Darryl T. Hamamoto

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.

Mechanism of Cancer Pain

Ongoing and breakthrough pain is a primary concern for the cancer patient. Although the etiology of cancer pain remains unclear, animal models of cancer pain have allowed investigators to unravel some of the cancer-induced neuropathologic processes that occur in the region of tumor growth and in the dorsal horn of the spinal cord. Within the cancer microenvironment, cancer and immune cells produce and secrete mediators that activate and sensitize primary afferent nociceptors. Pursuant to these peripheral changes, nociceptive secondary neurons in spinal cord exhibit increased spontaneous activity and enhanced responsiveness to three modes of noxious stimulation: heat, cold, and mechanical stimuli. As our understanding of the peripheral and central mechanisms that underlie cancer pain improves, targeted analgesics for the cancer patient will likely follow.

Bone formation on carbon nanotube composite

The effects of a layer-by-layer assembled carbon nanotube composite (CNT-comp) on osteoblasts in vitro and bone tissue in vivo in rats were studied. The effects of CNT-comp on osteoblasts were compared against the effects by commercially pure titanium (cpTi) and tissue culture dishes. Cell proliferation on the CNT-comp and cpTi were similar. However, cell differentiation, measured by alkaline phosphatase activity and matrix mineralization, was better on the CNT-comp. When implanted in critical-sized rat calvarial defect, the CNT-comp permitted bone formation and bone repair without signs of rejection or inflammation. These data indicate that CNT-comp may be a promising substrate for use as a bone implant or as a scaffold for tissue engineering.

Tumor-evoked hyperalgesia and sensitization of nociceptive dorsal horn neurons in a murine model of cancer pain.

Pain associated with cancer, particularly when tumors metastasize to bone, is often severe and debilitating. Better understanding of the neurobiological mechanisms underlying cancer pain will likely lead to the development of more effective treatments. The aim of this study was to characterize changes in response properties of nociceptive dorsal horn neurons following implantation of fibrosarcoma cells into and around the calcaneus bone, an established model of cancer pain. Extracellular electrophysiological recordings were made from wide dynamic range (WDR) and high threshold (HT) dorsal horn neurons in mice with tumor-evoked hyperalgesia and control mice. WDR and HT neurons were examined for ongoing activity and responses to mechanical, heat, and cold stimuli applied to the plantar surface of the hind paw. Behavioral experiments showed that mice exhibited hyperalgesia to mechanical and heat stimuli applied to their tumor-bearing hind paw. WDR, but not HT, nociceptive dorsal horn neurons in tumor-bearing mice exhibited sensitization to mechanical, heat, and cold stimuli and may contribute to tumor-evoked hyperalgesia. Specifically, the proportion of WDR neurons that exhibited ongoing activity and their evoked discharge rates were greater in tumor-bearing than in control mice. In addition, WDR neurons exhibited lower response thresholds for mechanical and heat stimuli, and increased responses to suprathreshold mechanical, heat, and cold stimuli. Our findings show that sensitization of WDR neurons contributes to cancer pain and supports the notion that the mechanisms underlying cancer pain differ from those that contribute to inflammatory and neuropathic pain.

The role of pH and osmolarity in evoking the acetic acid-induced wiping response in a model of nociception in frogs.

Acetic acid applied to the hindlimb of a frog evokes a vigorous wiping of the exposed skin. The aim of this study was to determine if acetic acid evokes this wiping response by decreasing subepidermal pH. Because acetic acid is hyperosmolar, a second aim was to determine if the osmolarity of acetic acid contributed to evoking the wiping response. In behavioral experiments, different acids or acetic acid/sodium acetate buffers at different pHs were used to evoke the wiping response. In separate experiments, subepidermal pH was measured in vitro while these same solutions were applied to samples of skin from frogs. The wiping response evoked by acetic acid was associated with a decrease in subepidermal pH to a level that has been shown to activate nociceptors. Interestingly, formic, oxalic, sulfuric, and hydrochloric acid evoked the wiping response without decreasing subepidermal pH. The osmolarity of acetic acid contributed to evoking the wiping response because buffers at subthreshold pHs evoked the wiping response. Also, the osmolarity required to evoke the wiping response depended upon the pH of the buffer. Thus, acetic acid and the buffers at pH 2.97 and 4.67 could evoke the wiping response by decreasing subepidermal pH. In contrast, formic, oxalic, sulfuric, and hydrochloric acid, as well as the buffers at pH 5.17 and 5.67, evoked the wiping response through another mechanism, perhaps by increasing subepidermal osmolarity. These studies demonstrate that both pH and osmolarity may contribute to nociception produced by algesic chemicals and may be important in inflammatory pain.

