Alternative Splicing of G Protein–Coupled Receptors: Relevance to Pain Management

Drugs that target G-protein coupled receptors (GPCRs) represent the primary treatment strategy for patients with acute and chronic pain; however, there is substantial individual variability in both the efficacy and adverse side effects associated with these drugs. Variability in drug responses is, in part, due to individuals’ diversity in alternative splicing of pain-relevant GPCRs. GPCR alternative splice variants often exhibit distinct tissue distribution patterns, drug binding properties, and signaling characteristics that may impact disease pathology as well as the size and direction of analgesic effects. Here, we review the importance of GPCRs and their known splice variants to the management of pain

Novel intrathecal and subcutaneous catheter delivery systems in the mouse

Catheter systems that permit targeted delivery of genes, molecules, ligands, and other agents represent an investigative tool critical to the development of clinically relevant animal models that facilitate the study of neurological health and disease. The development of new sustained catheter delivery systems to spinal and peripheral sites will reduce the need for repeated injections, while ensuring constant levels of drug in plasma and tissues

Mu Opioid Splice Variant MOR-1K Contributes to the Development of Opioid-Induced Hyperalgesia

A subset of the population receiving opioids for the treatment of acute and chronic clinical pain develops a paradoxical increase in pain sensitivity known as opioid-induced hyperalgesia. Given that opioid analgesics are one of few treatments available against clinical pain, it is critical to determine the key molecular mechanisms that drive opioid-induced hyperalgesia in order to reduce its prevalence. Recent evidence implicates a splice variant of the mu opioid receptor known as MOR-1K in the emergence of opioid-induced hyperalgesia. Results from human genetic association and cell signaling studies demonstrate that MOR-1K contributes to decreased opioid analgesic responses and produces increased cellular activity via Gs signaling. Here, we conducted the first study to directly test the role of MOR-1K in opioid-induced hyperalgesia

Nuclear factor-kappa B regulates pain and COMT expression in a rodent model of inflammation

Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed protein complex regulating the transcription of genes involved in inflammation and pain. Increased NF-κB activity in immune and nervous system cells is linked to several chronic pain conditions in humans as well as inflammation- and nerve injury-evoked pain in animals. A recent in vitro study further demonstrates that increased NF-κB activity in astrocytes decreases transcription of catechol-o-methyltransferase (COMT), an enzyme that inactivates catecholamines that cause pain. The purpose of the present study was to examine the relationship between systemic and astrocytic NF-κB activity, pain, and COMT expression in an animal model of inflammation. Results demonstrated that administration of the inflammatory stimulant complete Freund’s adjuvant (CFA) led to increased pain and decreased COMT protein expression in an NF-κB-dependent manner. Specifically, we found that rats and mice receiving intraplantar CFA exhibited increased behavioral responses to mechanical and thermal heat stimuli. CFA-evoked pain was blocked in rats receiving a pre-emptive systemic dose of the NF-κB inhibitor MG132 and exacerbated in IKKca mice with constitutive NF-κB activity in astrocytes. Furthermore, we observed NF-κB-linked reductions in COMT expression in midbrain at 6h and 1d following CFA in rats and at 1h and 1d in forebrain and midbrain following CFA in IKKca mice. Collectively, these results demonstrate that systemic and astrocytic NF-κB activity drive inflammatory pain and regulate the expression of COMT in forebrain and midbrain structures

Mu Opioid Splice Variant MOR-1K Contributes to the Development of Opioid-Induced Hyperalgesia.

A subset of the population receiving opioids for the treatment of acute and chronic clinical pain develops a paradoxical increase in pain sensitivity known as opioid-induced hyperalgesia. Given that opioid analgesics are one of few treatments available against clinical pain, it is critical to determine the key molecular mechanisms that drive opioid-induced hyperalgesia in order to reduce its prevalence. Recent evidence implicates a splice variant of the mu opioid receptor known as MOR-1K in the emergence of opioid-induced hyperalgesia. Results from human genetic association and cell signaling studies demonstrate that MOR-1K contributes to decreased opioid analgesic responses and produces increased cellular activity via Gs signaling. Here, we conducted the first study to directly test the role of MOR-1K in opioid-induced hyperalgesia.In order to examine the role of MOR-1K in opioid-induced hyperalgesia, we first assessed pain responses to mechanical and thermal stimuli prior to, during, and following chronic morphine administration. Results show that genetically diverse mouse strains (C57BL/6J, 129S6, and CXB7/ByJ) exhibited different morphine response profiles with corresponding changes in MOR-1K gene expression patterns. The 129S6 mice exhibited an analgesic response correlating to a measured decrease in MOR-1K gene expression levels, while CXB7/ByJ mice exhibited a hyperalgesic response correlating to a measured increase in MOR-1K gene expression levels. Furthermore, knockdown of MOR-1K in CXB7/ByJ mice via chronic intrathecal siRNA administration not only prevented the development of opioid-induced hyperalgesia, but also unmasked morphine analgesia.These findings suggest that MOR-1K is likely a necessary contributor to the development of opioid-induced hyperalgesia. With further research, MOR-1K could be exploited as a target for antagonists that reduce or prevent opioid-induced hyperalgesia

Strains exhibit divergent morphine-dependent analgesic and allodynic/hyperalgesic profiles with corresponding changes in <i>MOR-1K</i> gene expression.

<p>(A) A timeline of chronic morphine administration and the assessments of pain behavior and gene expression are shown. (B-E) Compared to C57BL/6J, 129S6 mice exhibit increased paw withdrawal thresholds and fail to develop allodynia or hyperalgesia. In contrast, CXB7/ByJ exhibit decreased paw withdrawal thresholds and increased allodynia and hyperalgesia. (F) 129S6 mice exhibit decreased <i>MOR-1K</i> gene expression levels on days 1 and 4, corresponding to their analgesic behavioral profile. In contrast, CXB7/ByJ mice exhibit increased <i>MOR-1K</i> gene expression levels on day 4, corresponding to their hyperalgesic behavioral profile. For behavioral graphs, blue background indicates a decrease in pain sensitivity (analgesia), and red background indicates an increase in pain sensitivity (allodynia/hyperalgesia). Panels B-E: <i>N</i> = 15-16/group. Data expressed as mean ± SEM. <i>***p<</i>0.001, <i>**p<</i>0.01, *<i>p</i><0.05 different from C57BL/6J. ^###<i>p<</i>0.001, ^##<i>p</i><0.01, ^#<i>p</i><0.05 different from 129S6. Panel F: <i>N</i> = 7/group. Data expressed as means ± SEM. *<i>p</i><0.05 different from baseline.</p

Strains exhibit divergent behavioral responses to mechanical stimuli at baseline.

<p>Compared to C57BL/6J mice, 129S6, and CXB7/ByJ mice exhibit differences in (A) paw withdrawal threshold, (B) the number of responses to repeated presentation of a noxious mechanical stimulus sensitivity, and (C) the number of responses to continuous thermal heat. <i>N</i> = 15-16/group. Data expressed as mean ± SEM. ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 different from C57BL/6J. ^###<i>p</i><0.001 different from 129S6.</p

CXB7/ByJ mice treated with exon 13 antisense siRNA fail to develop OIH.

<p>(A) A timeline of sustained i.t. siRNA delivery, chronic morphine administration, and assessments of pain behavior and gene expression is shown. CXB7/ByJ mice receiving antisense siRNA exhibit analgesia following morphine administration and cessation, and fail to exhibit (B) decreased paw withdrawal thresholds, or increased responses to (C) repeated presentation of an innocuous or (D) noxious mechanical stimulus. In contrast, mice receiving sense siRNA or sham exhibited analgesia following morphine administration, which was then followed by allodynia/hyperalgesia on days 5–7. (E) CXB7/ByJ mice receiving antisense siRNA exhibit decreased <i>MOR-1K</i> gene expression levels in the spinal cord as compared to those receiving sense siRNA. For all behavioral graphs, blue background indicates a decrease in pain sensitivity (analgesia), and red background indicates an increase in pain sensitivity (allodynia/hyperalgesia). Panels B-D: <i>N</i> = 6-9/group. Data expressed as mean ± SEM.***<i>*p</i><0.0001, ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05 different from Sham. Panel E: <i>N =</i> 3-4/group. **<i>p</i><0.01 different from Sense.</p

Novel intrathecal and subcutaneous catheter delivery systems in the mouse

BACKGROUND: Catheter systems that permit targeted delivery of genes, molecules, ligands, and other agents represent an investigative tool critical to the development of clinically relevant animal models that facilitate the study of neurological health and disease. The development of new sustained catheter delivery systems to spinal and peripheral sites will reduce the need for repeated injections, while ensuring constant levels of drug in plasma and tissues. NEW METHOD: Here, we introduce two novel catheter delivery systems in the mouse: the O’Buckley intrathecal catheter system for sustained delivery to the spinal region and a subcutaneous bifurcated catheter system for sustained drug delivery to both hindpaws. RESULTS: The O’Buckley intrathecal catheter system consistently distributed Evans Blue throughout the spinal cord, with the greatest concentration at the thoracic region, and with an 85% surgery success rate. The subcutaneous catheter system consistently distributed Evans Blue to the hindlimbs, with a 100% surgery success rate. COMPARISON TO EXISTING METHOD: The O’Buckley intrathecal catheter system accomplishes sustained drug delivery to the spinal region, with a 2-fold increase in surgery success rate, as compared to the traditional method. Our subcutaneous bifurcated catheter system accomplishes sustained drug delivery to both hindpaws, eliminating the need for repeated intraplantar injections. CONCLUSIONS: We have developed catheter systems that improve upon traditional methods in order to achieve sustained localized drug delivery to spinal tissues and to hindpaw tissues surrounding peripheral sciatic nerve terminals. These methods have a broad reach, and can be used to enhance behavioral, physiologic and mechanistic studies in mice