Tony L. Yaksh

Quantitative assessment of tactile allodynia in the rat paw

We applied and validated a quantitative allodynia assessment technique, using a recently developed rat surgical neuropathy model wherein nocifensive behaviors are evoked by light touch to the paw. Employing von Frey hairs from 0.41 to 15.1 g, we first characterized the percent response at each stimulus intensity. A smooth log-linear relationship was observed, with a median 50% threshold at 1.97 g (95% confidence limits, 1.12-3.57 g). Subsequently, we applied a paradigm using stimulus oscillation around the response threshold, which allowed more rapid, efficient measurements. Median 50% threshold by this up-down method was 2.4 g (1.81-2.76). Correlation coefficient between the two methods was 0.91. In neuropathic rats, good intra- and inter-observer reproducibility was found for the up-down paradigm; some variability was seen in normal rats, attributable to extensive testing. Thresholds in a sizable group of neuropathic rats showed insignificant variability over 20 days. After 50 days, 61% still met strict neuropathy criteria, using survival analysis. Threshold measurement using the up-down paradigm, in combination with the neuropathic pain model, represents a powerful tool for analyzing the effects of manipulations of the neuropathic pain state.

Studies in the Primate on the Analgetic Effects Associated with Intrathecal Actions of Opiates,α-Adrenergic Agonists and Baclofen

The effects of intrathecally administered opiates (morphine sulfate and meperidine), alpha-adrenergic agonists (clonidine and ST-91) and baclofen were examined on the shock titration threshold of macaque monkeys chronically prepared with intrathecal (I) or epidural (E) catheters. Spinal opiates produced a long-lasting analgesia which was antagonized by naloxone. The order of potency was I morphine greater than I meperidine greater than E meperidine greater than E morphine. Clonidine and ST-91, also produced a dose-dependent, long-lasting elevation in the shock titration threshold, antagonized by phentolamine, but not naloxone. L-baclofen, but not D-baclofen, resulted in a dose-dependent elevation of shock titration threshold, which was not antagonized by naloxone. Repeated administration at 24-h intervals over a 7-day period of morphine, clonidine or baclofen, resulted in a significant reduction in the analgetic effects of each drug. Cross tolerance between the three classes of agents was not observed. Intrathecal co-administration of inactive doses of ST-91 and morphine resulted in a near maximal increase in the shock titration threshold, which failed to show any significant tolerance over 21 days. Intrathecal ST-91 and morphine produced no change in either muscle strength, tendon reflexes, respiratory rate, urine formation, or the ability to locomote. Baclofen, in contrast, produced a dose-dependent decrease in muscle strength. That the intrathecal drugs did not produce anesthesia was demonstrated by their failure to block the avoidance response to ensuing ear shock cued by a light tactile stimulus applied to the hind paw. These results clearly indicate that a powerful analgesia can be produced by selectively activating adrenergic, opiate, and baclofenergic receptor systems in the spinal cord.

Spinal nitric oxide synthesis inhibition blocks NMDA-induced thermal hyperalgesia and produces antinociception in the formalin test in rats.

&NA; To assess the possible role of spinal nitric oxide (NO) synthesis in nociceptive processing, we examined the effect of intrathecal (i.t.) injection of arginine analogs that act as alternate substrates for NO synthase and thus inhibit NO production. NG‐nitro‐l‐arginine methyl ester (L‐NAME) and NG‐monomethyl‐l‐arginine (L‐NMMA) produced a dose‐dependent, stereospecific inhibition of the second phase (10–60 min; ED50, 135 and 246 nmol) of the formalin test with minimal effect on the first phase (0–9 min; ED50 > 1.1 &mgr;mol). The inhibitory action of L‐NAME was dose‐dependently reversed by i.t. L‐arginine (ID50, 4.9 &mgr;mol) but not by D‐arginine (ID50 > 14 &mgr;mol). The suppression of the second‐phase formalin response by L‐NAME was similar whether administered before or after formalin injection into the rat paw. Spinal administration of L‐NAME (370 nmol), but not D‐NAME (3.7 &mgr;mol), also blocked thermal hyperalgesia induced by i.t. injection of N‐methyl‐d‐aspartate (NMDA; 6.8 nmol). The effect of L‐NAME was reversed by L‐arginine (4.7 &bgr;mol) but not with D‐arginine (14 &mgr;zmol). None of the compounds, L‐NAME, D‐NAME or L‐arginine, when injected alone, had any effect on normal thermal response latencies or on the 52.5°C hot plate. These studies indicate that modulation of spinal NO synthesis can diminish the facilitated processing of afferent activity which is induced by a continued afferent barrage (second phase of the formalin test). This hyperalgesic component appears initiated by the activation of a spinal NMDA receptor that, through the generation of NO, leads to the observed augmented processing of afferent input and the associated hyperalgesic component of the subsequent pain behavior.

