Sarah M. Sweitzer

Antinociceptive action of a p38α MAPK inhibitor, SD-282, in a diabetic neuropathy model

&NA; Diabetes can induce a bewildering list of sensory changes, including alteration in pain sensitivity. Painful diabetic neuropathy is refractory to most common analgesics. This study examined the effect of a p38&agr; MAPK inhibitor, SD‐282, on mechanical allodynia, thermal hyperalgesia, and formalin‐evoked nociception in streptozotocin‐induced diabetic rats. Four‐week diabetic rats exhibited mechanical allodynia, decreased mechanical thresholds, and C‐ and A&dgr;‐fiber mediated thermal hyperalgesia. Mechanical and thermal responses were measured in diabetic rats following acute and repeated intraperitoneal administration of vehicle, 15 or 45 mg/kg SD‐282. Mechanical allodynia was reversed by acute and repeated administration of 15 and 45 mg/kg SD‐282. Repeated administration of 15 or 45 mg/kg SD‐282 prevented the exacerbation of C‐, but not A&dgr;‐fiber, mediated thermal hyperalgesia. Repeated administration of 45 mg/kg SD‐282 attenuated flinching behaviors during the quiescent period and the second phase of the formalin response in diabetic rats. Acute and repeated administration of 15 or 45 mg/kg SD‐282 had no effect on mechanical, thermal or formalin responses in age‐matched control rats. These results indicate a potential therapeutic value of p38&agr; MAPK inhibitors in the treatment of aberrant pain sensitivity produced by diabetes.

Exaggerated nociceptive responses on morphine withdrawal: roles of protein kinase C ε and γ

&NA; On withdrawal from opioids many patients experience a heightened sensitivity to stimuli and an exaggerated pain response. The phenomenon has been little studied in infants. We present evidence that in postnatal day 7 rats an exaggerated nociceptive ventral root response of spinal cords in vitro and withdrawal‐associated thermal hyperalgesia in vivo are dependent on protein kinase C (PKC), and we document the roles of PKC &egr; and &ggr; isozymes. In vitro, the slow ventral root potential (sVRP) is a nociceptive‐related response in spinal cord that is depressed by morphine and recovers to levels significantly above control on administration of naloxone. A broad‐spectrum PKC antagonist, GF109213X, blocked withdrawal hyperresponsiveness of the sVRP whereas an antagonist specific to Ca++‐dependent isozymes, Go69076, did not. Consistent with this finding, a specific peptide inhibitor of calcium‐independent PKC &egr;, but not an inhibitor of calcium‐dependent PKC &ggr;, blocked withdrawal hyperresponsiveness of the sVRP. Similarly, in vivo in 7‐day‐old rat pups, inhibition of PKC &egr;, but not PKC &ggr;, prevented thermal hyperalgesia precipitated by naloxone at 30 min post‐morphine. In contrast, thermal hyperalgesia during spontaneous withdrawal was inhibited by both PKC &egr; and &ggr; inhibitors. The consistency between the in vivo and in vitro findings with respect to naloxone‐precipitated withdrawal provides further evidence that the sVRP reflects nociceptive neurotransmission. In addition the difference between naloxone‐precipitated and spontaneous withdrawal in vivo suggests that in postnatal day 7 rats, morphine exposure produces an early phase of primary afferent sensitization dependent upon PKC &egr; translocation, followed by a later phase involving spinal sensitization mediated by PKC &ggr;.

Peripheral and central p38 MAPK mediates capsaicin-induced hyperalgesia

Abstract The stress‐activated mitogen‐activated protein kinase (MAPK) p38 is emerging as an important mediator of pain. The present study examined the possible involvement of peripheral and spinal p38 MAPK in capsaicin‐induced thermal hyperalgesia. Topical capsaicin produced phosphorylation of p38 MAPK in the skin from the affected hindpaw as well as the corresponding lumbar spinal cord in a time dependent manner. Topical capsaicin produced robust C‐fiber mediated thermal hyperalgesia that was inhibited by systemic, local peripheral, or central intrathecal pre‐treatment with the p38 MAPK inhibitor, SD‐282. Intraperitoneal SD‐282 (10–60 mg/kg) significantly and dose‐dependently attenuated capsaicin‐induced C‐fiber mediated thermal hyperalgesia. Similarly, 0.1–5 mg/kg subcutaneous SD‐282 in the hindpaw dose‐dependently attenuated capsaicin‐induced thermal hyperalgesia. Intrathecal administration of 1 μg SD‐282 was also anti‐hyperalgesic in this model. Functionally, SD‐282 decreased capsaicin‐induced release of calcitonin gene related peptide in an in vitro skin release assay, consistent with a role for p38 MAPK in peripheral nerve function. These results suggest that p38 MAPK plays a role in the development of hyperalgesic states, exerting effects both centrally in the spinal cord and peripherally in sensory C fibers.

