David C. Yeomans

Nociceptive responses to high and low rates of noxious cutaneous heating are mediated by different nociceptors in the rat: electrophysiological evidence

&NA; Several lines of evidence suggest that different classes of nociceptive afferents mediate the responses produced by different rates of noxious skin heating. More specifically, low skin heating rates evoke nociceptive responses that appear to be mediated by the activation of capsaicin‐sensitive C‐fiber nociceptors, whereas high skin heating rates appear to produce responses mediated by the activation of other nociceptors. This hypothesis was examined by both electrophysiological and behavioral experiments. This report describes the results of experiments designed to determine whether pharmacologic treatments that selectively alter the activity of C‐fiber nociceptive afferents also produce selective effects on foot withdrawal responses to either high or low rates of noxious foot heating. The results these experiments demonstrate that: (1) topical application of a low concentration of capsaicin, which sensitizes C‐fiber nociceptors, selectively decreased the latency of responses to low heating rates; (2) topical application of a high concentration of capsaicin, that desensitizes C‐fiber nociceptors, selectively increased the latency of responses to low heating rates; (3) low doses of systemic morphine, which selectively attenuate nociception produced by the activation of C‐fiber nociceptors, selectively increased response latencies for low skin heating rates. These results support the conclusion that foot withdrawal responses evoked by low skin heating rates are mediated by the activation of capsaicin‐sensitive C‐fiber nociceptors and foot withdrawal responses evoked by high skin heating rates are mediated by the activation of other nociceptors. This conclusion is supported by the results of the accompanying electrophysiological study which provides direct evidence that low rates of skin heating preferentially activate C‐fiber nociceptors while high rates of skin heating preferentially activate A&dgr; nociceptors.

The function of noradrenergic neurons in mediating antinociception induced by electrical stimulation of the locus coeruleus in two different sources of Sprague-Dawley rats

Although noradrenergic neurons in the nucleus locus coeruleus are known to project to the spinal cord, these neurons appear to innervate different regions of the spinal cord in Sprague-Dawley rats obtained from two different vendors. Recent anatomical studies demonstrated that the noradrenergic neurons in the locus coeruleus in Sasco Sprague-Dawley rats primarily innervate the ventral horn, whereas Harlan Sprague-Dawley rats have coeruleospinal projections that terminate in the dorsal horn of the spinal cord. This report describes the results of behavioral experiments that were designed to determine the functional significance of these anatomical differences. Electrical stimulation of neurons in the locus coeruleus produced antinociception in both Harlan and Sasco rats. The antinociception in Harlan rats was readily reversed by intrathecal injection of yohimbine, a selective alpha 2-adrenoceptor antagonist, or by phentolamine, a non-selective alpha 2-adrenoceptor antagonist. In contrast, these antagonists did not alter the antinociception produced by locus coeruleus stimulation in Sasco rats. Finally, the alpha 2-antagonist, idazoxan, did not alter the antinociceptive effect of locus coeruleus stimulation in either group of rats. These observations indicate that coeruleospinal noradrenergic neurons in Harlan and Sasco Sprague-Dawley rats have different physiological functions. Thus, electrical stimulation of noradrenergic neurons in the locus coeruleus that innervate the spinal cord dorsal horn (Harlan rats) produces antinociception, but stimulation of coeruleospinal noradrenergic neurons that project to the ventral horn (Sasco rats) does not produce antinociception. It is likely that genetic differences between these outbred stocks of rats account for the fundamental differences in the projections of coeruleospinal neurons and their function in controlling nociception.

ASIC3 in muscle mediates mechanical, but not heat, hyperalgesia associated with muscle inflammation

Abstract Peripheral initiators of muscle pain are virtually unknown, but likely key to development of chronic pain after muscle insult. The current study tested the hypothesis that ASIC3 in muscle is necessary for development of cutaneous mechanical, but not heat, hyperalgesia induced by muscle inflammation. Using mechanical and heat stimuli, we assessed behavioral responses in ASIC3−/− and ASIC3+/+ mice after induction of carrageenan muscle inflammation. ASIC3−/− mice did not develop cutaneous mechanical hyperalgesia after muscle inflammation when compared to ASIC3+/+ mice; heat hyperalgesia developed similarly between groups. We then tested if the phenotype could be rescued in ASIC3−/− mice by using a recombinant herpes virus vector to express ASIC3 in skin (where testing occurred) or muscle (where inflammation occurred). Infection of mouse DRG neurons with ASIC3‐encoding virus resulted in functional expression of ASICs. Injection of ASIC3‐encoding virus into muscle or skin of ASIC3−/− mice resulted in ASIC3 mRNA in DRG and protein expression in DRG and the peripheral injection site. Injection of ASIC3‐encoding virus into muscle, but not skin, resulted in development of mechanical hyperalgesia similar to that observed in ASIC3+/+ mice. Thus, ASIC3 in primary afferent fibers innervating muscle is critical to development of hyperalgesia that results from muscle insult.

