Kathleen A. Sluka

The DRASIC Cation Channel Contributes to the Detection of Cutaneous Touch and Acid Stimuli in Mice

Cation channels in the DEG/ENaC family are proposed to detect cutaneous stimuli in mammals. We localized one such channel, DRASIC, in several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and/or acid-evoked nociception. Disrupting the mouse DRASIC gene altered sensory transduction in specific and distinct ways. Loss of DRASIC increased the sensitivity of mechanoreceptors detecting light touch, but it reduced the sensitivity of a mechanoreceptor responding to noxious pinch and decreased the response of acid- and noxious heat-sensitive nociceptors. The data suggest that DRASIC subunits participate in heteromultimeric channel complexes in sensory neurons. Moreover, in different cellular contexts, DRASIC may respond to mechanical stimuli or to low pH to mediate normal touch and pain sensation.

Unilateral intramuscular injections of acidic saline produce a bilateral, long-lasting hyperalgesia.

This study characterizes an animal model of persistent mechanical hyperalgesia induced by repeated intramuscular injections of low pH saline. Saline at pH 4, 5, 6, or 7.2 was injected twice, 2 to 10 days apart, into the gastrocnemius muscle of rats. To quantify hyperalgesia, paw withdrawal latency to radiant heat (heat hyperalgesia) and withdrawal threshold to mechanical stimuli (mechanical hyperalgesia) were measured. Two unilateral injections of low pH saline, 5 days apart, caused a pH‐dependent bilateral mechanical, but not heat, hyperalgesia that lasted 30 days. Injections given 2 and 5 days apart produced a significantly greater mechanical hyperalgesia than injections given 10 days apart. Lidocaine injection into the gastrocnemius muscle or unilateral dorsal rhizotomy, 24 h after the second injection (pH 4), had no effect on the contralateral mechanical hyperalgesia. Minimal histopathology was observed in the injected muscle, and changes were similar between groups injected with pH 4 and pH 7.2. Thus, this new model of widespread, chronic muscle‐induced pain is unrelated to tissue damage and is not maintained by continued primary afferent input from the site of injury.

Chronic hyperalgesia induced by repeated acid injections in muscle is abolished by the loss of ASIC3, but not ASIC1.

&NA; Clinically, chronic pain and hyperalgesia induced by muscle injury are disabling and difficult to treat. Cellular and molecular mechanisms underlying chronic muscle‐induced hyperalgesia are not well understood. For this reason, we developed an animal model where repeated injections of acidic saline into one gastrocnemius muscle produce bilateral, long‐lasting mechanical hypersensitivity of the paw (i.e. hyperalgesia) without associated tissue damage. Since acid sensing ion channels (ASICs) are found on primary afferent fibers and respond to decreases in pH, we tested the hypothesis that ASICs on primary afferent fibers innervating muscle are critical to development of hyperalgesia and central sensitization in response to repeated intramuscular acid. Dorsal root ganglion neurons innervating muscle express ASIC3 and respond to acidic pH with fast, transient inward and sustained currents that resemble those of ASICs. Mechanical hyperalgesia produced by repeated intramuscular acid injections is prevented by prior treatment of the muscle with the non‐selective ASIC antagonist, amiloride, suggesting ASICs might be involved. ASIC3 knockouts do not develop mechanical hyperalgesia to repeated intramuscular acid injection when compared to wildtype littermates. In contrast, ASIC1 knockouts develop hyperalgesia similar to their wildtype littermates. Extracellular recordings of spinal wide dynamic range (WDR) neurons from wildtype mice show an expansion of the receptive field to include the contralateral paw, an increased response to von Frey filaments applied to the paw both ipsilaterally and contralaterally, and increased response to noxious pinch contralaterally after the second intramuscular acid injection. These changes in WDR neurons do not occur in ASIC3 knockouts. Thus, activation of ASIC3s on muscle afferents is required for development of mechanical hyperalgesia and central sensitization that normally occurs in response to repeated intramuscular acid. Therefore, interfering with ASIC3 might be of benefit in treatment or prevention of chronic hyperalgesia.

Unique role of dystroglycan in peripheral nerve myelination, nodal structure, and sodium channel stabilization.

