Rajan Radhakrishnan

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.

Transcutaneous electrical nerve stimulation (TENS) reduces chronic hyperalgesia induced by muscle inflammation

Abstract Transcutaneous electrical nerve stimulation (TENS) reduces pain through central mechanisms involving spinal cord and brainstem sites. Since TENS acts through central mechanisms, we hypothesized that TENS will reduce chronic bilateral hyperalgesia produced by unilateral inflammation when applied either ipsilateral or contralateral to the site of muscle inflammation. Sprague–Dawley rats were injected with carrageenan in the left gastrocnemius muscle belly. Mechanical withdrawal threshold was tested bilaterally before and 2 weeks after carrageenan injection. After testing withdrawal thresholds at 2 weeks, rats received TENS treatment either ipsilateral or contralateral to the site of inflammation. In each of these groups, rats were randomized to control (no TENS), low frequency (4 Hz), or high frequency (100 Hz) TENS treatment. TENS was applied for 20 min at sensory intensity under light halothane anesthesia. Mechanical withdrawal thresholds were re‐assessed after TENS or ‘no TENS’ treatment. Unilateral injection of carrageenan to the gastrocnemius muscle significantly reduced the mechanical withdrawal threshold (mechanical hyperalgesia) bilaterally 2 weeks later. Either low or high frequency TENS applied to the gastrocnemius muscle ipsilateral to the site of inflammation significantly reversed mechanical hyperalgesia, both ipsilateral and contralateral to the site of inflammation. Low or high frequency TENS applied to the gastrocnemius muscle contralateral to the site of inflammation also significantly reduced mechanical hyperalgesia, both ipsilateral and contralateral to the site of inflammation. Since ipsilateral or contralateral TENS treatments were effective in reducing chronic bilateral hyperalgesia in this animal model, we suggest that TENS act through modulating descending influences from supraspinal sites such as rostral ventromedial medulla (RVM).

Spinal 5-HT2 and 5-HT3 receptors mediate low, but not high, frequency TENS-induced antihyperalgesia in rats

Transcutaneous electrical nerve stimulation (TENS) is a form of non‐pharmacological treatment for pain. Involvement of descending inhibitory systems is implicated in TENS‐induced analgesia. In the present study, the roles of spinal 5‐HT and &agr;2‐adrenoceptors in TENS analgesia were investigated in rats. Hyperalgesia was induced by inflaming the knee joint with 3% kaolin–carrageenan mixture and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The (1) &agr;2‐adrenergic antagonist yohimbine (30 &mgr;g), (2) 5‐HT antagonist methysergide (5‐HT1 and 5‐HT2, 30 &mgr;g), one of the 5‐HT receptor subtype antagonists, (3) NAN‐190 (5‐HT1A, 15 &mgr;g), (4) ketanserin (5‐HT2A, 30 &mgr;g), (5) MDL‐72222 (5‐HT3, 12 &mgr;g), or (6) vehicle was administered intrathecally prior to TENS treatment. Low (4 Hz) or high (100 Hz) frequency TENS at sensory intensity was then applied to the inflamed knee for 20 min and PWL was determined. Selectivity of the antagonists used was confirmed using respective agonists administered intrathecally. Yohimbine had no effect on the antihyperalgesia produced by low or high frequency TENS. Methysergide and MDL‐72222 prevented the antihyperalgesia produced by low, but not high, frequency TENS. Ketanserin attenuated the antihyperalgesic effects of low frequency TENS whereas NAN‐190 had no effect. The results from the present study show that spinal 5‐HT receptors mediate low, but not high, frequency TENS‐induced antihyperalgesia through activation of 5‐HT2A and 5‐HT3 receptors in rats. Furthermore, spinal noradrenergic receptors are not involved in either low or high frequency TENS antihyperalgesia.

Spinal muscarinic receptors are activated during low or high frequency TENS-induced antihyperalgesia in rats

Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacological modality used clinically to relieve pain. Central involvement of serotonin and endogenous opioids are implicated in TENS-induced analgesia. Activation of spinal cholinergic receptors is antinociceptive and these receptors interact with opioid and serotonin receptors. In the current study, the possible involvement of spinal cholinergic receptors in TENS analgesia was investigated in rats. Hyperalgesia was induced by inflaming one knee joint with 3% kaolin-carrageenan and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The non-selective nicotinic antagonist mecamylamine (50 microg), non-selective muscarinic antagonist atropine (30 microg) or one of the muscarinic subtype antagonists: pirenzepine (M1, 10 microg), methoctramine (M2, 10 microg), 4-DAMP (M3, 10 microg), or saline was administered intrathecally just prior to TENS treatment. Low or high frequency TENS was then applied to the inflamed knee and PWL was determined again. Atropine, pirenzepine and 4-DAMP significantly attenuated the antihyperalgesic effects of low and high frequency TENS while mecamylamine and methoctramine had no effects, compared to saline control. The results show that TENS-induced antihyperalgesia is mediated partially by activation of spinal muscarinic receptors but not spinal nicotinic receptors. Further, the results also indicate that spinal M1 and M3 muscarinic receptor subtypes mediate the muscarinic component of TENS antihyperalgesia.

