Metastatic Infiltration of Nervous Tissue and Periosteal Nerve Sprouting in Multiple Myeloma-Induced Bone Pain in Mice and Human.

Multiple myeloma (MM) is a neoplasia of B plasma cells that often induces bone pain. However, the mechanisms underlying myeloma-induced bone pain (MIBP) are mostly unknown. Using a syngeneic MM mouse model, we show that periosteal nerve sprouting of calcitonin gene-related peptide (CGRP+) and growth associated protein 43 (GAP43+) fibers occurs concurrent to the onset of nociception and its blockade provides transient pain relief. MM patient samples also showed increased periosteal innervation. Mechanistically, we investigated MM induced gene expression changes in the dorsal root ganglia (DRG) innervating the MM-bearing bone of male mice and found alterations in pathways associated with cell cycle, immune response and neuronal signaling. The MM transcriptional signature was consistent with metastatic MM infiltration to the DRG, a never-before described feature of the disease that we further demonstrated histologically. In the DRG, MM cells caused loss of vascularization and neuronal injury, which may contribute to late-stage MIBP. Interestingly, the transcriptional signature of a MM patient was consistent with MM cell infiltration to the DRG. Overall, our results suggest that MM induces a plethora of peripheral nervous system alterations that may contribute to the failure of current analgesics and suggest neuroprotective drugs as appropriate strategies to treat early onset MIBP.SIGNIFICANCE STATEMENT Multiple myeloma (MM) is a painful bone marrow cancer that significantly impairs the quality of life of the patients. Analgesic therapies for myeloma-induced bone pain (MIBP) are limited and often ineffective, and the mechanisms of MIBP remain unknown. In this manuscript, we describe cancer-induced periosteal nerve sprouting in a mouse model of MIBP, where we also encounter metastasis to the dorsal root ganglia (DRG), a never-before described feature of the disease. Concomitant to myeloma infiltration, the lumbar DRGs presented blood vessel damage and transcriptional alterations, which may mediate MIBP. Explorative studies on human tissue support our preclinical findings. Understanding the mechanisms of MIBP is crucial to develop targeted analgesic with better efficacy and fewer side effects for this patient population.

Characterization of pain-related behaviors, changes in bone microarchitecture and sensory innervation induced by chronic cadmium exposure in adult mice.

Because of the relative lack of understanding of the neurobiological mechanisms that drive toxic effects of cadmium in bone, the purpose of this study was to characterize a preclinical model of chronic cadmium exposure. Adult male C57BL/6 J mice were exposed to cadmium 25 mg/L (as CdCl2) in drinking water for 16 weeks. During this time, pain-related behaviors including hindpaw mechanical sensitivity and vertical rears were evaluated every four weeks. We assessed changes in bone microarchitecture at the femoral neck and L5 vertebra by microcomputed tomography and quantified the density of nerve fibers expressing PGP 9.5 (a pan-neuronal marker) and CGRP (a marker of sensory nerve fibers subfamily) at the femoral neck and glabrous skin of the hindpaw using immunohistochemistry. Cadmium exposure produced mechanical hypersensitivity in both hindpaws along with decreased rearing activity (surrogate for musculoskeletal-related pain) without affecting the horizontal activity (a measure of locomotor behavior) in comparison to the control group. Intraperitoneal acute treatment with morphine and gabapentin reversed pain-related behaviors in cadmium-exposed mice. Furthermore, exposure to cadmium resulted in significant trabecular bone deterioration at the femoral neck and L5 vertebra. We also observed a significant reduction in the density of both CGRP+ and PGP 9.5+ nerve fibers in the femoral neck, but not in the hindpaw glabrous skin, suggesting tissue-dependent neurotoxicity. This model may help in developing a mechanism-based understanding of the factors that generate and maintain musculoskeletal pain and bone loss caused by chronic cadmium exposure and in translating these findings into new therapies for treating cadmium-induced bone toxicity.

Safety and efficacy of intravesical instillation of resiniferatoxin in healthy cats: A preliminary study.

