Inhibition of Fatty Acid Binding Proteins Elevates Brain Anandamide Levels and Produces Analgesia

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics

Inhibition of Tetrodotoxin-Resistant Sodium Current in Dorsal Root Ganglia Neurons Mediated by D1/D5 Dopamine Receptors

BACKGROUND: Dopaminergic fibers originating from area A11 of the hypothalamus project to different levels of the spinal cord and represent the major source of dopamine. In addition, tyrosine hydroxylase, the rate-limiting enzyme for the synthesis of catecholamines, is expressed in 8-10% of dorsal root ganglia (DRG) neurons, suggesting that dopamine may be released in the dorsal root ganglia. Dopamine has been shown to modulate calcium current in DRG neurons, but the effects of dopamine on sodium current and on the firing properties of small DRG neurons are poorly understood. RESULTS: The effects of dopamine and dopamine receptor agonists were tested on the tetrodotoxin-resistant (TTX-R) sodium current recorded from acutely dissociated small (diameter ≤ 25 μm) DRG neurons. Dopamine (20 μM) and SKF 81297 (10 μM) caused inhibition of TTX-R sodium current in small DRG neurons by 23% and 37%, respectively. In contrast, quinpirole (20 μM) had no effects on the TTX-R sodium current. Inhibition by SKF 81297 of the TTX-R sodium current was not affected when the protein kinase A (PKA) activity was blocked with the PKA inhibitory peptide (6–22), but was greatly reduced when the protein kinase C (PKC) activity was blocked with the PKC inhibitory peptide (19–36), suggesting that activation of D1/D5 dopamine receptors is linked to PKC activity. Expression of D1and D5 dopamine receptors in small DRG neurons, but not D2 dopamine receptors, was confirmed by Western blotting and immunofluorescence analysis. In current clamp experiments, the number of action potentials elicited in small DRG neurons by current injection was reduced by ~ 30% by SKF 81297. CONCLUSIONS: We conclude that activation of D1/D5 dopamine receptors inhibits TTX-R sodium current in unmyelinated nociceptive neurons and dampens their intrinsic excitability by reducing the number of action potentials in response to stimulus. Increasing or decreasing levels of dopamine in the dorsal root ganglia may serve to adjust the sensitivity of nociceptors to noxious stimuli

Transcriptomic evidence of a para-inflammatory state in the middle aged lumbar spinal cord

Abstract Background We have previously reported elevated expression of multiple pro-inflammatory markers in the lumbar spinal cord (LSC) of middle-aged male rats compared to young adults suggesting a para-inflammatory state develops in the LSC by middle age, a time that in humans is associated with the greatest pain prevalence and persistence. The goal of the current study was to examine the transcriptome-wide gene expression differences between young and middle aged LSC. Methods Young (3 month) and middle-aged (17 month) naïve Fisher 344 rats (n = 5 per group) were euthanized, perfused with heparinized saline, and the LSC were removed. Results ~70% of 31,000 coding sequences were detected. After normalization, ~ 1100 showed statistically significant differential expression. Of these genes, 353 middle-aged annotated genes differed by > 1.5 fold compared to the young group. Nearly 10% of these genes belonged to the microglial sensome. Analysis of this subset revealed that the principal age-related differential pathways populated are complement, pattern recognition receptors, OX40, and various T cell regulatory pathways consistent with microglial priming and T cell invasion and modulation. Many of these pathways substantially overlap those previously identified in studies of LSC of young animals with chronic inflammatory or neuropathic pain. Conclusions Up-modulation of complement pathway, microglial priming and activation, and T cell/antigen-presenting cell communication in healthy middle-aged LSC was found. Taken together with our previous work, the results support our conclusion that an incipient or para-inflammatory state develops in the LSC in healthy middle-aged adults

Neuroimmune and Neuropathic Responses of Spinal Cord and Dorsal Root Ganglia in Middle Age

<div><p>Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from aged-matched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFβ1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middle-aged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and T-cell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain.</p></div