Transcutaneous Electrical Nerve Stimulation at Both High and Low Frequencies Reduces Primary Hyperalgesia in Rats With Joint Inflammation in a Time-Dependent Manner

Background and Purpose Clinical studies of transcutaneous electrical nerve stimulation (TENS) have used a variety of outcome measures to assess its effectiveness, with conflicting results. It is possible that TENS is effective on some measures of pain and not on others. The purpose of this study was to test the hypothesis that TENS reduces primary hyperalgesia of the knee induced by joint inflammation. Subjects Male Sprague-Dawley rats were used in this study. Methods Inflammation of the knee joint was induced by intra-articular injection of a mixture of 3% kaolin and 3% carrageenan. Primary hyperalgesia was measured as the compression withdrawal threshold of the knee joint before and after the induction of inflammation (4 hours, 24 hours, and 2 weeks) and after sham TENS treatment, treatment with high-frequency TENS (100 Hz), or treatment with low-frequency TENS (4 Hz). Results The compression withdrawal threshold was significantly reduced at 4 hours, 24 hours, and 2 weeks after the induction of inflammation. Either high-frequency TENS or low-frequency TENS completely reversed the compression withdrawal threshold when applied at 24 hours or 2 weeks after the induction of inflammation but not when applied at 4 hours after the induction of inflammation. Discussion and Conclusion These data suggest that TENS inhibits primary hyperalgesia associated with inflammation in a time-dependent manner after inflammation has already developed during both acute and chronic stages.

TRPV1 is important for mechanical and heat sensitivity in uninjured animals and development of heat hypersensitivity after muscle inflammation

Summary TRPV1−/− mice do not develop heat hyperalgesia after muscle inflammation; hyperalgesia is restored by reexpression of TRPV1 into both skin and muscle of TRPV1−/− mice. Abstract Inflammatory thermal hyperalgesia is principally mediated through transient receptor potential vanilloid 1 (TRPV1) channels, as demonstrated by prior studies using models of cutaneous inflammation. Muscle pain is significantly different from cutaneous pain, and the involvement of TRPV1 in hyperalgesia induced by muscle inflammation is unknown. We tested whether TRPV1 contributes to the development of mechanical and heat hypersensitivity of the paw in TRPV1−/− mice after muscle inflammation. Because TRPV1−/− mice lack TRPV1 at the site of inflammation (muscle) and at the testing site (paw), we do not know whether TRPV1 is important as a mediator of nociceptor sensitization in the muscle or as a heat sensor in the paw. Using recombinant herpesviruses, we reexpressed TRPV1 in TRPV1−/− mice in primary afferents innervating skin, muscle, or both to determine which sites were important for the behavioral deficits. Responses to repeated application of noxious mechanical stimuli to the hind paw were enhanced in TRPV1−/− mice; this was restored by reexpression of TRPV1 into skin. Withdrawal latencies to noxious heat were increased in TRPV1−/− mice; normal latencies were restored by reexpression of TRPV1 in both skin and muscle. Heat hypersensitivity induced by muscle inflammation did not develop in TRPV1−/− mice; mechanical hypersensitivity was similar between TRPV1−/− and TRPV1+/+ mice. Heat hypersensitivity induced by muscle inflammation was restored by reexpression of TRPV1 into both muscle and skin of TRPV1−/− mice. These results suggest that TRPV1 serves as both a mediator of nociceptor sensitization at the site of inflammation and as a heat sensor at the paw.

Increased glutamate and decreased glycine release in the rostral ventromedial medulla during induction of a pre-clinical model of chronic widespread muscle pain

Two injections of acidic saline into the gastrocnemius muscle produce long-lasting hyperalgesia that is initiated and maintained by changes in the rostroventromedial medulla (RVM). Potential underlying mechanisms could be increased release of excitatory neurotransmitters and/or reduced release of inhibitory neurotransmitters, in the RVM. We tested this hypothesis by measuring concentrations of aspartate, glutamate and glycine in response to the first and second injection of acidic saline and compared to intramuscular injections of normal saline using microdialysis with HPLC analysis. We show a significant increase in aspartate and glutamate during the second acidic saline injection compared to normal saline injections or the first injection of acidic saline. There were also long-lasting decreases in glycine concentrations in the RVM in response to both the first and second injection of acidic saline. It is possible that disinhibition after the first injection leads to long-lasting neuronal changes that allow a greater release of excitatory neurotransmitters after the second injection. We hypothesize that increased release of excitatory neurotransmitters in the RVM drives the release of excitatory neurotransmitters in the spinal cord, central sensitization and the consequent hyperalgesia.

Models of muscle pain: carrageenan model and acidic saline model.

Carrageenan or acidic saline injected unilaterally into the gastrocnemius muscle or triceps muscle produces a robust and long‐lasting hyperalgesia in rats and mice, which is reversible with systemic administration of opioid or anti‐inflammatory drugs. This unit describes detailed protocols for inducing and measuring hyperalgesia, and provides information on validation of these models. These models are useful for assessing new compounds for their analgesic activity in muscular pain.