Dystroglycan is a central component of the dystrophin-glycoprotein complex implicated in the pathogenesis of several neuromuscular diseases. Although dystroglycan is expressed by Schwann cells, its normal peripheral nerve functions are unknown. Here we show that selective deletion of Schwann cell dystroglycan results in slowed nerve conduction and nodal changes including reduced sodium channel density and disorganized microvilli. Additional features of mutant mice include deficits in rotorod performance, aberrant pain responses, and abnormal myelin sheath folding. These data indicate that dystroglycan is crucial for both myelination and nodal architecture. Dystroglycan may be required for the normal maintenance of voltage-gated sodium channels at nodes of Ranvier, possibly by mediating trans interactions between Schwann cell microvilli and the nodal axolemma.

Behavioral and immunohistochemical changes in an experimental arthritis model in rats

&NA; An experimental arthritis induced by injection of kaolin and carrageenan into the knee joint resulted in a temporal relationship between glutamate dorsal horn content and paw withdrawal latency (PWL) which was positively correlated. Limping, guarding, increased response to heat stimuli (hyperalgesia) and altered staining patterns for glutamate (GLU), substance P (SP), and calcitonin gene‐related peptide (CGRP) were monitored in the awake behaving arthritic rat over a 1 week time course. A decrease in PWL occurred on the side ipsilateral to the inflamed knee as early as 4 h after the induction of arthritis indicating the animals are hyperalgesic. The PWL remained decreased through the first 24 h. Computer‐assisted quantification of the density of immunohistochemical staining indicated the content of GLU, SP and CGRP was altered differentially throughout the time course of the arthritis. The changes observed for all three substances occurred across the entire superficial dorsal horn. There was an initial depletion of SP followed by an increase in both SP and CGRP content which was maintained through 1 week. The GLU content was increased during the hyperalgesic period. The GLU changes followed the same time course and were positively correlated with the changes in PWL. In a small group of animals injected with kaolin and carrageenan, hyperalgesia did not develop. In this group of animals, no change in dorsal horn GLU or SP content occurred. Rather, there was an increase in CGRP content in the middle portion of the superficial dorsal horn which is the termination site of knee joint afferents. These data indicate that the development of heat hyperalgesia is dependent on GLU and possibly SP. Since inflammation of the knee joint does not involve the foot pad, the heat hyperalgesia observed during the first 24 h following induction of arthritis represents a central neuronal sensitization.

Unilateral carrageenan injection into muscle or joint induces chronic bilateral hyperalgesia in rats

Chronic musculoskeletal pain is a major clinical problem and there is a general lack of animal models to study this condition. Carrageenan is commonly used to produce short‐lasting acute inflammation and hyperalgesia in animal models. However, the potential of carrageenan to produce chronic, long‐lasting hyperalgesia has not been evaluated. In the present study, we investigated the long‐term effects of carrageenan injected into joint or muscle in rats. Rats were injected with 0.3, 1 or 3% carrageenan in one knee joint or gastrocnemius muscle and hyperalgesia to mechanical (measured as decreased withdrawal threshold) and heat (measured as decreased withdrawal latency) stimuli of both paws assessed before and at varying times after injection, through 8 weeks. Histological changes were examined only after injection of 3% carrageenan. Three percent carrageenan injected in the muscle or knee produced hyperalgesia to mechanical and heat stimuli ipsilaterally, which lasted 7–8 weeks and spread to the contralateral side 1–2 weeks after injection. One percent carrageenan injected to the knee joint or gastrocnemius muscle, produced hyperalgesia that was shorter‐lasting and remained ipsilateral; 0.3% carrageenan injected into the knee joint or gastrocnemius muscle had no effect. Three percent carrageenan injected into the skin surrounding the knee joint did not produce hyperalgesia. A similar pattern of inflammatory changes was observed histologically for both the joint and muscle tissues. Acute inflammation was observed for the first 24 h with edema and neutrophilic infiltration evident as early as 4 h. At 1 week, the inflammation converted to primarily a macrophage response with scattered mast cells. The data suggest that animals injected with 1 or 3% carrageenan in the knee joint or gastrocnemius muscle could be used as models of acute inflammation through 24 h and chronic inflammation after 1 week. Furthermore, 3% carrageenan injected into deep tissues produces hyperalgesia that spreads to the contralateral side, at the same time period as the inflammation transforms from acute to chronic.

