Lois J. Kehl

A new animal model for assessing mechanisms and management of muscle hyperalgesia

&NA; Musculoskeletal pain is one of the most frequent symptoms for which medical assistance is sought. Yet, the majority of our knowledge regarding pain physiology is based on studies of cutaneous tissue. Comparatively little is known about activation of visceral, joint and perhaps least of all, musculoskeletal nociceptors although clinically‐treated pain originates principally in these structures. Studies elucidating the mechanisms of muscle hyperalgesia have been hampered by the lack of an animal model that permits the evaluation of hypotheses using behavioral, biochemical, pharmacological, anatomical and molecular experimental approaches. Here we describe an animal model of muscle hyperalgesia we recently developed that permits such multidisciplinary investigation. This model employs the intramuscular injection of carrageenan, a chemical stimulus which evokes a well recognized model of cutaneous inflammation and is reported to activate muscle nociceptors. Intramuscular carrageenan evokes a time‐ and dose‐dependent reduction in forelimb grip force that is anatomically specific. The carrageenan‐evoked reduction in grip force is blocked by the &mgr;‐opioid agonist levorphanol in a dose‐dependent, stereoselective and naltrexone‐reversible manner. This behavioral dependent measure is also significantly reversed by agents used clinically to treat muscle pain, indomethacin and dexamethasone, as well as the non‐competitive N‐methyl‐D‐aspartate receptor antagonist MK801. Finally, evidence that reduction in grip force is in part mediated by small, unmyelinated afferents is provided by the demonstration that neonatal capsaicin treatment significantly reduced carrageenan‐evoked behavioral hyperalgesia (˜45% reduction) and reduced muscle content of immunoreactive CGRP (˜60% reduction) relative to control levels. Collectively, these findings provide converging lines of evidence for the validity of this animal model to investigate mechanisms involved in the development of muscle hyperalgesia.

A cannabinoid agonist differentially attenuates deep tissue hyperalgesia in animal models of cancer and inflammatory muscle pain.

&NA; Pain associated with cancer and chronic musculoskeletal disorders can be difficult to control. We used murine models of cancer and inflammatory muscle pain to examine whether the cannabinoid receptor agonist WIN55,212–2 reduces hyperalgesia originating in deep tissues. C3H/He mice were anesthetized and implanted with osteolytic NCTC clone 2472 cells into the humeri or injected with 4% carrageenan into the triceps muscles of both forelimbs. At the time of peak hyperalgesia, WIN55,212–2 (1–30 mg/kg) or vehicle was administered intraperitoneally and forelimb grip force was measured 0.5–24 h later. WIN55,212–2 produced time‐ and dose‐related antihyperalgesia in both models. A 10 mg/kg dose of WIN55,212–2 fully reversed carrageenan‐evoked muscle hyperalgesia. However, 30 mg/kg of WIN55,212–2 attenuated tumor‐evoked hyperalgesia only ∼50%. After controlling for the difference in magnitude of hyperalgesia between the two models, WIN55,212–2 was still more potent at reducing hyperalgesia in the inflammatory model. In the cancer pain model, the antihyperalgesic effect of WIN55,212–2 was partially blocked by pretreatment with the selective CB1 (SR141716A) but not the CB2 (SR144528) receptor antagonist. In contrast, both antagonists blocked antihyperalgesic effects of WIN55,212–2 on carrageenan‐evoked muscle hyperalgesia. Catalepsy and loss of motor coordination, known side effects of cannabinoids, did not account for the antihyperalgesia produced by WIN55,212–2. These data show that cannabinoids attenuate deep tissue hyperalgesia produced by both cancer and inflammatory conditions. Interestingly, cannabinoids differentially modulated carrageenan‐ and tumor‐evoked hyperalgesia in terms of potency and receptor subtypes involved suggesting that differences in underlying mechanisms may exist between these two models of deep tissue pain.