Comparison of the Antinociceptive Effects of Pre- and Posttreatment with Intrathecal Morphine and MK801, an NMDA Antagonist, on the Formalin Test in the Rat

Current thinking emphasizes that protracted small afferent input can evoke mechanisms that mediate a significant potentiation of spinal nociceptive processing and that this facilitory component has a unique pharmacology. To investigate the behavioral parallels of this spinal facilitation, we evaluated the effects of pre- and post-treatment of intrathecal morphine (mu agonist) and MK801 (N-methyl-D-aspartate [NMDA] antagonist) on the formalin test. Intraplantar formalin resulted in a biphasic appearance of flinching behavior (phase 1 = 0-5 min; phase 2 = 10-60 min). Morphine and MK801 were administered intrathecally 15 min before formalin injection in the pretreatment study and 9 min after formalin injection in the posttreatment study. Pretreatment with intrathecal morphine produced comparable dose-dependent suppressions of the phase 1 and phase 2 behaviors (ED50 = 0.5 micrograms [95% CI = 0.3-0.9] and 0.3 micrograms [95% CI = 0.1-0.7], respectively). Posttreatment with morphine also resulted in comparable suppression of the phase 2 response (ED50 = 0.2 micrograms [95% CI = 0.1-0.3]). At the highest dose of intrathecal morphine (10 micrograms), an almost complete suppression of formalin-evoked behavior was observed. Pretreatment with MK801 inhibited the second-phase response more strongly than the first-phase response (ED50 = 1.6 micrograms [95% CI = 0.5-5.7] vs. 0.1 microgram [95% CI = 0.3 - 0.4], respectively). In contrast, posttreatment with the highest dose of MK801 had no effect on the phase 2 response.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of p38 mitogen‐activated protein kinase in spinal microglia is a critical link in inflammation‐induced spinal pain processing

We examined the effect of p38 mitogen‐activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immuncytochemisty revealed that phosphorylated p38 MAPK‐immunoreactive cells were predominantly present in laminae I–IV of the dorsal horn. Double‐staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co‐localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD‐282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo‐oxygenase‐2 and inflammation‐induced appearance of Fos‐positive neurons, was blocked by pretreatment, but not post‐treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation‐induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.

Spinal pharmacology of thermal hyperesthesia induced by constriction injury of sciatic nerve. Excitatory amino acid antagonists

&NA; This study evaluated the effects of spinally administered excitatory amino acid antagonists on the thermal hyperesthetic state induced by unilateral partial ligation of the sciatic nerve in the rat. The measured response was the latency to paw withdrawal of each hind paw after application of a focused heat lamp on the plantar surface of the paw through a glass plate upon which the animal stood. In this work, antagonists (MK801, Dl‐2‐amino‐5‐phosphonovalerate, ketamine) of the Symbol receptor (NMDA), the glycine potentiation site at the NMDA receptor (5‐chloro‐indole‐2‐carboxylic acid) and non‐NMDA receptor (kynurenic acid: g‐D‐glutamylaminomethyl sulphonate) were injected through chronically implanted lumbar intrathecal catheters in normal rats (no lesions) and in rats with unilateral constriction injury. In the normal rat study, NMDA and non‐NMDA antagonists had little effect upon paw withdrawal latency at intrathecal doses which did not produce readily detectable motor weakness. In the hyperesthetic rat study, NMDA antagonists would temporarily eliminate the hyperesthetic state at doses below those which altered the response latency of the normal paw or which altered motor function. These results suggested that spinal NMDA receptors play an important role in the hyperesthetic state induced by peripheral nerve injury. Figure. No caption available

Pharmacology of the Spinal Action of Ketorolac, Morphine, ST-91, U50488H, and L-PIA on the Formalin Test and an Isobolographic Analysis of the NSAID Interaction

Background:Noxious cutaneous stimuli enhance spinal excitability. The behavioral correlate to this response is found in the rat formalin test, in which formalin injection into the hindpaw evokes signs of nociception (flinching and licking of the injected paw) with acute (phase 1) and delayed-hyperalgesic (phase 2) components. Methods:The effect of intrathecal morphine (a µ agonist), U50488H (a κ agonist), ST-91 (an α2 agonist), L-PIA (an adenosine A1 agonist), and ketorolac (a nonsteroidal antiinflammatory drug, or NSAID), were examined in rats undergoing the formalin test. Spinal interactions between ketorolac and the µ, κ, α2, and adenosine A1 agonists were assessed using isobolographic analysis. Results:Morphine and ST-91 caused a dose-dependent suppression of phase 1 and phase 2 of the formalin test, while U50488H and L-PIA had little effect on phase 1, but caused dose-dependent depression of phase 2. Intrathecal ketorolac inhibited the phase 2 response, but had limited effect on phase 1. The isobolographic analysis revealed a significant synergy (with fractional dose ratios of less than 1) between ketorolac and morphine or ST-91 for phase 1 and phase 2, but only an additive interaction was found between ketorolac and L-PIA or U50488H. Conclusions:These observations offer systematic support for the powerful interaction between NSAIDs and opioids and certain other analgesics in clinical pain states. These studies also demonstrate that spinal synergy is not a common property of all interactions. Thus, the NSAID synergy appears to occur with agents that exert a concurrent action both preand postsynaptic to the primary afferents.

