRNA but not protein were proportional to basal α1-adrenoceptor protein levels. Skin cells play an important role in persistent inflammation in CRPS. Potentially, a positive feedback loop between α1-adrenoceptors and IL-6 production in skin cells contributes to this inflammatory state.

Descending projections from neurons in the rostral ventromedial medulla (RVM) make synapses within the superficial dorsal horn (SDH) of the spinal cord that are involved in the modulation of nociception, the development of chronic pain and itch, and an important analgesic target for opioids. This projection is primarily inhibitory, but the relative contribution of GABAergic and glycinergic transmission is unknown and there is limited knowledge about the SDH neurons targeted. Additionally, the details of how spinal opioids mediate analgesia remain unclear, and no study has investigated the opioid modulation of this synapse. We address this using ex vivo optogenetic stimulation of RVM fibres in conjunction with whole-cell patch-clamp recordings from the SDH in spinal cord slices. We demonstrate that both GABAergic and glycinergic neurotransmission is employed and show that SDH target neurons have diverse morphological and electrical properties, consistent with both inhibitory and excitatory interneurons. Then, we describe a subtype of SDH neurons that have a glycine-dominant input, indicating that the quality of descending inhibition across cells is not uniform. Finally, we discovered that the kappa-opioid receptor agonist U69593 presynaptically suppressed most RVM-SDH synapses. By contrast, the mu-opioid receptor agonist DAMGO acted both pre- and post-synaptically at a subset of synapses, and the delta-opioid receptor agonist deltorphin II had little effect. These data provide important mechanistic information about a descending control pathway that regulates spinal circuits. This information is necessary to understand how sensory inputs are shaped and develop more reliable and effective alternatives to current opioid analgesics. Abstract figure legend We combined ex vivo optogenetic stimulation of RVM fibres with whole cell electrophysiology of SDH neurons to investigate the final synapse in a key descending pain modulatory pathway. We demonstrate that both glycine and GABA mediate signalling at the RVM-SDH synapse, that the SDH targets of RVM projections have diverse electrical and morphological characteristics, and that presynaptic inhibition is directly and consistently achieved by kappa opioid agonists. Opioid receptors shown are sized relative to the proportion of neurons that responded to its specific agonists (81 and 84percent of DF and non-DF neurons responded to kappa opioid receptor agonists, respectively. Responses that occurred in <255 percentage of neurons are not indicated here). This article is protected by copyright. All rights reserved.