Joint manipulation reduces hyperalgesia by activation of monoamine receptors but not opioid or GABA receptors in the spinal cord

&NA; Joint manipulation has long been used for pain relief. However, the underlying mechanisms for manipulation‐related pain relief remain largely unexplored. The purpose of the current study was to determine which spinal neurotransmitter receptors mediate manipulation‐induced antihyperalgesia. Rats were injected with capsaicin (50 &mgr;l, 0.2%) into one ankle joint and mechanical withdrawal threshold measured before and after injection. The mechanical withdrawal threshold decreases 2 h after capsaicin injection. Two hours after capsaicin injection, the following drugs were administered intrathecally: bicuculline, blocks &ggr;‐aminobutyric acid (GABAA) receptors; naloxone, blocks opioid receptors; yohimbine blocks, &agr;2‐adrenergic receptors; and methysergide, blocks 5‐HT1/2 receptors. In addition, NAN‐190, ketanserin, and MDL‐72222 were administered to selectively block 5‐HT1A, 5‐HT2A, and 5‐HT3 receptors, respectively. Knee joint manipulation was performed 15 min after administration of drug. The knee joint was flexed and extended to end range of extension while the tibia was simultaneously translated in an anterior to posterior direction. The treatment group received three applications of manipulation, each 3 min in duration separated by 1 min of rest. Knee joint manipulation after capsaicin injection into the ankle joint significantly increases the mechanical withdrawal threshold for 45 min after treatment. Spinal blockade of 5‐HT1/2 receptors with methysergide prevented, while blockade of &agr;2‐adrenergic receptors attenuated, the manipulation‐induced antihyperalgesia. NAN‐190 also blocked manipulation‐induced antihyperalgesia suggesting that effects of methysergide are mediated by 5‐HT1A receptor blockade. However, spinal blockade of opioid or GABAA receptors had no effect on manipulation induced‐antihyperalgesia. Thus, the antihyperalgesia produced by joint manipulation appears to involve descending inhibitory mechanisms that utilize serotonin and noradrenaline.

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.

Neural changes in acute arthritis in monkeys. I. Parallel enhancement of responses of spinothalamic tract neurons to mechanical stimulation and excitatory amino acids

Somatosensory neurons of the spinal cord, including projection neurons, become hyperexcitable to mechanical stimuli during the development of experimental arthritis in rats and cats and hence are suggested to participate in the generation of arthritic hyperalgesia in humans. The experiments described here show a potentiation of the responses of spinothalamic tract (STT) neurons in monkeys during the development of an acute arthritis. The results demonstrate that the responses of STT neurons to mechanical stimuli and to iontophoretically applied excitatory amino acids (EAAs), particularly those acting at non-N-methyl-D-aspartate (non-NMDA) receptors, become enhanced during the development of inflammation produced by intra-articular injection of kaolin and carrageenan. Since the enhancement of both responses follows a similar time course, the results of this work suggest a role for EAAs in the hyperalgesia associated with arthritis and hence may provide a possible pharmacologic target for alleviation and/or prevention of arthritic pain.

Treatment with either high or low frequency TENS reduces the secondary hyperalgesia observed after injection of kaolin and carrageenan into the knee joint.

&NA; For years, physical therapists have been utilizing a variety of modalities, including transcutaneous electrical nerve stimulation (TENS), in an attempt to manage pain associated with inflammation. However, the data on clinical effectiveness is conflicting and the neurophysiological mechanism of action is not known. The purpose of this study was to investigate the effects of high and low frequency TENS on the secondary hyperalgesia that occurs after joint inflammation. Secondary hyperalgesia is thought to reflect changes in central neurons and is thus a measure of activity of central neurons. This study utilized the kaolin and carrageenan model of knee joint inflammation and measured the effects of TENS treatment on paw withdrawal latency to radiant heat (secondary hyperalgesia), spontaneous pain behaviors and joint circumference. Either high (100 Hz) or low (4 Hz) frequency TENS was applied to the knee joint for 20 min after the development of hyperalgesia. Both high and low frequency TENS resulted in a reversal of the hyperalgesia immediately following treatment. The effects of high frequency TENS lasted through at least 24 h while those of low frequency TENS lasted through 12 h. There was no effect of TENS on spontaneous pain behaviors or joint swelling when compared to controls. Thus, TENS appears to be more effective in reducing referred pain (or secondary hyperalgesia) without affecting guarding or splinting of the affected limb. Thus, clinically, the choice to use TENS may depend on patient symptoms; specifically TENS should be effective in reducing referred or radiating pain.