Kelley F. Kitto

Involvement of nitric oxide in spinally mediated hyperalgesia in the mouse.

Intrathecal injection of N-methyl-D-aspartate (NMDA) induces a short duration hyperalgesia in mice. An inhibitor of nitric oxide synthase (NOS), N omega-nitro-L-arginine methyl ester (L-NAME), administered either systemically or intrathecally, blocked the NMDA-induced hyperalgesia. This effect was partially reversed by the NOS substrate, L-arginine. Intrathecal hemoglobin mimicked the effects of L-NAME. Intrathecal injection of the NO-donating compounds, sodium nitroprusside (SNP) and hydroxylamine, resulted in a hyperalgesia that lasted 3 h and was reduced by coadministration of hemoglobin. Thus, nitric oxide production appears to mediate NMDA-induced hypersensitivity and may contribute to other forms of centrally induced hyperalgesia.

The α2a Adrenergic Receptor Subtype Mediates Spinal Analgesia Evoked by α2 Agonists and Is Necessary for Spinal Adrenergic–Opioid Synergy

Agonists acting at α2 adrenergic and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord; this synergy may also contribute to the potency and efficacy of spinally administered morphine. The lack of subtype-selective pharmacological agents has previously impeded the definition of the adrenergic receptor subtype(s) mediating these effects. We therefore exploited a genetically modified mouse line expressing a point mutation (D79N) in the α2a adrenergic receptor (α2aAR) to investigate the role of the α2aAR in α2 agonist-evoked analgesia and adrenergic–opioid synergy. In the tail-flick test, intrathecal administration of UK 14,304, a nonsubtype-selective α2AR agonist, had no analgesic effect in D79N mice, whereas the analgesic potency of morphine (intrathecal) in this assay was not affected by the mutation. The mutation also decreased α2-agonist-mediated spinal analgesia and blocked the synergy seen in wild-type mice with both the δ-opioid agonist deltorphin II and the μ-opioid agonist [d-ALA2,N-Me-Phe4,Gly-ol5]-Enkephalin (DAMGO) in the substance P behavioral test. In addition, the potency of spinally administered morphine was decreased in this test, suggesting that activation of descending noradrenergic systems impinging on the α2aAR contributes to morphine-induced spinal inhibition in this model. These results demonstrate that the α2aAR subtype is the primary mediator of α2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids. Thus, combination therapies targeting the α2aAR and opioid receptors may prove useful in maximizing the analgesic efficacy of opioids while decreasing total dose requirements.

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.

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.

Moxonidine, a selective imidazoline/α2 adrenergic receptor agonist, synergizes with morphine and deltorphin II to inhibit substance P-induced behavior in mice

Abstract The &agr;2 adrenergic receptor (AR) class of catecholamine/imidazoline (I) agonists, such as norepinephrine and clonidine, produce spinal antinociceptive synergy when co‐administered with opioids. We have observed that intrathecally administered moxonidine, a selective I1/&agr;2 (AR) agonist, produces antinociception. The present experiments tested moxonidine for ability to synergize with morphine, deltorphin II, and DAMGO (Tyr‐D‐Ala‐NMe‐Phe‐Gly(ol)) to inhibit substance P‐elicted nociceptive behavior in Institute of Cancer Research mice. Moxonidine, morphine, deltorphin II, and DAMGO inhibited substance P‐elicited nociceptive behavior with full efficacy. Effective dose 50% (ED50) values were calculated and equi‐effective dose ratios of the combinations moxonidine‐morphine, moxonidine‐deltorphin II, and moxonidine‐DAMGO were determined. The interactions were tested by isobolographic analysis. The observed ED50 values of the combinations were statistically compared against their respective calculated theoretical additive ED50 values. The combinations of moxonidine‐morphine and moxonidine‐deltorphin II resulted in significant leftward shifts in the dose‐response curves compared to those of each agonist administered separately. The ED50 values of the dose‐response curves of these combinations were significantly less than the corresponding calculated theoretical additive ED50 values; these results indicated that moxonidine synergizes with both morphine and deltorphin II. In contrast, combining moxonidine with DAMGO did not increase the potencies of the agonists (in combination) when compared to the potencies of each agonist administered separately. These results indicated that the moxonidine‐DAMGO interaction is subadditive. Collectively, these data demonstrate that moxonidine combined with some opioid agonists produces spinal antinociceptive synergy. Spinally administered moxonidine‐opioid combinations may prove an effective therapeutic strategy to manage pain.

