Alberto Subieta

A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat

P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22–92 nM) and was highly selective (IC50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 μmol/kg s.c.) reduced complete Freunds adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

Changes in tissue pH and temperature after incision indicate acidosis may contribute to postoperative pain

Background:Incisional pain is a common form of acute pain. Previously, the authors studied persistent pain behaviors caused by incisions, using animal models for postoperative pain. In this study, the authors measured tissue pH and hind paw temperature before and after incision to understand factors that may activate and sensitize nociceptors in the incision. Methods:Rats underwent a plantar incision, a gastrocnemius muscle incision, or a cutaneous paraspinal incision. For the hind paw incision, pain behaviors were measured. Tissue pH was measured using a pH-sensitive needle electrode in halothane-anesthetized rats. The pH in the incision was compared to a corresponding control site on the contralateral side of the rat or to the sham-operated group. Results:Plantar tissue pH was 7.16 ± 0.04 in sham-operated rats. Ten minutes after plantar incision, tissue pH was decreased to 6.91 ± 0.20 (P < 0.05), and this decrease was sustained through 60 min after incision, when pH was 6.99 ± 0.06 (P < 0.05). Tissue pH values were 6.95, 6.90, 6.89, and 6.95 (P < 0.05 vs. sham) 4 h and 1, 2, and 4 days after incision, respectively. On postoperative day 7, when plantar pH was same as for the control side (7.13 ± 0.05), guarding behavior, heat responses, and responses to mechanical stimuli recovered. Outside the incised area in the hind paw, tissue pH was normal. Tissue pH was significantly correlated with all pain behaviors. In the gastrocnemius muscle, tissue pH was 7.14 ± 0.7 in the sham-operated side. Ten minutes after incision, tissue pH was 6.54 ± 0.12 (P < 0.05), and muscle pH remained decreased through 60 min after gastrocnemius incision when pH was 6.76 ± 0.17 (P < 0.05). Tissue pH was also significantly decreased (P < 0.05) on day 1 (6.96 vs. 7.20) and day 4 (7.06 vs. 7.18) after gastrocnemius incision but was not reduced on postoperative day 8 (7.11 vs. 7.15). A paraspinal incision also decreased tissue pH in the hairy skin of the rat compared with the preincision value. Hind paw skin temperature did not change after incision. Conclusion:A decrease in pH occurs immediately after incision and is sustained for at least 4 days. During the period of decreased tissue pH, pain behaviors are evident. When the tissue pH returns to normal, pain behaviors are diminished. The decreased pH is localized at the incision site and not to areas surrounding the incision. Decreased pH likely contributes to nociceptor sensitization and pain related behaviors after incision. The magnitude of the pH change varies among tissues. An increase in hind paw skin temperature does not play a role in these pain-related behaviors.

Increased nerve growth factor after rat plantar incision contributes to guarding behavior and heat hyperalgesia.

&NA; Acutely, nerve growth factor (NGF) exerts profound effects on nociceptive transmission and produces pain and hyperalgesia. In the present study, we sought to determine the tissue levels and role of NGF after a plantar incision. A substantial increase in NGF protein expression occurred in skin 4‐h, 1‐day and 2‐days and 5‐days after incision comparing contralateral uninjured skin. Plantar incision did not change NGF levels in the tibial nerve and L4‐L6 dorsal root ganglia. The therapeutic effect of a monoclonal antibody against endogenous NGF was evaluated by intraperitoneal administration of a single preoperative dose of anti‐NGF. Of three different doses of anti‐NGF used, the highest dose 2.8 mg/kg anti‐NGF attenuated or almost abolished guarding pain scores at 4‐h, 1‐day (>80% decrease) and 2‐days after incision. This effect is dose dependent in that lower doses (1, 0.1 mg/kg) were effective only at 1‐day after incision. Anti‐NGF also attenuated heat hyperalgesia at 1‐day and 2‐days after incision when the highest dose was used. However, treatment by anti‐NGF did not affect C‐fibers sensitized 1‐day after incision in a glabrous skin‐tibial nerve in vitro preparation. In conclusion, increased NGF was present in skin after plantar incision. NGF contributes to some incision‐induced pain behaviors, guarding and heat hyperalgesia. Anti‐NGF did not affect the extent of sensitization of C‐fibers observed in vitro.

