Kenneth M. Hargreaves

Antihyperalgesic effects of spinal cannabinoids

Cannabinoids have been widely reported to produce antinociception in models such as tail flick and hot plate. However, their role in modulating thermal hyperalgesia is unknown. The potency of some drugs, such as the opioids, increases during hyperalgesia. Thus, we evaluated whether there is a change in the effectiveness of intrathecal cannabinoids with hyperalgesia. Additionally, we evaluated whether cannabinoids could inhibit capsaicin-evoked neurosecretion from isolated rat spinal cord. Our results indicate that 1 fmol anandamide (i.t.) completely blocked carrageenan-induced thermal hyperalgesia. However, anandamide at doses as high as 100 pmol had no effect on thermal latencies in normal animals. Additionally, anandamide inhibited K+- as well as capsaicin-evoked immunoreactive calcitonin gene-related peptide release. Finally, cannabinoid receptors were identified in sensory neurons. Collectively, these results indicate that there is an increased effectiveness of modulation of thermal nociceptive thresholds by spinal cannabinoids during hyperalgesia. This antihyperalgesic effect may be the result of cannabinoid-induced inhibition of neurosecretion from certain primary afferent fibers.

Opioid analgesia at peripheral sites: a target for opioids released during stress and inflammation?

The peripheral analgesic effects of opiates were evaluated in a rat model of inflammation. The experimental design excluded a potential central nervous system site of action for the observed analgesia. After the injection of carrageenan (CARRA) in the plantar surface of both hind paws, an opiate was injected into one paw and saline was injected into the other paw. The inflamed paws injected with the μ-agonist, fentanyl (0.3 μg) or the k-agonist, ethylketocyclazocine (10 μg) were significantly less hyperalgesic (P < 0.001 and P < 0.01, respectively) than were the contralateral inflamed paws injected with saline. At these doses, fentanyl and ethylketocyclazocine were devoid of systemic effects. Another μ-agonist, levorphanol (20, 40, 80, or 160 μg) and dextrorphan (160 μg), its dextrorotatory isomer, were used next to evaluate opioid specificity. Levorphanol produced a dose-related blockade of CARRA-induced hyperalgesia (P < 0.005). In contrast, 160 μg of dextrorphan was inactive. These results demonstrate that local administration of opiates into an inflamed paw produces a dose-related, stereospecific analgesia restricted to the injected area.

Glutamate participates in the peripheral modulation of thermal hyperalgesia in rats

While the effects of excitatory amino acids have been well characterized in the central nervous system, relatively little is known about their possible modulation of elements responsible for hyperalgesia within peripheral tissue. The presented experiments demonstrate that the intraplantar (i.pl.) injection of L-glutamate (30 nmol) evokes a thermal hyperalgesic response in the paw withdrawal latencies of normal rats which is stereospecific. In addition, the i.pl. injection of either the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 nmol) or the competitive alpha-amino-3-hydroxy-4-methyl-5-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)(100 nmol) into hindpaws inflamed with carrageenan significantly reduced the thermal hyperalgesic response in rats. Collectively, these results suggest that excitatory amino acids activate a peripheral target which facilitates a hyperalgesic behavioural response to thermal stimulation via a receptor mediated process.

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freunds adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Opiates suppress carrageenan-induced edema and hyperthermia at doses that inhibit hyperalgesia

This study determined whether opiates alter vascular components of inflammation (hyperthermia, edema and plasma extravasation) in addition to the suppression of hyperalgesia. Rats were administered carrageenan into one hind paw and saline into the other hind paw, followed by i.p. injection of morphine (0.2-5.0 mg/kg) or saline at 60 min, and testing at 90 min after hind paw injections. Morphine produced a dose-dependent reduction in carrageenan-induced hyperalgesia (17-53%), hyperthermia (39-53%) and edema (24-36%). Morphine treatment did not alter the temperatures of the contralateral saline-injected paws, indicating that opiate suppression of hyperthermia was not confounded by alterations in systemic body temperature or blood flow. The opiate effects on inflammation were stereospecific since levorphanol (1 mg/kg), but not dextrorphan (1 mg/kg), suppressed carrageenan-evoked hyperalgesia, hyperthermia and edema. Pre-treatment with naltrexone (1.5 mg/kg) blocked the effects of a 5 mg/kg dose of morphine sulfate on hyperalgesia, hyperthermia and edema. In a separate study, i.v. injection of morphine sulfate (2 mg/kg) reduced plasma extravasation by 41% (P less than 0.01). Morphine administration resulted in significantly greater increases in paw withdrawal latencies in the inflamed (38-139%) than the contralateral, saline-treated paws (4-19%). The results indicate that opiates exert a moderate, though significant, reduction in the vascular signs of inflammation in addition to their reduction of hyperalgesia. The mechanisms for this vascular effect involve inhibition of both vasodilation (as indicated by a decrease in hyperthermia) and inhibition of vascular permeability. In addition, opiates exhibit enhanced antinociceptive effects in inflamed paws, even when compared to uninjured paws in the same animal.