Antinociception induced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group of the rat

&NA; Recent anatomical evidence indicates that the pontine A7 catecholamine cell group provides the major noradrenergic innervation of the spinal cord dorsal horn (laminae I–IV). The experiments described in this report were designed to determine if these neurons modulate nociception at the level of the spinal cord. To this end, the antinociceptive effect of electrical stimulation applied at various sites along several tracks through the dorsolateral pontine tegmentum was determined in lightly anesthetized rats. The latency of the withdrawal response of the hind feet to noxious radiant thermal stimulation applied to the dorsal surface was used as a measure of nociception. The results indicated that the most potent and consistent antinociception was produced at sites near the A7 cell group. In addition, intrathecal injection of &agr;‐noradrenergic antagonists blocked the antinociception produced by electrical stimulation at sites near the A7 group. These observations indicate that the antinociception produced by stimulation near the A7 cell group was mediated by spinally projecting noradrenergic neurons. The results of these experiments provide evidence that pontospinal noradrenergic neurons located in the A7 cell group are important components of the descending neuronal system that modulates nociception.

Selective Reduction of Second Pain Sensations by Systemic Morphine in Humans

&NA; A variety of forms of painful stimulation were delivered to human subjects in order to determine whether therapeutic dosages of systemic morphine might produce significant attenuation of some forms of phasic pain that are tolerable for experimental usage. Consistent with previous reports, simple application of thermal or electrical energy to the skin (for 3 sec) produced sensations of pain that were not significantly reduced by prior administration of morphine. Similarly, subjects that were trained to focus their attention on the magnitude of the immediate (first) pain sensation evoked by brief electrical or mechanical stimulation did not report reduction by morphine of pain attributed to conduction in myelinated peripheral nociceptors. In contrast, the magnitude of late (second) pain sensations produced by brief pulses of electrical, thermal or mechanical stimuli to the same subjects was consistently reduced significantly by doses of 5 or 10 mg of morphine. The simplest interpretation of the effect on second pain intensity is that morphine preferentially attenuates input from unmyelinated nociceptors. This conclusion was reinforced by an experiment in which chemicals were applied to the skin. Morphine reduced pain produced by capsaicin (presumed to selectively excite unmyelinated peripheral afferents) but did not diminish pain elicited by bradykinin (presumed to excite A&dgr; and C nociceptors). Comparing long duration pains from chemical stimulation (lasting in excess of 5 min) with briefer pains elicited by 50 msec to 3 sec of stimulation did not support the notion that morphine acts selectively on tonic pain. Also, after‐sensations that could be discerned following second pain were not eliminated by morphine, and paired pulse facilitation of first pain sensations remained after administration of morphine, indicating that temporal summation is not preferentially reduced. Regardless of duration, frequency or latency, pain arising exclusively from unmyelinated nociceptors was attenuated substantially, but other elicited sensations were not reliably affected. For example, detection thresholds for warmth were unaffected by morphine, demonstrating that input from all unmvelinated afferents is not reduced.

Characterization of the foot withdrawal response to noxious radiant heat in the rat

&NA; The rat foot withdrawal response to noxious radiant heat has been used as a model of nociception that is particularly useful for measurements of unilateral changes in nociceptive responses. The purpose of these studies was to characterize the foot withdrawal response to graded rates of noxious skin heating. Response latencies and both surface and subsurface temperatures produced by 6 different intensities of radiant heat were measured to determine whether response latency is an appropriate measure of nociceptive threshold. With constant intensity heating, the temperature of the skin surface increased as logarithmic function of time, while subsurface temperature increased linearly with time. In contrast, a heating function that linearly increased the temperature at the skin surface increased the subsurface temperature as an exponential function of time. These results and published reports of nociceptive afferent recordings which used similar skin heating parameters, indicate that nociceptive foot withdrawal responses occur at about the same skin temperature as the activation of nociceptors. These results also indicate that since constant intensity heating produces linear increases in the subsurface temperature, then response latency can be used as an accurate measure of changes in nociceptive threshold produced by drug treatments. These observations lead to the conclusion that the foot withdrawal response latency is a valid and useful measure of nociceptive threshold in rodents.

Morphine reduces local cytokine expression and neutrophil infiltration after incision

BackgroundInflammation and nociceptive sensitization are hallmarks of tissue surrounding surgical incisions. Recent studies demonstrate that several cytokines may participate in the enhancement of nociception near these wounds. Since opioids like morphine interact with neutrophils and other immunocytes, it is possible that morphine exerts some of its antinociceptive action after surgical incision by altering the vigor of the inflammatory response. On the other hand, keratinocytes also express opioid receptors and have the capacity to produce cytokines after injury. Our studies were directed towards determining if opioids alter cytokine production near incisions and to identify cell populations responsible for producing these cytokines.ResultsA murine incisional model was used to measure the effects of acute morphine administration (0.1–10 mg/kg) on nociceptive thresholds, neutrophil infiltration and cytokine production in hind paw skin 30 minutes and 2 hours after incision. Incised hind paws displayed profound allodynia which was reduced by morphine (0.1–10 mg/kg) in the 2 hours following incision. Skin samples harvested from these mice showed enhanced levels of 5 cytokines: IL-1β, IL-6, tumor necrosis factor alpha (TNFα), granulocyte colony stimulating factor (G-CSF) and keratinocyte-derived cytokine (KC). Morphine reduced these incision-stimulated levels. Separate analyses measuring myeloperoxidase (MPO) and using immunohistochemistry demonstrated that morphine dose-dependently reduced the infiltration of neutrophils into the peri-incisional tissue. The dose of morphine required for reduction of cytokine accumulation, however, was below that required for inhibition of peri-incisional neutrophil infiltration. Additional immunohistochemical studies revealed wound edge keratinocytes as being an important source of cytokines in the acute phase after incision.ConclusionAcute morphine administration of doses as low as 0.1 mg/kg reduces peri-incisional cytokine expression. A reduction in neutrophil infiltration does not provide a complete explanation for this effect, and keratinocytes may be responsible for some incision area cytokine production. These studies suggest that morphine may alter the inflammatory milieu of incisional wounds, but these alterations do not likely contribute significantly to analgesia in the acute setting.