Differential Opioid Inhibition of C- and Aδ- Fiber Mediated Thermonociception After Stimulation of the Nucleus Raphe Magnus

Although the importance of the nucleus raphe magnus in descending inhibitory control of nociception is clear, it is not known whether these effects are equivalent for different types of nociception. Thus, we examined the differential inhibition of behavioral responses evoked by A&dgr; or C fiber thermonociceptor activation by electrical stimulation of nucleus raphe magnus neurons as well as the involvement of different classes of opiate receptors in this inhibition. In general, it was necessary to apply twice as much current to the nucleus raphe magnus to produce criterion antinociception for A&dgr; mediated versus C fiber mediated nociceptive responses. Intrathecal administration of the nonselective opioid receptor antagonist, naltrexone, or the &dgr;1 opioid receptor antagonist, naltrindole, attenuated both A&dgr; and C fiber antinociception induced by nucleus raphe magnus stimulation with similar efficacy. In contrast, intrathecal administration of naloxonazine, a &mgr; specific opioid receptor antagonist, or naltriben, a &dgr;2 specific opioid receptor antagonist, preferentially attenuated nucleus raphe magnus induced antinociception for C fiber responses when compared with A&dgr; mediated responses. These findings suggest that nociception evoked by the activation of A&dgr; or C fiber nociceptors is under pharmacologically distinguishable descending control from the nucleus raphe magnus.

Afferent fiber-selective shift in opiate potency following targeted opioid receptor knockdown.

&NA; Spinal application of opiates is the cornerstone of potent analgesia. In the present study, opiate analgesia was investigated after cutaneous application of a recombinant herpes simplex virus type‐1 (HSV‐1) encoding &mgr;‐opioid receptor (&mgr;OR) cDNA in reverse orientation with respect to the human cytomegalovirus early enhancer‐promoter. Hind paw application of this recombinant vector was used in order to attenuate expression of the &mgr;OR in primary afferents and determine whether recombinant vector application would differentially affect the antinociceptive effects of the specific &mgr;OR agonist, [d‐Ala2,N‐MePhe4,Gly‐ol5] enkephalin (DAMGO), on behavioral responses mediated by C‐ and A&dgr;‐thermonociceptors. The recombinant vector encoding the Escherichia coli lacZ gene marker, KHZ, served as a control virus. Dorsal hind paw surfaces of female Swiss‐Webster mice were treated with one of these two viruses (1×108 pfu, 10 &mgr;l) or vehicle (uninfected). Immunohistochemistry and quantitative image analyses revealed decreased &mgr;OR expression in the superficial dorsal horns ipsilateral to hind paws treated with AMOR, but not KHZ. To add, behavioral foot withdrawal latencies of AMOR‐ and KHZ‐treated hind paws demonstrated dose‐dependent antinociception after intrathecal DAMGO administration. However, cutaneous application of dorsal hind paw surfaces treated with AMOR, but not KHZ, caused a rightward shift in the C‐fiber dose–response, thus, indicating a loss of potency of intrathecal DAMGO. Loss or diminution of DAMGO potency during A&dgr;‐fiber‐mediated responses was not observed. These immunohistochemistry and behavioral results of novel, recombinant HSV‐1 vector &mgr;OR ‘knock‐down’ in nociceptor afferent fibers provide additional evidence for presynaptic localization of &mgr;ORs on central C‐, but not A&dgr;‐terminals.

Mechanical allodynia and thermal hyperalgesia upon acute opioid withdrawal in the neonatal rat

&NA; Upon withdrawal from opioids many patients experience a heightened sensitivity to stimuli and an exaggerated pain response. We present evidence that neonatal rats exhibit allodynia and hyperalgesia on acute opiate withdrawal. Postnatal 7 and 21 day rats were used to approximately model a full term human infant and a human child, respectively. The opiate antagonist naloxone was used to precipitate withdrawal at 30 or 120 min after a single acute administration of morphine. Alternatively, rats were allowed to undergo spontaneous withdrawal. Behavioral manifestations of withdrawal syndrome were not observed when naloxone was administered at 30 min post‐morphine, but were present when withdrawal was precipitated at 120 min. Spontaneous and precipitated withdrawal from a single acute administration of morphine produced mechanical allodynia and thermal hyperalgesia in postnatal day 7 rats and mechanical allodynia in postnatal day 21 rats. A higher dose of morphine was required to produce mechanical allodynia in postnatal day 21 versus 7 rats but this increase was independent of the analgesic efficacy of morphine at these two ages. The present work illustrates the need to examine the phenomenon of hypersensitivity upon opioid withdrawal in the human pediatric population.