Pain-related behaviors and neurochemical alterations in mice expressing sickle hemoglobin: modulation by cannabinoids

Sickle cell disease causes severe pain. We examined pain-related behaviors, correlative neurochemical changes, and analgesic effects of morphine and cannabinoids in transgenic mice expressing human sickle hemoglobin (HbS). Paw withdrawal threshold and withdrawal latency (to mechanical and thermal stimuli, respectively) and grip force were lower in homozygous and hemizygous Berkley mice (BERK and hBERK1, respectively) compared with control mice expressing human hemoglobin A (HbA-BERK), indicating deep/musculoskeletal and cutaneous hyperalgesia. Peripheral nerves and blood vessels were structurally altered in BERK and hBERK1 skin, with decreased expression of mu opioid receptor and increased calcitonin gene-related peptide and substance P immunoreactivity. Activators of neuropathic and inflammatory pain (p38 mitogen-activated protein kinase, STAT3, and mitogen-activated protein kinase/extracellular signal-regulated kinase) showed increased phosphorylation, with accompanying increase in COX-2, interleukin-6, and Toll-like receptor 4 in the spinal cord of hBERK1 compared with HbA-BERK. These neurochemical changes in the periphery and spinal cord may contribute to hyperalgesia in mice expressing HbS. In BERK and hBERK1, hyperalgesia was markedly attenuated by morphine and cannabinoid receptor agonist CP 55940. We show that mice expressing HbS exhibit characteristics of pain observed in sickle cell disease patients, and neurochemical changes suggestive of nociceptor and glial activation. Importantly, cannabinoids attenuate pain in mice expressing HbS.

Tumor implantation in mouse humerus evokes movement-related hyperalgesia exceeding that evoked by intramuscular carrageenan.

&NA; In this paper we compare two innovative models of movement‐related pain: tumor‐induced nociception following implantation of fibrosarcoma cells into bone and muscle inflammation‐induced nociception following injection of the irritant carrageenan into muscle. Importantly, using the grip force test, an assay of movement‐related hyperalgesia, both non‐malignant and malignant pain are examined in parallel. Movement‐related hyperalgesia, known clinically as a specific type of ‘breakthrough pain’, is a common feature of bone cancer and is thought to be a predictor of poor response to conventional analgesic pharmacotherapy (Bruera et al., 1995, J. Pain Symptom. Manage. 10 (1995) 348; Mercadaute et al., 1992, Pain 50 (1992) 151; Pain 81 (1999) 129). Implantation of NCTC 2472 sarcoma cells in both humeri or injection of carrageenan (4%) in both triceps of C3H/He mice produced apparent forelimb hyperalgesia that was not associated with mechanical hyperalgesia in the forepaw, whereas carrageenan at 6 and 8% did evoke significant cutaneous hyperalgesia of the forepaw as well. Control groups receiving implants of vehicle or no treatment at all did not manifest this forelimb hyperalgesia. B6C3/F1 mice implanted with non‐lysis‐inducing G3.26 melanoma cells or vehicle did not manifest significant hyperalgesia when compared to B6C3/F1 mice receiving fibrosarcoma cells, indicating a dependence on bone involvement for induction of hyperalgesia in this model. Histological examination at days 3, 7, and 10 post‐implantation showed a clear correlation of tumor growth‐induced bone destruction with behavioral hyperalgesia. Morphine was more potent in decreasing the maximal hyperalgesia induced by carrageenan than that induced by tumor implantation. Acutely administered morphine (3–100 mg/kg, i.p.) attenuated peak hyperalgesia of carrageenan‐injected mice (ED50 6.9 mg/kg) and tumor‐bearing mice (ED50 23.9 mg/kg) in a dose‐related manner with a difference in potency of 3.5. Tumor‐implanted mice with a level of hyperalgesia comparable to that induced by carrageenan required almost three times more morphine (ED50 18.5 mg/kg) for equivalent attenuation of forelimb hyperalgesia. These animal models of movement‐related hyperalgesia may aid in discerning the peripheral and central mechanisms underlying pain that accompanies bone metastases and distinguishing it from the pain associated with muscular inflammation. Importantly, they may also aid in predicting differences in analgesic efficacy in different types of musculoskeletal pain.

Experimental animal models of muscle pain and analgesia.

KEHL, L. J., and C. A. FAIRBANKS. Experimental animal models of muscle pain and analgesia. Exerc. Sport Sci. Rev., Vol. 31, No. 4, pp. 188–194, 2003. Muscle pain is a prevalent clinical problem but can be difficult to treat because relatively little is known about nervous system mechanisms that mediate and modulate it. This review profiles four new animal models of muscle and deep tissue pain currently being used to elucidate mechanisms of muscle pain and analgesia.

Tolerance develops to the effect of lipopolysaccharides on movement-evoked hyperalgesia when administered chronically by a systemic but not an intrathecal route.