Effect of anti-NGF antibodies in a rat tibia fracture model of complex regional pain syndrome type I

&NA; Tibia fracture in rats evokes chronic hindpaw warmth, edema, allodynia, and regional osteopenia resembling the clinical characteristics of patients with complex regional pain syndrome type I (CRPS I). Nerve growth factor (NGF) has been shown to support nociceptive and other types of changes found in neuropathic pain models. We hypothesized that anti‐NGF antibodies might reduce one or more of the CRPS I‐like features of the rat fracture model. For our studies one distal tibia of each experimental rat was fractured and casted for 4 weeks. The rats were injected with anti‐NGF or vehicle at days 17 and 24 post‐fracture. Nociceptive testing as well as assessment of edema and hindpaw warmth were followed during this period. Molecular and biochemical techniques were used to follow cytokine, NGF and neuropeptide levels in hindpaw skin and sciatic nerves. Lumbar spinal cord Fos immunostaining was performed. Bone microarchitecture was measured using microcomputed tomography (μCT). We found that tibia fracture upregulated NGF expression in hindpaw skin and tibia bone along with sciatic nerve neuropeptide content. We also found nociceptive sensitization, enhanced spinal cord Fos expression, osteopenia and enhanced cytokine content of hindpaw skin on the side of the fracture. Anti‐NGF treatment reduced neuropeptide levels in sciatic nerve and reduced nociceptive sensitization. There was less spinal cord Fos expression and bone loss in the anti‐NGF treated animals. Conversely, anti‐NGF did not decrease hindpaw edema, warmth or cytokine production. Collectively, anti‐NGF reduced some but not all signs characteristic of CRPS illustrating the complexity of CRPS pathogenesis and NGF signaling.

The noradrenergic innervation of the spinal cord: differences between two substrains of Sprague-Dawley rats determined using retrograde tracers combined with immunocytochemistry

We have recently described the spinal cord terminations of noradrenergic neurons located in the A5, A6 and A7 cell groups. However, recent reports from another laboratory, using similar experimental methods, have described results that are profoundly different. The present experiments were designed to determine whether these discrepant results are due to fundamental differences between the substrains of rats used in the conflicting experiments. To this end, retrograde tract tracing experiments were done using Sprague-Dawley rats from either Sasco, Inc. or Harlan Sprague-Dawley, Inc. The results indicate that noradrenergic neurons in the pontine catecholamine cell groups exhibit remarkably different spinal cord projections in these two substrains of Sprague-Dawley derived rats.

Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: a microdialysis study.

Abstract Animal studies have documented a critical role for cytokines in cell signaling events underlying inflammation and pain associated with tissue injury. While clinical reports indicate an important role of cytokines in inflammatory pain, methodological limitations have made systematic human studies difficult. This study examined the utility of a human in vivo bioassay combining microdialysis with multiplex immunoassay techniques for measuring cytokine arrays in tissue. The first experiment measured cytokines in interstitial fluid collected from non‐inflamed and experimentally inflamed skin (UVB). The effects of noxious heat on cytokine release were also assessed. The second experiment examined whether anti‐hyperalgesic effects of the COX‐inhibitor ibuprofen were associated with decreased tissue levels of the pro‐inflammatory cytokines IL‐1β and IL‐6. In the first experiment, inflammation significantly increased IL‐1β, IL‐6, IL‐8, IL‐10, G‐CSF, and MIP‐1β. Noxious heat but not experimental inflammation significantly increased IL‐7 and IL‐13. In the second experiment, an oral dose of 400 and 800 mg ibuprofen produced similar anti‐hyperalgesic effects suggesting a ceiling effect. Tissue levels of IL‐1β and IL‐6 were not affected after the 400 mg dose but decreased significantly (44 ± 32% and 38 ± 13%) after the 800 mg dose. These results support the utility of explored method for tracking cytokines in human tissue and suggest that anti‐hyperalgesic and anti‐inflammatory effects of ibuprofen are at least partially dissociated. The data further suggest that high clinical doses of ibuprofen exert anti‐inflammatory effects by down‐regulating tissue cytokine levels. Explored human bioassay is a promising tool for studying the pathology and pharmacology of inflammatory and chronic pain conditions.