GABAB receptors on central terminals of C-afferents mediate intersegmental Aδ-afferent evoked hypoalgesia

The current study tested the hypothesis that repetitive activation of sciatic Aδ‐afferents evokes a saphenous C‐afferent hypoalgesia mediated by pre‐synaptic GABAB receptors. Tonic activation of sciatic Aδ‐afferents was produced by cutaneous application of dimethyl sulfoxide (DMSO) followed by repetitive thermal activation of Aδ‐afferents on the dorsolateral hind paw. The tonic activation of sciatic Aδ‐afferents produced hypoalgesia in saphenous C‐afferents. Intrathecal administration of the GABAB receptor antagonist, saclofen, attenuated saphenous hypoalgesia demonstrating at least partial mediation by central GABAB receptors. To determine if this central GABAB receptor activation occurs at pre‐synaptic primary afferent terminals or postsynaptic spinal cord neurons, the dorsal hind paws of mice were infected with a recombinant herpes simplex virus type 1 (HSV‐1) designed to selectively knock down expression of the GABAB1a receptor subunit (PAGB1a) in primary afferents or a control virus encoding the E. coli lacZ gene (PZ). Four weeks after infection, GABAB receptor immunoreactivity in the superficial dorsal horns ipsilateral to PAGB1a application was reduced and hypoalgesia in saphenous C‐afferents was attenuated when compared to PZ‐infected mice. These findings indicate an intersegmental, sciatic Aδ‐afferent‐evoked hypoalgesic effect on saphenous C‐afferent responses that is mediated by pre‐synaptic GABAB receptors on the terminals of those C‐afferents.

Acute and chronic ethanol exacerbates formalin pain in neonatal rats

We have previously reported that withdrawal from acute ethanol (EtOH) exposure lowers mechanical thresholds in post-natal day 7 (P7) and post-natal day 21 (P21) rats. The present study tested the hypothesis that daily administration of 4 g/kg 15% EtOH for 5 days in rats during the human developmental equivalent of the third trimester, but not at a later time in development, would alter mechanical thresholds and formalin-induced pain behaviors. A transient decrease in mechanical thresholds (allodynia) was observed in P7 rats upon withdrawal from repeated EtOH between P3 and P7. When challenged with intraplantar formalin on P11, rats exposed to acute or chronic EtOH had enhanced phase II pain behaviors. In contrast to chronic EtOH administration to rats between P3 and P7, prolonged mechanical allodynia was observed in P21 rats upon withdrawal from chronic EtOH between P17 and P21. Formalin responses were unchanged in P25 rats exposed to acute or chronic EtOH. The affects of EtOH on somatosensory processing are dependent upon the age at which exposure occurs.

Hyperresponsiveness on washout of volatile anesthetics from isolated spinal cord compared to withdrawal from ethanol

We performed experiments in spinal cords isolated from neonatal rats to probe the mechanisms responsible for hyperresponsiveness of the population excitatory evoked potential (pEPSP) observed on washout of the volatile anesthetics halothane and isoflurane (1 minimal alveolar anesthetic concentration equivalent, MAC) compared with that observed after an anesthetic concentration of ethanol. After 30 min exposure to each anesthetic and washout, pEPSP area increased to levels significantly more than control (P < 0.01–0.001). Exposure to a very small (0.025 MAC) concentration of isoflurane over the same period itself produced a similarly exaggerated pEPSP (P < 0.05) in the continued presence of the drug, suggesting that the phenomenon is a direct excitatory effect of the small concentrations of anesthetic on washout, unlike the true withdrawal observed with ethanol. Isoflurane, but not halothane, significantly increased the amount of potassium-stimulated release of the excitatory neurotransmitters glutamate, aspartate, and substance P, suggesting the hyperresponsiveness for that drug is the result of a presynaptically mediated increase in transmitter release. A broad spectrum specific protein kinase C inhibitor, GF109203X, blocked ethanol withdrawal hyperresponsiveness but not hyperresponsiveness after halothane. If the behavioral symptoms of emergence from anesthesia are based on excitatory actions similar to those observed in the spinal cord, the results show that they represent direct excitatory actions rather than withdrawal and are attributable to direct actions on ion channels or receptors, rather than indirect effects mediated by protein kinase C.

Acute and chronic ethanol does not affect incisional pain in neonatal rats

We have previously found that in post-natal day 7 rats withdrawal from acute and chronic ethanol (EtOH) exposure lowers mechanical thresholds during withdrawal and exacerbates spontaneous pain responses to an inflammatory injury 4 days post-withdrawal. These findings suggested alterations in somatosensory pathways following EtOH exposure during the third trimester developmental equivalent. In this study we wanted to determine whether EtOH exposure during the third trimester equivalent exacerbates mechanical allodynia and thermal hyperalgesia produced by an incisional model of post-operative pain at post-natal day 21. The extent and duration of mechanical allodynia and thermal hyperalgesia following incision was measured and found to be unaffected by prior EtOH exposure.