High Doses of Spinal Morphine Produce a Nonopiate Receptormediated Hyperesthesia: Clinical and Theoretic Implications

In rats with chronically implanted intrathecal catheters, high concentrations of morphine (3 μl of 50 mg/ml: 150 μg) yielded a reliable and striking syndrome of pain behavior that involved intermittent bouts of biting and scratching at the dermatomes innervated by levels of the spinal cord proximal to the catheter tip. In addition, during intervals between bouts of agitation, the animals displayed a clear, marked hyperesthesia where an otherwise innocuous stimuli (brush stroke) evoked significant signs of discomfort and consequent aggressive behavior. These effects were exaggerated rather than reversed by high doses of naltrexone. The effect, perfectly mimicked by a considerably lower dose of morphine-3-glucuronide (15 μg) or the glycine antagonist strychnine (30 μg), was not produced by equimolar concentrations of sodium sulfate, glucuronide, methadone, or sufentanil. In halothane-anesthetized cats, light brushing of the hindpaw and tail or low-intensity stimulation of the sciatic nerves resulted in prominent elevations in blood pressure and pupil diameter following the intrathecal administration of high concentrations (50 mg/ml; 0.1 ml) of morphine sulfate. This effect, exaggerated by naloxone, was produced by a lower concentration of intrathecal morphine-3-glucuronide (5 mg/ml; 0.1 ml) but not by intrathecal saline. These results suggest the possibility that the effects of high doses of morphine may be characterized by a nonopiate receptor-mediated effect that alters the coding of sensory information in the spinal cord. The authors speculate that high concentrations of spinal opiates, as may be employed in tolerant terminal-cancer patients, could exert an action that physiologically antagonizes the analgesic effects otherwise mediated by the action of morphine on the spinal opiate receptor.

Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli

We have examined the stability and sources of variation within the nociceptive model of rat hind paw withdrawal from an under-glass radiant stimulus (Hargreaves et al., 1988) using a system where stimulus intensity and floor temperature can be controlled and reproducibly changed. The current study demonstrates that: (i) increased stimulus intensity with a fixed surface temperature is associated with a monotonic decrease in mean response latency and its variance; (ii) for a fixed stimulus intensity, the mean paw withdrawal latency and variance increased as the glass floor temperature is lowered from 30 degrees C to room temperature (25 degrees C). Using subcutaneously-implanted thermocouples and a 30 degrees C glass surface, the subcutaneous paw temperature observed at an interval corresponding to the time at which the animal displayed a paw withdrawal did not differ across multiple heating rates (41-42.5 degrees C). This finding is in agreement with human studies of pain thresholds and C-fiber activity. These studies emphasize the importance of maintaining a fixed surface temperature to reduce experimental variability and the utility of this apparatus across multiple stimulus intensities to define agonist efficacy.

Effect of continuous intrathecal infusion of ω-conopeptides, N-type calcium-channel blockers, on behavior and antinociception in the formalin and hot-plate tests in rats

&NA; The effect of continuous intrathecal infusion of &ohgr;‐conopeptides in the rat was examined to determine whether antinociception, as measured on the formalin and hot‐plate (52.5°C) tests, was altered and whether tolerance developed with chronic infusion of these agents. Infusion of 0.030 and 0.003 nmol/h SNX‐111 and 0.290 nmol/h SNX‐239 was performed for either 2 days (‘acute’) or 7 days (‘chronic’) was compared to the effect of 20 nmol/h morphine or saline. Both doses of SNX‐111 and SNX‐239 produced a significant reduction of the response to the hot‐plate and formalin tests at both 2 and 7 days of infusion compared to saline infusion. In contrast, morphine only produced a significant effect on day 2, but not on infusion day 7, indicating that tolerance had developed. The effect of SNX‐111 was reversible, as shown by a return to nociceptive responses similar to saline‐infused rats 2 days after the minipumps had been disconnected after a 7‐day infusion period. These data indicate that chronic infusion of &ohgr;‐conopeptides that block N‐type voltage‐sensitive calcium channels produce a powerful antinociception, with minimal development of tolerance.