Objectives: To evaluate the safety of intravesical application of resiniferatoxin (RTX) in healthy cats and its effects on calcitonin gene-related peptide (CGRP) and substance P (SP) produced by C-fibers. Methods: Seven adult female cats received either 25 mL of saline (control; n = 1), or intravesical RTX at 5, 25, or 50 µg in 25 mL of saline to a final concentration of 0.2 µg/mL (318 nM), 1 µg/mL (1,591 nM), and 2 µg/mL (3,181 nM) (n = 2 per group). The treatment was instilled into the urinary bladder for 20 min. Plasma concentrations of RTX were measured at 0, 0.5, 1, and 4 h. Physical exam, complete blood count, and serum biochemical analysis were performed on day 0, 7, and 14. After 14 days, the sacral dorsal root ganglia (DRG) and the urinary bladder were harvested for histological and immunofluorescence analysis. Results: Intravesical RTX was well tolerated and plasma concentrations were below the quantifiable limits except for one cat receiving 1 µg/mL. Mild to moderate histopathological changes, including epithelial changes, edema, and blood vessel proliferation, were observed at lower doses (0.2 and 1 µg/mL), and were more severe at the higher dose (2 µg/mL). C-fiber ablation was observed in the urinary bladder tissue at all doses, as shown by an apparent reduction of both CGRP and SP immunoreactive axons. Conclusion: A dose of 25 µg (1 µg/mL) of RTX instilled in the urinary bladder of healthy cats appeared to decrease the density of SP and CGRP nerve axons innervating bladder and induced moderate changes in the bladder tissue.

Cadmium exposure negatively affects the microarchitecture of trabecular bone and decreases the density of a subset of sympathetic nerve fibers innervating the developing rat femur.

Cadmium (Cd) is toxic to the skeletal system resulting in bone loss and pain. We aimed at determining the effect of chronic Cd exposure on bone density and microarchitecture along with changes in the density of a subset of sensory and sympathetic nerve fibers innervating the developing rat femur. Newborn male Wistar rats were injected daily for 49 days with CdCl2 (1 mg/kg i.p.) or saline solution (control group). At the day of sacrifice, levels of Cd in the right femur, liver and kidney were determined by atomic absorption spectrophotometry. Additionally, microCT followed by immunohistochemical analyses were performed in the left femur. Results showed Cd accumulation in trabecular bone neared levels seen in liver and kidney. Cd concentration in cortical bone was significantly lower versus trabecular bone. MicroCT analysis revealed that Cd-exposed rats had a significant decrease in trabecular bone parameters at the distal femoral metaphysis; however, most of the cortical bone parameters were not significantly affected. Cd-exposed rats showed a significant loss of TH+ sympathetic nerve fibers, but not of CGRP+ sensory nerve fibers, at the level of bone marrow of the femoral diaphysis as compared to control rats. This study shows that Cd negatively affects bone density and microarchitecture of trabecular bone and decreases the density of sympathetic nerve fibers innervating rat femur. Future studies are warranted to determine the toxigenic mechanisms of Cd on sympathetic nerves and how sympathetic denervation influences bone loss in animals exposed to Cd.

Blockade of the colony-stimulating factor-1 receptor reverses bone loss in osteoporosis mouse models.

BACKGROUND: Mice lacking either colony-stimulating factor-1 (CSF-1) or its receptor, CSF-1R, display osteopetrosis. Accordingly, genetic deletion or pharmacological blockade of CSF-1 prevents the bone loss associated with estrogen deficiency. However, the role of CSF-1R in osteoporosis models of type-1 diabetes (T1D) and ovariectomy (OVX) has not been examined. Thus, we evaluated whether CSF-1R blockade would relieve the bone loss in a model of primary osteoporosis (female mice with OVX) and a model of secondary osteoporosis (female with T1D) using micro-computed tomography. METHODS: Female ICR mice at 10 weeks underwent OVX or received five daily administrations of streptozotocin (ip, 50 mg/kg) to induce T1D. Four weeks after OVX and 14 weeks after first injection of streptozotocin, mice received an anti-CSF-1R (2G2) antibody (10 mg/kg, ip; once/week for 6 weeks) or vehicle. At the last day of antibody administration, mice were sacrificed and femur and tibia were harvested for micro-computed tomography analysis. RESULTS: Mice with OVX had a significant loss of trabecular bone at the distal femoral and proximal tibial metaphysis. Chronic treatment with anti-CSF-1R significantly reversed the trabecular bone loss at these anatomical sites. Streptozotocin-induced T1D resulted in significant loss of trabecular bone at the femoral neck and cortical bone at the femoral mid-diaphysis. Chronic treatment with anti-CSF-1R antibody significantly reversed the bone loss observed in mice with T1D. CONCLUSION: Our results demonstrate that blockade of CSF-1R signaling reverses bone loss in two different mouse models of osteoporosis.