SR 141716A, a cannabinoid receptor antagonist, produces hyperalgesia in untreated mice

Antinociceptive effects of cannabinoids are well documented. However, the physiological role of endogenous cannabinoids in nociception is unknown. We evaluated the effects of the cannabinoid receptor antagonist SR 141716A (N-piperidino-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide) on mouse hot plate latencies. Intrathecal injection of SR 141716A evoked a significant thermal hyperalgesia. These results suggest that the cannabinoid system tonically regulates thermal nociceptive thresholds. Furthermore, the absence of this regulation results in hyperalgesia suggesting that hypoactivity of this system may be involved in certain types of chronic pain.

Comparison of nonsteroidal anti-inflammatory drugs, ibuprofen and flurbiprofen, with methylprednisolone and placebo for acute pain, swelling, and trismus

Pain, swelling, loss of function, and hyperthermia are acute postoperative sequelae of inflammation due to tissue injury during surgical procedures. Pharmacologic strategies for minimizing the clinical manifestations of surgical trauma are often directed toward blocking the formation or inhibiting the effects of the biochemical mediators of acute inflammation. This study compared two nonsteroidal anti-inflammatory drugs (NSAIDs), flurbiprofen and ibuprofen, with a prototype glucocorticoid, methylprednisolone, in two replicate placebo-controlled studies for suppression of inflammation due to the surgical removal of impacted third molars. The results indicate that NSAIDs produce greater initial analgesia than do steroids, whereas steroids result in greater suppression of swelling and less loss of function. Examination of the pooled data from the two studies indicates that NSAID pretreatment results in a modest suppression of swelling in comparison with placebo. These data suggest that the acute analgesic effects of NSAIDs in the oral surgery model are due to suppression of a nociceptive process, presumably prostaglandin formation, rather than a generalized anti-inflammatory effect.

Peripheral CGRP release as a marker for neurogenic inflammation: a model system for the study of neuropeptide secretion in rat paw skin

&NA; The local release of pro‐inflammatory neuropeptides in the periphery has been associated with the development of neurogenic inflammation. However, there is an increasing number of reports demonstrating tissue‐dependent differences regarding the mechanisms engaged by these neuropeptides to initiate and maintain the inflammatory response in the target tissue. Since skin is often involved in tissue injury, the present studies were designed to develop a model for assessing cutaneous peptide secretion as a marker for neurogenic inflammation in skin tissue. Calcitonin gene‐related peptide (CGRP), as one of several neuropeptides known to be involved in neurogenic inflammation, was chosen to study capsaicin‐induced effects on peripheral neurosecretion. The corial surface of the hairy skin of a rat hindlimb was superfused in vitro, and the basal and capsaicin‐evoked peripheral release of immunoreactive CGRP (iCGRP) was measured using a radioimmunoassay. The main objectives of these studies were to characterize the various properties of this release including dose‐dependency, exocytosis and receptor‐mediation as well as the effects of acute and long‐term capsaicin desensitization. Capsaicin significantly and dose‐dependently increased the release of iCGRP at concentrations ranging from 3 to 300 &mgr;M. Omission of calcium ions or treatment with the competitive capsaicin receptor antagonist capsazepine completely inhibited the capsaicin‐induced iCGRP release. Superfusion of the skin with 100 &mgr;M capsaicin following a conditioning stimulation with capsaicin at concentrations ranging from 0.3 to 100 &mgr;M led to an acute, dose‐dependent desensitization of the CGRP response. In addition, chronic desensitization following the neonatal injection of capsaicin completely abolished the acute iCGRP response to capsaicin. The method described here should prove to be a valuable tool for the evaluation of the processes regulating the peripheral, cutaneous release of pro‐inflammatory neuropeptides. This strategy, therefore, may lead to a better understanding of the mechanisms involved in the development and maintenance of neurogenic inflammation, particularly in the skin.

Corticotropin-releasing factor (CRF) produces analgesia in humans and rats

The analgesic activity of corticotropin releasing factor (CRF) was determined in a clinical model and in the rat hot plate test. Patients administered CRF reported significantly less postoperative pain than patients pretreated with placebo. In rats, injection of CRF resulted in a significant analgesia which was comparable in both intensity and duration to a 300 times greater molar dose of morphine. These findings suggest that endogenous CRF may play a physiologic role in modulating pain when released under conditions of stress.

Bradykinin is increased during acute and chronic inflammation: therapeutic implications.

Bradykinin is a potent pain‐producing substance, yet little is known about its role in inflammation. The present study measured circulating levels of immunoreactive bradykinin in a clinical model of acute inflammation (oral surgery) and chronic inflammation (rheumatoid arthritis) and in the rat carrageenan model of inflammation. The effects of a kallikrein inhibitor (soybean trypsin inhibitor) on blocking bradykinin synthesis in vitro and its analgesic actions in the rat model were also evaluated. Levels of immunoreactive bradykinin increased threefold to fourfold during oral surgery. Levels were twofold to threefold greater in patients with rheumatoid arthritis compared with control subjects. Levels of immunoreactive bradykinin increased twofold in rats during carrageenan inflammation. Soybean trypsin inhibitor blocked synthesis of bradykinin in vitro and possessed analgesic activity in rats. The results indicate that the bradykinin system is activated during inflammation. Kallikrein inhibitors may represent a new class of analgesic/antiinflammatory drugs.