Differential Effect of Capsaicin Treatment on Pain-Related Behaviors After Plantar Incision

UNLABELLED We evaluated the effect of infiltration of dilute solutions of capsaicin, administered before plantar incision, on 3 pain-related behaviors: guarding pain, heat-withdrawal latency, and mechanical-withdrawal threshold. Perineural application of capsaicin was also studied and the appearance of the wound was also evaluated. Dilute solutions of capsaicin .025% and .10% were infiltrated in the plantar region 1 day before incision. In another group of rats, perineural capsaicin (1%) was applied to the nerves innervating the plantar aspect of the rat hindpaw. Rats were then tested for pain-related behaviors before and after plantar incision and then daily thereafter. Wound appearance was graded and histopathology was evaluated. Infiltration with capsaicin reduced guarding pain and heat hyperalgesia after plantar incision; there were minimal effects on mechanical responses. Perineural-capsaicin application produced a similar result. Both capsaicin infiltration and perineural-capsaicin application impaired wound apposition. Histologic evaluation also confirmed impaired wound apposition after capsaicin infiltration. In conclusion, dilute solutions of capsaicin have differential effects on pain-related behaviors after plantar incision. Based on the antinociception produced by capsaicin both via infiltration and perineural injection, the effect on wound appearance was likely related to its inhibitory effects on pain behaviors and was not necessarily a local effect of the drug. PERSPECTIVE This study demonstrated that capsaicin infiltration before plantar incision produced an analgesic effect that depended upon the stimulus modality tested. When evaluating novel treatments for postoperative pain, studies using a single stimulus modality may overlook an analgesic effect by not examining a variety of stimuli.

Doxycycline Reduces Early Neurologic Impairment after Cerebral Arterial Air Embolism in the Rabbit

Background Previous studies indicate leukocytes play a role in the pathogenesis of cerebral arterial air embolism. Because doxycycline inhibits numerous leukocyte activities, the authors hypothesized doxycycline would decrease neurologic impairment after cerebral arterial air embolism. Methods New Zealand White rabbits anesthetized with methohexital received either intravenous saline (n = 7) or 10 mg/kg doxycycline (n = 7) 1 h before administration of 100 micro liter/kg of air into the internal carotid artery. Somatosensory-evoked potentials (SSEPs) were recorded at 30-min intervals for the next 2 h. After the final recording, the anesthetic was discontinued, and animals recovered. Animals were neurologically evaluated 4 h after air embolism on a scale of 0 (normal) to 99 (coma) points. Results At 4 h, doxycycline animals had lesser neurologic impairment (46 +/- 23; median, 41) than animals that received saline (77 +/- 20; median, 81); P = 0.007. SSEP amplitude was greater in the doxycycline group at 60, 90, and 120 min after air embolism; P = 0.001, 0.006, 0.026, respectively. SSEP amplitudes at 30, 60, 90, and 120 min inversely correlated with 4 h neurologic impairment; tau = -0.43, -0.75, -0.85, -0.79, respectively. Conclusions Doxycycline decreased electrophysiologic and neurologic abnormalities after cerebral air embolism. Because groups could be distinguished electrophysiologically as soon as 1 h after air embolism and because SSEP amplitude inversely correlated with neurologic impairment, doxycycline appears to inhibit a key early ([approximately] 1 h) process in the pathophysiology of cerebral air embolism.

Alterations in Cardiac Parasympathetic Indices in STZ-Induced Diabetic Rats

Autonomic neuropathy involving parasympathetic innervation is a complication of diabetes mellitus. Biochemical and morphological indices of the parasympathetic innervation of the heart were investigated in rats after diabetes mellitus was induced with streptozocin (STZ). Choline acetyltransferase (CAT) activity was used as a biochemical marker for parasympathetic innervation. Total CAT activity within the hearts of diabetic rats was unchanged after 1 and 2 wk of diabetes and was significantly reduced after 4, 8, and 12 wk. Morphological changes within the cardiac portion of the parasympathetic innervation were assessed at 8 wk when CAT activity was decreased. In diabetic rats, there was a reduction in both cardiac ganglion cell size and number. In contrast, in insulin-treated STZ-induced diabetic rats, ganglion cells were similar in size and number to those in a control group given 3-O-methylglucose to prevent induction of diabetes mellitus by STZ. Thus, diabetes mellitus is associated with alterations in cardiac parasympathetic innervation in rats, and supplemental insulin protects against these changes. These alterations may contribute to impaired parasympathetic neural control of the heart in diabetes mellitus.

Epidural tezampanel, an AMPA/kainate receptor antagonist, produces postoperative analgesia in rats.

BACKGROUND:We evaluated the epidural administration of tezampanel, a non-N-methyl-d-aspartate receptor antagonist, in a rat model for postoperative pain. We sought to determine if this drug affects nociception when administered epidurally by testing its effects on responses to heat in normal rats. The effects of epidural tezampanel on pain-related behaviors in rats that underwent plantar incision were also studied. METHODS:Rats were anesthetized and epidural catheters were placed. One day after epidural catheterization, the baseline heat withdrawal latency was measured. Epidural tezampanel or morphine was tested for analgesia by examining their effects against heat withdrawal latency. Motor function was also tested. Comparisons to subcutaneous drug administration were made. Other rats underwent plantar incision after epidural catheterization to assess pain behavior caused by incision. The effects of epidural tezampanel on the cumulative pain scoring, based on guarding, the withdrawal threshold to von Frey filament application, and the withdrawal latency to heat, were measured. The effects of epidural tezampanel on arterial blood pressure and heart rate were also tested. RESULTS:Both epidural morphine and epidural tezampanel increased withdrawal latency to heat. Only subcutaneous morphine affected heat withdrawal latency. After plantar incision, epidural tezampanel decreased the median guarding pain score, increased the heat withdrawal latency and increased the mechanical withdrawal threshold indicating analgesic effects. Arterial blood pressure and heart rate did not change after epidural drug administration. CONCLUSION:These experiments demonstrate that epidural administration of tezampanel produces analgesia to heat, motor side effects in some rats, and reduces pain behaviors caused by incision. No systemic analgesia was apparent using the largest dose.