Intraplantar injection of hyaluronic acid at low pH into the rat hindpaw produces tissue acidosis and enhances withdrawal responses to mechanical stimuli

&NA; Application of buffers covering a range of acidic pH values activates and sensitizes nociceptors and produces pain. The purpose of this study was to determine whether a range of acidic pH in tissue produces mechanical hyperalgesia. Tissue acidosis was produced in the hindpaw of the rat by intraplantar injections of hyaluronic acid (HA) adjusted to pH 7.4, 6.0, 5.0, 4.0 or 3.0. Mechanical hyperalgesia was assessed by evaluating responses to application of a von Frey monofilament to the plantar surface before and after injection of HA. In separate experiments, magnitude of tissue acidosis produced by injection of HA was determined by measuring pH of intraplantar tissue using a pH microelectrode. Although needle stick alone produced mechanical hyperalgesia, intraplantar injections of HA at pH 6.0 or 5.0 produced significantly greater mechanical hyperalgesia. In contrast, mechanical hyperalgesia produced by injection of HA at pH 7.4, 4.0 or 3.0 was not different from that produced by needle stick. Although injection of HA at low pH produced tissue acidosis in a pH dependent manner, only a narrow range of tissue acidosis (pH=6.38–6.00) produced mechanical hyperalgesia. Our data suggest that tissue acidosis induces mechanical hyperalgesia; however, the range of tissue pH that produces this effect is limited.

Tumor-Evoked Sensitization of C Nociceptors: A Role for Endothelin

Primary and metastatic cancers that effect bone are frequently associated with pain. Sensitization of primary afferent C nociceptors innervating tissue near the tumor likely contributes to the chronic pain and hyperalgesia accompanying this condition. This study focused on the role of the endogenous peptide endothelin-1 (ET-1) as a potential peripheral algogen implicated in the process of cancer pain. Electrophysiological response properties, including ongoing activity and responses evoked by heat stimuli, of C nociceptors were recorded in vivo from the tibial nerve in anesthetized control mice and mice exhibiting mechanical hyperalgesia following implantation of fibrosarcoma cells into and around the calcaneus bone. ET-1 (100 microM) injected into the receptive fields of C nociceptors innervating the plantar surface of the hind paw evoked an increase in ongoing activity in both control and tumor-bearing mice. Moreover, the selective ETA receptor antagonist, BQ-123 (3 mM), attenuated tumor-evoked ongoing activity in tumor-bearing mice. Whereas ET-1 produced sensitization of C nociceptors to heat stimuli in control mice, C nociceptors in tumor-bearing mice were sensitized to heat, and their responses were not further increased by ET-1. Importantly, administration of BQ-123 attenuated tumor-evoked sensitization of C nociceptors to heat. We conclude that ET-1 at the tumor site contributes to tumor-evoked excitation and sensitization of C nociceptors through an ETA receptor mediated mechanism.

SYM 2081, an agonist that desensitizes kainate receptors, attenuates capsaicin and inflammatory hyperalgesia

Excitatory amino acids acting at non-NMDA receptors contribute to transmission of nociceptive information. SYM 2081 ((2S,4R)-4-methyl glutamic acid) desensitizes kainate receptors, one subtype of non-NMDA receptors, to subsequent release of excitatory amino acids and thus may attenuate transmission of nociceptive information. To determine if SYM 2081 can prevent development of hyperalgesia, SYM 2081 (10, 50 or 100 mg/kg, i.p.) was administered prior to injection of capsaicin into the hindpaw of rats, which produces mechanical and heat hyperalgesia. To determine if SYM 2081 can reduce ongoing inflammatory hyperalgesia, SYM 2081 (10 or 100 mg/kg, i.p.) was administered after development of carrageenan-evoked hyperalgesia. Intraplantar injection of capsaicin produced an increase in hindpaw withdrawal frequency to mechanical stimuli (from 4+/-2 to 41+/-7%; mean+/-S.E.M.) and a decrease in withdrawal latency to heat (from 12.3+/-0.3 to 5.9+/-0.4 s) in rats that received vehicle. In contrast, rats that received SYM 2081 (100 mg/kg) prior to injection of capsaicin exhibited a lower hindpaw withdrawal frequency (18+/-4%) and a longer withdrawal latency (7.7+/-0.5 s). Intrathecal (1-100 microg/5 microl), but not intraplantar (10 or 100 microg/50 microl), injection of SYM 2081 attenuated the development of capsaicin-evoked heat hyperalgesia suggesting that SYM 2081s antihyperalgesic effects were due to its central effects. Furthermore, SYM 2081 completely reversed ongoing carrageenan-evoked mechanical hyperalgesia and partially (approximately 50%) reversed ongoing heat hyperalgesia. The present study demonstrates that administration of a high-potency ligand that selectively desensitizes kainate receptors attenuates the development of mechanical and heat hyperalgesia and attenuates ongoing inflammatory hyperalgesia.

Inhibition of Inactive States of Tetrodotoxin-Sensitive Sodium Channels Reduces Spontaneous Firing of C-Fiber Nociceptors and Produces Analgesia in Formalin and Complete Freund’s Adjuvant Models of Pain

While genetic evidence shows that the Nav1.7 voltage-gated sodium ion channel is a key regulator of pain, it is unclear exactly how Nav1.7 governs neuronal firing and what biophysical, physiological, and distribution properties of a pharmacological Nav1.7 inhibitor are required to produce analgesia. Here we characterize a series of aminotriazine inhibitors of Nav1.7 in vitro and in rodent models of pain and test the effects of the previously reported “compound 52” aminotriazine inhibitor on the spiking properties of nociceptors in vivo. Multiple aminotriazines, including some with low terminal brain to plasma concentration ratios, showed analgesic efficacy in the formalin model of pain. Effective concentrations were consistent with the in vitro potency as measured on partially-inactivated Nav1.7 but were far below concentrations required to inhibit non-inactivated Nav1.7. Compound 52 also reversed thermal hyperalgesia in the complete Freund’s adjuvant (CFA) model of pain. To study neuronal mechanisms, electrophysiological recordings were made in vivo from single nociceptive fibers from the rat tibial nerve one day after CFA injection. Compound 52 reduced the spontaneous firing of C-fiber nociceptors from approximately 0.7 Hz to 0.2 Hz and decreased the number of action potentials evoked by suprathreshold tactile and heat stimuli. It did not, however, appreciably alter the C-fiber thresholds for response to tactile or thermal stimuli. Surprisingly, compound 52 did not affect spontaneous activity or evoked responses of Aδ-fiber nociceptors. Results suggest that inhibition of inactivated states of TTX-S channels, mostly likely Nav1.7, in the peripheral nervous system produces analgesia by regulating the spontaneous discharge of C-fiber nociceptors.

Intramuscular pH in Myofascial Pain Syndrome of the Masticatory Muscles

Objective: Myofascial pain syndrome [MPS] of the masticatory muscles is characterized by regional muscle pain and the presence of hypersensitive trigger points. The pathophysiological mechanisms that underlie MPS are poorly understood. Sustained contracture of muscle fibers may produce ischemia and localized acidosis that can excite nociceptors. The objective of this study was to compare pH in the most and the least painful areas of the masseter muscle in subjects with and without MPS of the masticatory muscles. Methods: Symptoms and signs of MPS, pressure-pain thresholds [PPT], and intramuscular pH were determined in 14 female subjects who met the criteria for MPS according to the Research Diagnostic Criteria for Temporomandibular Disorders and 14 female age-matched control subjects. Results: Subjects with MPS reported more clinical symptoms and more clinical signs of MPS than did healthy normal control [HNC] subjects. Subjects with MPS had significantly lower PPTs at the most painful site [97.4 ± 9.5 kPa] than did HNC subjects at either the most [143.0 ± 17.7 kPa] or the least [161.1 ± 15.5 kPa] painful sites. However, there were no significant differences in intramuscular pH between the least painful [7.40 ± 0.17] and the most painful [7.22 ± 0.08] sites in subjects with MPS or in HNC subjects [least painful site, 7.30 ± 0.09; most painful site, 7.25 ± 0.09]. Moreover, there was no significant correlation between PPT and intramuscular pH. Conclusion: These results suggest that intramuscular pH does not contribute to pain in the masseter muscle associated with MPS of the masticatory muscles.