Chronic administration of Cl-amidine, a pan-peptidylarginine deiminase inhibitor, does not reverse bone loss in two different murine models of osteoporosis.

Recent in vitro studies have shown a role for the peptidyl-arginine deiminases (PADs) in bone resorption. However, it is unknown whether these enzymes are involved in bone loss in vivo. Thus, we evaluated the antiresorptive effect of a pan-PAD inhibitor in two murine models of osteoporosis: (a) primary osteoporosis induced by ovariectomy (OVX); and (b) secondary osteoporosis associated to Type-1 diabetes induced by streptozotocin (STZ, 50 mg/kg, i.p., five daily administrations). Five weeks after OVX and 15 weeks after injections of STZ, mice received daily administrations of Cl-amidine (3 or 10 mg/kg, i.p.) or vehicle for 30 consecutive days. At the end of the treatment, femur and vertebra were harvested for microCT analysis. Blood samples were collected for determination of antibodies against cyclic citrullinated peptides (anti-CCP) by enzyme-linked immunosorbent assay. Serum levels of anti-CCP antibodies from diabetic mice were not significantly different compared to control mice. However, a significant loss of both trabecular bone at the femoral neck and cortical bone at the femoral diaphysis was found in diabetic mice, and Cl-amidine did not reverse the diabetes-induced bone loss. Mice with OVX had significantly lower serum levels of anti-CCP compared to mice with sham surgery. OVX resulted in significant loss of both trabecular bone at the L5 vertebra and distal femoral metaphysis. Cl-amidine did not block the OVX-induced bone loss. Our results suggest that chronic treatment with Cl-amidine at the doses and period of time administered is not long enough to inhibit bone loss in two different murine models of osteoporosis.

The effect of androgen excess on maternal metabolism, placental function and fetal growth in obese dams.

Pregnant women with polycystic ovary syndrome (PCOS) are often overweight or obese. To study the effects of maternal androgen excess in obese dams on metabolism, placental function and fetal growth, female C57Bl6J mice were fed a control (CD) or a high fat/high sucrose (HF/HS) diet for 4-10 weeks, and then mated. On gestational day (GD) 15.5-17.5, dams were injected with dihydrotestosterone (CD-DHT, HF/HS-DHT) or a vehicle (CD-Veh, HF/HS-Veh). HF/HS dams had higher fat content, both before mating and on GD18.5, with no difference in glucose homeostasis, whereas the insulin sensitivity was higher in DHT-exposed dams. Compared to the CD groups, the livers from HF/HS dams weighed more on GD18.5, the triglyceride content was higher, and there was a dysregulation of liver enzymes related to lipogenesis and higher mRNA expression of Fitm1. Fetuses from HF/HS-Veh dams had lower liver triglyceride content and mRNA expression of Srebf1c. Maternal DHT exposure, regardless of diet, decreased fetal liver Pparg mRNA expression and increased placental androgen receptor protein expression. Maternal diet-induced obesity, together with androgen excess, affects maternal and fetal liver function as demonstrated by increased triglyceride content and dysfunctional expression of enzymes and transcription factors involved in de novo lipogenesis and fat storage.

Streptozocin-induced type-1 diabetes mellitus results in decreased density of CGRP sensory and TH sympathetic nerve fibers that are positively correlated with bone loss at the mouse femoral neck.

Type-1 diabetes mellitus (T1DM) results in loss of innervation in some tissues including epidermis and retina; however, the effect on bone innervation is unknown. Likewise, T1DM results in pathological bone loss and increased risk of fracture. Thus, we quantified the density of calcitonin gene-related peptide (CGRP+) sensory and tyrosine hydroxylase (TH+) sympathetic nerve fibers and determined the association between the innervation density and microarchitecture of trabecular bone at the mouse femoral neck. Ten weeks-old female mice received 5 daily administrations of streptozocin (i.p. 50mg/kg) or citrate (control group). Twenty weeks later, femurs were analyzed by microCT and processed for immunohistochemistry. Confocal microscopy analysis revealed that mice with T1DM had a significant loss of both CGRP+ and TH+ nerve fibers in the bone marrow at the femoral neck. Likewise, microCT analysis revealed a significant decrease in the trabecular bone mineral density (tBMD), bone volume/total volume ratio (BV/TB), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) in mice with T1DM as compared to control mice. Analysis of correlation revealed a positive and significant association between density of CGRP+ or TH+ nerve fibers with tBMD, BV/TV, Tb.Th and Tb.Sp, but not with trabecular number (there was a positive association only for CGRP+) and degree of anisotropy (DA). This study suggests an interaction between sensory and sympathetic nervous system and T1DM-induced bone loss. Identification of the factors involved in the loss of CGRP+ sensory and TH+ sympathetic fibers and how they regulate bone loss may result in new avenues to treat T1DM-related osteoporosis.

High-fat diet exacerbates pain-like behaviors and periarticular bone loss in mice with CFA-induced knee arthritis.

OBJECTIVE: Our aim was to quantify nociceptive spontaneous behaviors, knee edema, proinflammatory cytokines, bone density, and microarchitecture in high-fat diet (HFD)-fed mice with unilateral knee arthritis. METHODS: ICR male mice were fed either standard diet (SD) or HFD starting at 3 weeks old. At 17 weeks, HFD and SD mice received intra-articular injections either with Complete Freund's Adjuvant (CFA) or saline into the right knee joint every 7 days for 4 weeks. Spontaneous pain-like behaviors and knee edema were assessed for 26 days. At day 26 post-first CFA injection, serum levels of IL-1ß, IL-6, and RANKL were measured by ELISA, and microcomputed tomography analysis of knee joints was performed. RESULTS: HFD-fed mice injected with CFA showed greater spontaneous pain-like behaviors of the affected extremity as well as a decrease in the weight-bearing index compared to SD-fed mice injected with CFA. Knee edema was not significantly different between diets. HFD significantly exacerbated arthritis-induced bone loss at the distal femoral metaphysis but had no effect on femoral diaphyseal cortical bone. HFD did not modify serum levels of proinflammatory cytokines. CONCLUSIONS: HFD exacerbates pain-like behaviors and significantly increases the magnitude of periarticular trabecular bone loss in a murine model of unilateral arthritis.

Targeting cells of the myeloid lineage attenuates pain and disease progression in a prostate model of bone cancer.

Tumor cells frequently metastasize to bone where they can generate cancer-induced bone pain (CIBP) that can be difficult to fully control using available therapies. Here, we explored whether PLX3397, a high-affinity small molecular antagonist that binds to and inhibits phosphorylation of colony-stimulating factor-1 receptor, the tyrosine-protein kinase c-Kit, and the FMS-like tyrosine kinase 3, can reduce CIBP. These 3 targets all regulate the proliferation and function of a subset of the myeloid cells including macrophages, osteoclasts, and mast cells. Preliminary experiments show that PLX3397 attenuated inflammatory pain after formalin injection into the hind paw of the rat. As there is an inflammatory component in CIBP, involving macrophages and osteoclasts, the effect of PLX3397 was explored in a prostate model of CIBP where skeletal pain, cancer cell proliferation, tumor metastasis, and bone remodeling could be monitored in the same animal. Administration of PLX3397 was initiated on day 14 after prostate cancer cell injection when the tumor was well established, and tumor-induced bone remodeling was first evident. Over the next 6 weeks, sustained administration of PLX3397 attenuated CIBP behaviors by approximately 50% and was equally efficacious in reducing tumor cell growth, formation of new tumor colonies in bone, and pathological tumor-induced bone remodeling. Developing a better understanding of potential effects that analgesic therapies have on the tumor itself may allow the development of therapies that not only better control the pain but also positively impact disease progression and overall survival in patients with bone cancer.

Chronic oral or intraarticular administration of docosahexaenoic acid reduces nociception and knee edema and improves functional outcomes in a mouse model of Complete Freund's Adjuvant-induced knee arthritis.

INTRODUCTION: Clinical and preclinical studies have shown that supplementation with ω-3 polyunsaturated fatty acids (ω-3 PUFAs) reduce joint destruction and inflammation present in rheumatoid arthritis (RA). However, the effects of individual ω-3 PUFAs on chronic arthritic pain have not been evaluated to date. Thus, our aim in this study was to examine whether purified docosahexaenoic acid (DHA, an ω-3 PUFA) reduces spontaneous pain-related behavior and knee edema and improves functional outcomes in a mouse model of knee arthritis. METHODS: Unilateral arthritis was induced by multiple injections of Complete Freund's Adjuvant (CFA) into the right knee joints of male ICR adult mice. Mice that received CFA injections were then chronically treated from day 15 until day 25 post-initial CFA injection with oral DHA (10, 30 and 100 mg/kg daily) or intraarticular DHA (25 and 50 µg/joint twice weekly). Spontaneous flinching of the injected extremity (considered as spontaneous pain-related behavior), vertical rearing and horizontal exploratory activity (considered as functional outcomes) and knee edema were assessed. To determine whether an endogenous opioid mechanism was involved in the therapeutic effect of DHA, naloxone (NLX, an opioid receptor antagonist, 3 mg/kg subcutaneously) was administered in arthritic mice chronically treated with DHA (30 mg/kg by mouth) at day 25 post-CFA injection. RESULTS: The intraarticular CFA injections resulted in increasing spontaneous flinching and knee edema of the ipsilateral extremity as well as worsening functional outcomes as time progressed. Chronic administration of DHA, given either orally or intraarticularly, significantly improved horizontal exploratory activity and reduced flinching behavior and knee edema in a dose-dependent manner. Administration of NLX did not reverse the antinociceptive effect of DHA. CONCLUSIONS: To the best of our knowledge, this report is the first to demonstrate DHA's antinociceptive and anti-inflammatory effects as individual ω-3 PUFAs following sustained systemic and intraarticular administration in a mouse model of CFA-induced knee arthritis. The results suggest that DHA treatment may offer a new therapeutic approach to alleviate inflammation as well as a beneficial effect on pain-related functional disabilities in RA patients.

Semi-mechanistic modeling of the interaction between the central and peripheral effects in the antinociceptive response to lumiracoxib in rats.

The model-based approach was undertaken to characterize the interaction between the peripheral and central antinociceptive effects exerted by lumiracoxib. The effects of intraplantar and intrathecal administrations and of fixed ratio combinations of lumiracoxib simultaneously administered by these two routes were evaluated using the formalin test in rats. Pain-related behavior data, quantified as the number of flinches of the injected paw, were analyzed using a population approach with NONMEM 7. The pain response during the first phase of the formalin test, which was insensitive to lumiracoxib, was modeled using a monoexponential decay. The second phase, which was sensitive to lumiracoxib, was described incorporating synthesis and degradation processes of pain mediators that were recruited locally after tissue injury. Upregulation at the local level and in the central nervous system (CNS) was set to be proportional to the predicted levels of pain mediators in the local (injured) compartment. Results suggest a greater role of upregulated COX-2(Local) in generating the pain response compared to COX-2(CNS). Drug effects were described as inhibition of upregulated COX-2. The model adequately described the time course of nociception after formalin injection in the absence or presence of lumiracoxib administered locally and/or spinally. Data suggest that the overall response is the additive outcome of drug effects at the peripheral and central compartments, with predominance of peripheral mechanisms. Application of modeling opens new perspectives for understanding the overall mechanism of action of analgesic drugs.

Pathophysiology and medical treatment of pain in fibrous dysplasia of bone.

One of the most common complications of fibrous dysplasia of bone (FD) is bone pain. Usual pain killers are often of inadequate efficacy to control this bone pain. The mechanism of bone pain in FD remains uncertain, but by analogy with bone tumors one may consider that ectopic sprouting and formation of neuroma-like structures by sensory and sympathetic nerve fibers also occur in the dysplastic skeleton. Bone pain has been reported in up to 81% of adults and 49% of children. It affects predominantly the lower limbs and the spine. The degree of pain is highly variable and adults reports more pain than children. Bisphosphonates have been shown to reduce bone pain in uncontrolled studies. Their influence on bone strength remains unknown. In a randomized trial testing alendronate, bone pain was not significantly improved. Another trial assessing the effect of risedronate is ongoing. Possible future therapies include tocilizumab, denosumab and drugs targeting nerve growth factor and its receptor TrkA.

Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice.

INTRODUCTION: Although the prevalence of arthritis dramatically increases with age, the great majority of preclinical studies concerning the mechanisms that drive arthritic joint pain have been performed in young animals. One mechanism hypothesized to contribute to arthritic pain is ectopic nerve sprouting; however, neuroplasticity is generally thought to be greater in young versus old nerves. Here we explore whether sensory and sympathetic nerve fibers can undergo a significant ectopic nerve remodeling in the painful arthritic knee joint of geriatric mice. METHODS: Vehicle (saline) or complete Freund's adjuvant (CFA) was injected into the knee joint of 27- to 29-month-old female mice. Pain behaviors, macrophage infiltration, neovascularization, and the sprouting of sensory and sympathetic nerve fibers were then assessed 28 days later, when significant knee-joint pain was present. Knee joints were processed for immunohistochemistry by using antibodies raised against CD68 (monocytes/macrophages), PECAM (endothelial cells), calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kDa (NF200; sensory nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), and growth-associated protein 43 (GAP43; nerve fibers undergoing sprouting). RESULTS: At 4 weeks after initial injection, CFA-injected mice displayed robust pain-related behaviors (which included flinching, guarding, impaired limb use, and reduced weight bearing), whereas animals injected with vehicle alone displayed no significant pain-related behaviors. Similarly, in the CFA-injected knee joint, but not in the vehicle-injected knee joint, a remarkable increase was noted in the number of CD68+ macrophages, density of PECAM+ blood vessels, and density and formation of neuroma-like structures by CGRP+, NF200+, and TH+ nerve fibers in the synovium and periosteum. CONCLUSIONS: Sensory and sympathetic nerve fibers that innervate the aged knee joint clearly maintain the capacity for robust nerve sprouting and formation of neuroma-like structures after inflammation/injury. Understanding the factors that drive this neuroplasticity, whether this pathologic reorganization of nerve fibers contributes to chronic joint pain, and how the phenotype of sensory and sympathetic nerves changes with age may provide pharmacologic insight and targets for better controlling aging-related joint pain.

Neuroplasticity of sensory and sympathetic nerve fibers in a mouse model of a painful arthritic joint.

OBJECTIVE: Many forms of arthritis are accompanied by significant chronic joint pain. This study was undertaken to investigate whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathologic reorganization. METHODS: A painful arthritic knee joint was produced by injection of Freund's complete adjuvant (CFA) into the knee joint of young adult mice. CFA-injected mice were then treated systemically with vehicle or anti-NGF antibody. Pain behaviors were assessed, and at 28 days following the initial CFA injection, the knee joints were processed for immunohistochemistry analysis using antibodies against calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kd (NF200; sensory nerve fibers), growth-associated protein 43 (GAP-43; sprouted nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), CD31 (endothelial cells), or CD68 (monocyte/macrophages). RESULTS: In CFA-injected mice, there was a significant increase in the density of CD68+ macrophages, CD31+ blood vessels, and CGRP+, NF200+, GAP-43+, and TH+ nerve fibers in the synovium, as well as a significant increase in joint pain-related behaviors. None of these findings were observed in sham-injected mice. Administration of anti-NGF reduced these pain-related behaviors and the ectopic sprouting of nerve fibers, but had no significant effect on the increase in density of CD31+ blood vessels or CD68+ macrophages. CONCLUSION: These findings demonstrate that ectopic sprouting of sensory and sympathetic nerve fibers occurs in the painful arthritic joint and may be involved in the generation and maintenance of arthritic pain.

The effect of aging on the density of the sensory nerve fiber innervation of bone and acute skeletal pain.