Transient changes in the synthesis of nitric oxide result in long-term as well as short-term changes in acetic acid-induced writhing in mice

&NA; A single injection of nitric oxide (NO) synthase (NOS) inhibitors prevents the development of persistent hyperalgesia induced by various manipulations, suggesting that NO precipitates long‐term changes in nociception. We examined the possibility that inhibition of NOS may also be sufficient to produce long‐term decreases in nociceptive assays, such as writhing, that are known to be sensitive to the short‐term effects of NOS inhibitors. We characterized short‐ and long‐term effects of NOS inhibitors, N&ohgr;‐nitro‐L‐arginine (L‐NAME) or 7‐nitro indazole (7‐NI) injected intrathecally (i.t.) in mice on acetic acid‐induced writhing. Doses of L‐NAME that had no effect on hot plate or tail flick latencies inhibited writhing (0.01–30 nmol) as well as spinal nNOS activity (5 and 100 nmol) when injected i.t. 60–90 min before testing. Anti‐nociception was not mimicked by D‐NAME but was prevented by co‐administration with the NO precursor, L‐arginine. Injection i.t. of 7‐NI (30 min), a selective inhibitor of neuronal NOS (nNOS), inhibited NOS activity in the spinal cord and produced anti‐nociception, confirming that writhing is sensitive to inhibition of nNOS. Although the acute action of both NOS inhibitors dissipated completely by 3–6 h, a delayed and prolonged inhibition of writhing was again observed 24 h after L‐NAME (5–100 nmol), a time when spinal NOS activity was no longer inhibited by L‐NAME (5 and 100 nmol) or 7‐NI (25 nmol). This novel effect appears to be initiated by the transient inhibition of nNOS as delayed anti‐nociception was mimicked by 7‐NI at doses (10–100 nmol) that no longer inhibited spinal nNOS (25 nmol) at 24 h. Co‐administration with L‐arginine prevented the delayed (24 h) anti‐nociceptive effects of L‐NAME (30 nmol). L‐Arginine (30 and 100 nmol) was without effect on nociception when administered alone 60 min or 24 h prior to testing. Together these data indicate that brief changes in the activity of nNOS induce both long‐ as well as short‐term changes in nociception.

Proteomic Analysis Uncovers Novel Actions of the Neurosecretory Protein VGF in Nociceptive Processing

Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression after nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-inducible protein VGF (nonacronymic), a putative neuropeptide precursor. In a rodent model of nerve injury, VGF levels were increased within 24 h in both injured and uninjured DRG neurons, and the increase persisted for at least 7 d. VGF was also upregulated 24 h after hindpaw inflammation. To determine whether peptides derived from proteolytic processing of VGF participate in nociceptive signaling, we examined the spinal effects of AQEE-30 and LQEQ-19, potential proteolytic products shown previously to be bioactive. Each peptide evoked dose-dependent thermal hyperalgesia that required activation of the mitogen-activated protein kinase p38. In addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intrathecally and in the BV-2 microglial cell line when applied in vitro. In summary, our results demonstrate rapid upregulation of VGF in sensory neurons after nerve injury and inflammation and activation of microglial p38 by VGF peptides. Therefore, VGF peptides released from sensory neurons may participate in activation of spinal microglia after peripheral tissue injury.

Parenterally administered kainic acid induces a persistent hyperalgesia in the mouse and rat.

Nociceptive primary afferent C-fibers express a subset of glutamate receptors that are sensitive to kainic acid. Thus, we tested the possibility that activation of these receptors alters nociception. Intraperitoneal (i.p.) injection of kainic acid induced a persistent thermal hyperalgesia, when tested using the hot plate (mice) and tail flick (mice and rats) assays, and mechanical hyperalgesia when tested using von Frey monofilaments (rats), but had no effect on acetic acid-induced chemical nociception (mice). When administered i. p., 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an (R, S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid HBr/kainate (AMPA/KA) antagonist, completely blocked hyperalgesia. When injected intrathecally (i.t.), kainic acid itself failed to induce hyperalgesia and AMPA/KA antagonists given i.t. also failed to attenuate the hyperalgesic effect of kainic acid administered i.p. , indicating that the spinal cord is not the primary site of action. Kainic acid injected subcutaneously in the back of mice decreased response latencies in the hot plate and tail flick assays, indicating that hyperalgesia is achieved by a variety of parenteral routes of injection. Histological evaluation of rat spinal cord and dorsal root ganglia revealed no neurodegenerative changes 24 h after kainic acid. Together these data suggest that a persistent hyperalgesia results from the transient activation of AMPA/KA receptors that are located outside the spinal cord, perhaps on the distal projections of primary afferent fibers.