&NA; Single exposures to lipopolysaccharides (LPS) produce deep tissue pain in humans and cutaneous hyperalgesia in rodents. While tolerance develops to many effects of LPS, sensitization to hyperalgesia is documented after a single injection. To determine the effect of long‐term exposure to LPS, we explored the chronic effect of LPS on movement‐evoked pain using a new assay based on grip force in mice. We found that a single systemic injection of LPS (i.p. or s.c.) induced a dose‐related decrease in forelimb grip force responses beginning 6–8 h after injection and peaking between 9 and 24 h. The consequence of LPS is likely hyperalgesia rather than weakness as these decreases were rapidly attenuated by either 10 mg/kg of morphine i.p. or 10 &mgr;g of morphine injected intrathecally (i.t.). Complete tolerance to this hyperalgesia developed after repeated injections of LPS at doses of 0.9 mg/kg i.p. or 5 mg/kg s.c. Tolerance began after a single injection and was fully developed after as few as four injections of 5 mg/kg of LPS delivered s.c. The concentration of circulating LPS 5 h after a single parenteral injection was less in LPS‐tolerant mice than naïve controls, suggesting that tolerance may result from a more efficient clearance of LPS from the circulation. Injected i.t., LPS also induced hyperalgesia, however, tolerance did not develop to multiple injections by this route. There was no cross‐tolerance between s.c. and i.t. injections of LPS. These data indicate that decreases in grip force are a sensitive measure of LPS‐induced movement‐evoked hyperalgesia and that tolerance develops to parenteral but not central hyperalgesic effects of LPS.

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.

A Quantitative Evaluation of Inflammatory Cells in Human Temporomandibular Joint Tissues From Patients With and Without Implants

PURPOSE Previous studies made only qualitative assessments of immune cell responses to temporomandibular joint (TMJ) implant wear debris. The aim of this study was to perform a quantitative comparison of inflammatory cell types in TMJ tissues with implant wear debris, TMJ tissues with a history of disc pathology without implant debris, and TMJ tissues from normal control subjects. MATERIALS AND METHODS TMJ tissues were collected from the following 3 groups of subjects: 1) individuals with failed TMJ implants (implant group, n = 10), 2) patients with TMJ disc pathology but no history of implant placement (nonimplant surgery group, n = 10), and normal cadaveric tissues with no history of surgery (control group, n = 10). Tissue sections (5 microm) from all subjects were stained with hematoxylin-eosin, after which cell counts were done for 2 types of inflammatory cells: multinucleated giant cells and lymphocytes. Mean inflammatory cell counts from the 3 groups were compared by use of a 1-way analysis of variance procedure and Bonferroni adjustment to maintain an overall type I error rate of .05. RESULTS Implant group tissues contained significantly more inflammatory cells than tissues from the nonimplant surgery and control groups (P < .0001). Multinucleated giant cells were only present in implant group tissues. Although the high number of multinucleated giant cells present in the implant group obscured a total count of lymphocytes for that group, lymphocyte cell counts were still significantly greater (P < .005) in implant group tissues than in tissues from the other 2 groups. CONCLUSIONS Our data provide quantitative confirmation that the presence of Proplast-Teflon implant (Vitek, Houston, TX) wear debris is associated with a significant increase in the number of local multinucleated giant cells and lymphocytes.

Evidence for a Role of Nerve Injury in Painful Intervertebral Disc Degeneration: A Cross-Sectional Proteomic Analysis of Human Cerebrospinal Fluid

Intervertebral disc degeneration (DD) is a cause of low back pain (LBP) in some individuals. However, although >30% of adults have DD, LBP only develops in a subset of individuals. To gain insight into the mechanisms underlying nonpainful versus painful DD, human cerebrospinal fluid (CSF) was examined using differential expression shotgun proteomic techniques comparing healthy control participants, subjects with nonpainful DD, and patients with painful DD scheduled for spinal fusion surgery. Eighty-eight proteins were detected, 27 of which were differentially expressed. Proteins associated with DD tended to be related to inflammation (eg, cystatin C) regardless of pain status. In contrast, most differentially expressed proteins in DD-associated chronic LBP patients were linked to nerve injury (eg, hemopexin). Cystatin C and hemopexin were selected for further examination using enzyme-linked immunosorbent assay in a larger cohort. While cystatin C correlated with DD severity but not pain or disability, hemopexin correlated with pain intensity, physical disability, and DD severity. This study shows that CSF can be used to study mechanisms underlying painful DD in humans, and suggests that while painful DD is associated with nerve injury, inflammation itself is not sufficient to develop LBP. PERSPECTIVE CSF was examined for differential protein expression in healthy control participants, pain-free adults with asymptomatic intervertebral DD, and LBP patients with painful intervertebral DD. While DD was related to inflammation regardless of pain status, painful degeneration was associated with markers linked to nerve injury.