Carolyn A. Fairbanks

Spinal analgesic actions of the new endogenous opioid peptides endomorphin-1 and -2

TWO highly-selective μ-opioid receptor agonists, endomorphin-1 and -2, were recently purified from bovine brain and are postulated to be endogenous μ-opioid receptor ligands. We sought to determine the effects of these ligands at the spinal level in mice. Endomorphin1 and -2 produced short acting, naloxone-sensitive antinociception in the tail flick test and inhibited the behavior elicited by intrathecally injected substance P. Both endomorphin-1 and -2 were anti-allodynic in the dynorphin-induced allodynia model. Although acute tolerance against both endomorphins developed rapidly, endomorphin-1 required a longer pretreatment time before tolerance was observed. We conclude that the endomorphins are potent spinal antinociceptive and anti-allodynic agents and that they or related compounds may prove therapeutically useful as spinal analgesics.

Spinal delivery of analgesics in experimental models of pain and analgesia

Systemic administration of analgesics can lead to serious adverse side effects compromising therapeutic benefit in some patients. Information coding pain transmits along an afferent neuronal network, the first synapses of which reside principally in the spinal cord. Delivery of compounds to spinal cord, the intended site of action for some analgesics, is potentially a more efficient and precise method for inhibiting the pain signal. Activation of specific proteins that reside in spinal neuronal membranes can result in hyperpolarization of secondary neurons, which can prevent transmission of the pain signal. This is one of the mechanisms by which opioids induce analgesia. The spinal cord is enriched in such molecular targets, the activation of which inhibit the transmission of the pain signal early in the afferent neuronal network. This review describes the pre-clinical models that enable new target discovery and development of novel analgesics for site-directed pain management.

Agmatine: clinical applications after 100 years in translation.

Agmatine (decarboxylated arginine) has been known as a natural product for over 100 years, but its biosynthesis in humans was left unexplored owing to long-standing controversy. Only recently has the demonstration of agmatine biosynthesis in mammals revived research, indicating its exceptional modulatory action at multiple molecular targets, including neurotransmitter systems, nitric oxide (NO) synthesis and polyamine metabolism, thus providing bases for broad therapeutic applications. This timely review, a concerted effort by 16 independent research groups, draws attention to the substantial preclinical and initial clinical evidence, and highlights challenges and opportunities, for the use of agmatine in treating a spectrum of complex diseases with unmet therapeutic needs, including diabetes mellitus, neurotrauma and neurodegenerative diseases, opioid addiction, mood disorders, cognitive disorders and cancer.

Transcutaneous electrical nerve stimulation activates peripherally located alpha-2A adrenergic receptors

&NA; The alpha2A and alpha2C adrenergic receptor (AR) subtypes mediate antinociception when activated by the endogenous ligand norepinephrine. These receptors also produce antinociceptive synergy when activated concurrently with opioid receptor activation. The involvement of the opioid receptors in the mechanisms governing transcutaneous electrical nerve stimulation (TENS) has been well described. While spinal alpha‐2 ARs do not appear to be involved in TENS antihyperalgesia in rats, the noradrenergic analgesic system also involves supraspinal and peripheral sites. Thus, a broader evaluation of the potential contribution of alpha‐2 AR to TENS is warranted. The current study compared the antihyperalgesic efficacy of high (100 Hz) and low (4 Hz) frequency TENS in mutant mice lacking a functional alpha2A AR against their respective wildtype counterparts. The degree of secondary heat hyperalgesia induced by intra‐articular injection of carrageenan/kaolin (3%) mixture did not differ among the experimental groups. However, the antihyperalgesia induced by both low and high frequency TENS was significantly diminished in alpha2A mutant mice compared to controls. The alpha2 adrenergic receptor selective antagonist, SK&F 86466, reversed TENS‐mediated antihyperalgesia when delivered intra‐articularly, but not when delivered intrathecally or intracerebroventricularly. These data suggest that peripheral alpha2 ARs contribute, in part, to TENS antihyperalgesia. This pharmacodynamic response is consistent with previous anatomical observations that alpha2A ARs are expressed on primary afferent neurons and macrophages near injured tissue.