As humans age there is a decline in most sensory systems including vision, hearing, taste, smell, and tactile acuity. In contrast, the frequency and severity of musculoskeletal pain generally increases with age. To determine whether the density of sensory nerve fibers that transduce skeletal pain changes with age, calcitonin gene related peptide (CGRP) and neurofilament 200 kDa (NF200) sensory nerve fibers that innervate the femur were examined in the femurs of young (4-month-old), middle-aged (13-month-old) and old (36-month-old) male F344/BNF1 rats. Whereas the bone quality showed a significant age-related decline, the density of CGRP(+) and NF200(+) nerve fibers that innervate the bone remained remarkably unchanged as did the severity of acute skeletal fracture pain. Thus, while bone mass, quality, and strength undergo a significant decline with age, the density of sensory nerve fibers that transduce noxious stimuli remain largely intact. These data may in part explain why musculoskeletal pain increases with age.

Breast cancer-induced bone remodeling, skeletal pain, and sprouting of sensory nerve fibers.

UNLABELLED: Breast cancer metastasis to bone is frequently accompanied by pain. What remains unclear is why this pain tends to become more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the breast cancer bearing bone undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of human breast cancer cells (MDA-MB-231-BO) into the femoral intramedullary space of female athymic nude mice induces sprouting of calcitonin gene-related peptide (CGRP(+)) sensory nerve fibers. Nearly all CGRP(+) nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA(+)) and growth-associated protein-43 (GAP43(+)). This ectopic sprouting occurs in periosteal sensory nerve fibers that are in close proximity to breast cancer cells, tumor-associated stromal cells, and remodeled cortical bone. Therapeutic treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. The present data suggest that the breast cancer cells and tumor-associated stromal cells express and release NGF, which drives bone pain and the pathological reorganization of nearby CGRP(+)/TrkA(+)/GAP43(+) sensory nerve fibers. PERSPECTIVE: Therapies that block breast cancer pain by reducing the tumor-induced pathological sprouting and reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive breast cancer pain and lead to more effective therapies for attenuating this chronic pain state.

Sustained blockade of neurotrophin receptors TrkA, TrkB and TrkC reduces non-malignant skeletal pain but not the maintenance of sensory and sympathetic nerve fibers.

Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.

Administration of a tropomyosin receptor kinase inhibitor attenuates sarcoma-induced nerve sprouting, neuroma formation and bone cancer pain.

Pain often accompanies cancer and most current therapies for treating cancer pain have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomyosin receptor kinase A (TrkA) has become an attractive target for attenuating chronic pain. In the present report, we use a mouse model of bone cancer pain and examine whether oral administration of a selective small molecule Trk inhibitor (ARRY-470, which blocks TrkA, TrkB and TrkC kinase activity at low nm concentrations) has a significant effect on cancer-induced pain behaviors, tumor-induced remodeling of sensory nerve fibers, tumor growth and tumor-induced bone remodeling. Early/sustained (initiated day 6 post cancer cell injection), but not late/acute (initiated day 18 post cancer cell injection) administration of ARRY-470 markedly attenuated bone cancer pain and significantly blocked the ectopic sprouting of sensory nerve fibers and the formation of neuroma-like structures in the tumor bearing bone, but did not have a significant effect on tumor growth or bone remodeling. These data suggest that, like therapies that target the cancer itself, the earlier that the blockade of TrkA occurs, the more effective the control of cancer pain and the tumor-induced remodeling of sensory nerve fibers. Developing targeted therapies that relieve cancer pain without the side effects of current analgesics has the potential to significantly improve the quality of life and functional status of cancer patients.

Pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain.

Pain frequently accompanies cancer. What remains unclear is why this pain frequently becomes more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the tumor-bearing tissue undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of canine prostate cancer cells into mouse bone induces a remarkable sprouting of calcitonin gene-related peptide (CGRP(+)) and neurofilament 200 kDa (NF200(+)) sensory nerve fibers. Nearly all sensory nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA(+)). This ectopic sprouting occurs in sensory nerve fibers that are in close proximity to colonies of prostate cancer cells, tumor-associated stromal cells and newly formed woven bone, which together form sclerotic lesions that closely mirror the osteoblastic bone lesions induced by metastatic prostate tumors in humans. Preventive treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. Interestingly, reverse transcription PCR analysis indicated that the prostate cancer cells themselves do not express detectable levels of mRNA coding for NGF. This suggests that the tumor-associated stromal cells express and release NGF, which drives the pathological reorganization of nearby TrkA(+) sensory nerve fibers. Therapies that prevent this reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive cancer pain and lead to more effective control of this chronic pain state.