Single intrathecal administration of the transcription factor decoy AYX1 prevents acute and chronic pain after incisional, inflammatory, or neuropathic injury

Summary AYX1 is a DNA decoy designed to specifically inhibit, in the spinal cord and dorsal root ganglia network, the trauma‐induced transcription factor EGR1 responsible for long‐term neuronal hyper‐excitability. A single intrathecal administration of AYX1 around the time of surgery or trauma prevents acute and chronic pain in animal models of inflammatory, incisional, bone, and neuropathic pain. ABSTRACT The persistence of pain after surgery increases the recovery interval from surgery to a normal quality of life. AYX1 is a DNA‐decoy drug candidate designed to prevent post‐surgical pain following a single intrathecal injection. Tissue injury causes a transient activation of the transcription factor EGR1 in the dorsal root ganglia–dorsal horn network, which then triggers changes in gene expression that induce neuronal hypersensitivity. AYX1 is a potent, specific inhibitor of EGR1 activity that mimics the genomic EGR1‐binding sequence. Administered in the peri‐operative period, AYX1 dose dependently prevents mechanical hypersensitivity in models of acute incisional (plantar), inflammatory (CFA), and chronic neuropathic pain (SNI) in rats. Furthermore, in a knee surgery model evaluating functional measures of postoperative pain, AYX1 improved weight‐bearing incapacitance and spontaneous rearing compared to control. These data illustrate the potential clinical therapeutic benefits of AYX1 for preventing the transition of acute to chronic post‐surgical pain.

Somatosensory Evoked Potentials Correlate With Neurological Outcome in Rabbits Undergoing Cerebral Air Embolism

BACKGROUND AND PURPOSE Somatosensory evoked potentials (SSEPs) have been used as an outcome measure in models of cerebral air embolism despite the lack of studies correlating SSEPs with other measures of neurological injury. We examined the relationship between SSEPs and neurological impairment in the setting of cerebral air embolism. METHODS Anesthetized New Zealand White rabbits received either 0, 50, 100, or 150 microL/kg of air into the internal carotid artery. SSEPs were recorded at intervals for the subsequent 2 hours. After the final recording the anesthetic was discontinued, and the animals recovered. Animals were neurologically evaluated at 3 and 24 hours after cerebral air embolism on a scale of zero (normal) to 97 (coma) points. RESULTS There was a clear relationship between the dose of air and 2-hour SSEP amplitude (P = .00003). SSEP amplitudes at 2 hours were inversely correlated with neurological impairment scores at 3 hours (r = -0.71, P < .0001). SSEP amplitudes at 2 hours were less in animals that died (11 +/- 16%; n = 9) than in those that survived to 24 hours (53 +/- 20%; n = 9) (P = .0008). CONCLUSIONS These results support SSEPs as an index of neurological impairment in this model of cerebral air embolism.

Ketoprofen Produces Modality-Specific Inhibition of Pain Behaviors in Rats After Plantar Incision

BACKGROUND: Postoperative pain remains a significant problem despite optimal treatment with current drugs. Nonsteroidal antiinflammatory drugs reduce inflammation and provide analgesia but are associated with adverse side effects. METHODS: We tested low doses (0.5–5 mg/kg) of parenteral ketoprofen against pain-related behaviors after plantar incision in rats. To further evaluate the potential sites of action of ketoprofen in our model, a novel, sustained-release microparticle formulation of ketoprofen was placed into the wound, and tested for its effects on pain behaviors. Intrathecal ketoprofen (150 μg) was also studied. Plasma samples were assayed for drug concentrations. RESULTS: We found that low doses of parenterally administered ketoprofen produced a modality-specific effect on pain behaviors; guarding after incision was decreased, whereas no inhibition of exaggerated responses to heat or mechanical stimuli was evident. Very low doses, 0.5 mg/kg, could produce inhibition of guarding. The locally applied sustained-release ketoprofen-eluting microparticles and intrathecally administered ketoprofen also produced a modality-specific effect on pain behaviors after incision, inhibiting only guarding. Plasma levels of ketoprofen after parenteral or local administration were in the range of therapeutic blood levels in postoperative patients. CONCLUSIONS: This study demonstrates that ketoprofen is an effective analgesic for nonevoked guarding in rats after plantar incision. There was no effect on mechanical or heat responses, which highlights the importance of multiple-modality testing of pain behaviors for drug evaluation. We found efficacy at doses used clinically in postoperative patients.