Disruption of nNOS-PSD95 protein-protein interaction inhibits acute thermal hyperalgesia and chronic mechanical allodynia in rodents

Background and purpose:  Post‐synaptic density protein 95 (PSD95) contains three PSD95/Dosophilia disc large/ZO‐1 homology domains and links neuronal nitric oxide synthase (nNOS) with the N‐methyl‐D‐aspartic acid (NMDA) receptor. This report assesses the effects of disruption of the protein–protein interaction between nNOS and PSD95 on pain sensitivity in rodent models of hyperalgesia and neuropathic pain.

Pharmacological Profiles of Alpha 2 Adrenergic Receptor Agonists Identified Using Genetically Altered Mice and Isobolographic Analysis

Endogenous, descending noradrenergic fibers impose analgesic control over spinal afferent circuitry mediating the rostrad transmission of pain signals. These fibers target alpha 2 adrenergic receptors (alpha(2)ARs) on both primary afferent terminals and secondary neurons, and their activation mediates substantial inhibitory control over this transmission, rivaling that of opioid receptors which share a similar pattern of distribution. The terminals of primary afferent nociceptive neurons and secondary spinal dorsal horn neurons express alpha(2A)AR and alpha(2C)AR subtypes, respectively. Spinal delivery of these agents serves to reduce their side effects, which are mediated largely at supraspinal sites, by concentrating the drugs at the spinal level. Targeting these spinal alpha(2)ARs with one of five selective therapeutic agonists, clonidine, dexmedetomidine, brimonidine, ST91 and moxonidine, produces significant antinociception that can work in concert with opioid agonists to yield synergistic antinociception. Application of several genetically altered mouse lines had facilitated identification of the primary receptor subtypes that likely mediate the antinociceptive effects of these agents. This review provides first an anatomical description of the localization of the three subtypes in the central nervous system, second a detailed account of the pharmacological history of each of the six primary agonists, and finally a comprehensive report of the specific interactions of other GPCR agonists with each of the six principal alpha(2)AR agonists featured.

Developmental shift of vanilloid receptor 1 (VR1) terminals into deeper regions of the superficial dorsal horn: correlation with a shift from TrkA to Ret expression by dorsal root ganglion neurons

The cloned vanilloid receptor VR1 can be activated by capsaicin and by thermal stimuli. The pattern of nerve terminals that contain VR1 in adult rat spinal cord does not correspond to axons that arise from a single subset of dorsal root ganglion neurons. Thus, we postulated that the basis underlying this complexity might be better understood from a developmental perspective. First, using capsaicin‐induced hyperalgesia as a measure of VR1 function, we found that vanilloid receptors were functional as early as postnatal day 10 (P10), although hyperalgesia was of longer duration in adult. Interestingly, the appearance of VR1 protein in terminals of dorsal root ganglion neurons shifts over this postnatal period. From embryonic day 16 to P20, the majority of VR1 protein in the spinal cord was observed in lamina I. As animals matured, VR1 protein became more abundant in lamina II, particularly in the inner portion. Consistent with these observations, the number of dorsal root ganglion neurons coexpressing VR1 and isolectin B4 binding sites doubled while the number of neurons that had both VR1 and substance P remained relatively constant from P2 to P10. In peripheral processes, the number of VR1‐positive nerve fibres and terminals in cutaneous structures in postnatal day 10 was half of that in adults. We also show that the association of VR1 with Ret is the reciprocal of the association of VR1 with Trk A. These results suggest that neurotrophins may regulate the extent to which populations of dorsal root ganglion neurons express VR1.

Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture

BackgroundNeuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.ResultsIntravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.ConclusionsThe present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.

Protein Kinase C Mediates the Synergistic Interaction Between Agonists Acting at α2-Adrenergic and Delta-Opioid Receptors in Spinal Cord

Coactivation of spinal α2-adrenergic receptors (ARs) and opioid receptors produces antinociceptive synergy. Antinociceptive synergy between intrathecally administered α2AR and opioid agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the α2AARs are coexpressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the α2AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through coadministration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Coadministration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, whereas the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, using a thermal nociceptive test, demonstrating the PKC dependence of CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and α2AR agonists in the spinal cord.

Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse

Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.) or intravenous (i.v.) delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir) was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG) neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir) primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at 6 weeks compared to 3 weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation.