The majority of myelinated and unmyelinated sensory nerve fibers that innervate bone express the tropomyosin receptor kinase A.

Although skeletal pain is a leading cause of chronic pain and disability, relatively little is known about the specific populations of nerve fibers that innervate the skeleton. Recent studies have reported that therapies blocking nerve growth factor (NGF) or its cognate receptor, tropomyosin receptor kinase A (TrkA) are efficacious in attenuating skeletal pain. A potential factor to consider when assessing the analgesic efficacy of targeting NGF-TrkA signaling in a pain state is the fraction of NGF-responsive TrkA+ nociceptors that innervate the tissue from which the pain is arising, as this innervation and the analgesic efficacy of targeting NGF-TrkA signaling may vary considerably from tissue to tissue. To explore this in the skeleton, tissue slices and whole mount preparations of the normal, adult mouse femur were analyzed using immunohistochemistry and confocal microscopy. Analysis of these preparations revealed that 80% of the unmyelinated/thinly myelinated sensory nerve fibers that express calcitonin gene-related peptide (CGRP) and innervate the periosteum, mineralized bone and bone marrow also express TrkA. Similarly, the majority of myelinated sensory nerve fibers that express neurofilament 200 kDa (NF200) which innervate the periosteum, mineralized bone and bone marrow also co-express TrkA. In the normal femur, the relative density of CGRP+, NF200+ and TrkA+ sensory nerve fibers per unit volume is: periosteum>bone marrow>mineralized bone>cartilage with the respective relative densities being 100:2:0.1:0. The observation that the majority of sensory nerve fibers innervating the skeleton express TrkA+, may in part explain why therapies that block NGF/TrkA pathway are highly efficacious in attenuating skeletal pain.

Blockade of nerve sprouting and neuroma formation markedly attenuates the development of late stage cancer pain.

For many patients, pain is the first sign of cancer and, while pain can be present at any time, the frequency and intensity of pain tend to increase with advancing stages of the disease. Thus, between 75 and 90% of patients with metastatic or advanced-stage cancer will experience significant cancer-induced pain. One major unanswered question is why cancer pain increases and frequently becomes more difficult to fully control with disease progression. To gain insight into this question we used a mouse model of bone cancer pain to demonstrate that as tumor growth progresses within bone, tropomyosin receptor kinase A (TrkA)-expressing sensory and sympathetic nerve fibers undergo profuse sprouting and form neuroma-like structures. To address what is driving the pathological nerve reorganization we administered an antibody to nerve growth factor (anti-NGF). Early sustained administration of anti-NGF, whose cognate receptor is TrkA, blocks the pathological sprouting of sensory and sympathetic nerve fibers, the formation of neuroma-like structures, and inhibits the development of cancer pain. These results suggest that cancer cells and their associated stromal cells release nerve growth factor (NGF), which induces a pathological remodeling of sensory and sympathetic nerve fibers. This pathological remodeling of the peripheral nervous system then participates in driving cancer pain. Similar to therapies that target the cancer itself, the data presented here suggest that, the earlier therapies blocking this pathological nerve remodeling are initiated, the more effective the control of cancer pain.

Capsaicin-sensitive sensory nerve fibers contribute to the generation and maintenance of skeletal fracture pain.

Although skeletal pain can have a marked impact on a patient's functional status and quality of life, relatively little is known about the specific populations of peripheral nerve fibers that drive non-malignant bone pain. In the present report, neonatal male Sprague-Dawley rats were treated with capsaicin or vehicle and femoral fracture was produced when the animals were young adults (15-16 weeks old). Capsaicin treatment, but not vehicle, resulted in a significant (>70%) depletion in the density of calcitonin-gene related peptide positive (CGRP(+)) sensory nerve fibers, but not 200 kDa neurofilament H positive (NF200(+)) sensory nerve fibers in the periosteum. The periosteum is a thin, cellular and fibrous tissue that tightly adheres to the outer surface of all but the articulated surface of bone and appears to play a pivotal role in driving fracture pain. In animals treated with capsaicin, but not vehicle, there was a 50% reduction in the severity, but no change in the time course, of fracture-induced skeletal pain-related behaviors as measured by spontaneous flinching, guarding and weight bearing. These results suggest that both capsaicin-sensitive (primarily CGRP(+) C-fibers) and capsaicin-insensitive (primarily NF200(+) A-delta fibers) sensory nerve fibers participate in driving skeletal fracture pain. Skeletal pain can be a significant impediment to functional recovery following trauma-induced fracture, osteoporosis-induced fracture and orthopedic surgery procedures such as knee and hip replacement. Understanding the specific populations of sensory nerve fibers that need to be targeted to inhibit the generation and maintenance of skeletal pain may allow the development of more specific mechanism-based therapies that can effectively attenuate acute and chronic skeletal pain.

A quantitative analysis of the sensory and sympathetic innervation of the mouse pancreas.

Pain from pancreatitis or pancreatic cancer can be both chronic and severe although little is known about the mechanisms that generate and maintain this pain. To define the peripheral sensory and sympathetic fibers involved in transmitting and modulating pancreatic pain, immunohistochemistry and confocal microscopy were used to examine the sensory and sympathetic innervation of the head, body and tail of the normal mouse pancreas. Myelinated sensory fibers were labeled with an antibody raised against 200 kD neurofilament H (clone RT97), thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and post-ganglionic sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase (TH). RT97, CGRP, and TH immunoreactive fibers were present in parenchyma of the head, body and tail of the pancreas with the relative density of both RT97 and CGRP expressing fibers being head>body>tail, whereas for TH, a relatively even distribution was observed. In all three regions of the pancreas, RT97 fibers were associated mainly with large blood vessels, the CGRP fibers were associated with the large- and medium-sized blood vessels and the TH were associated with the large- and medium-sized blood vessels as well as capillaries. In addition to this extensive set of sensory and sympathetic nerve fibers that terminate in the pancreas, there were large bundles of en passant nerve fibers in the dorsal region of the pancreas that expressed RT97 or CGRP and were associated with the superior mesenteric plexus. These data suggest the pancreas receives a significant sensory and sympathetic innervation. Understanding the factors and disease states that sensitize and/or directly excite the nerve fibers that terminate in the pancreas as well as those that are en passant may aid in the development of therapies that more effectively modulate the pain that frequently accompanies diseases of the pancreas, such as pancreatitis and pancreatic cancer.

Bone cancer pain and the role of RANKL/OPG.

Cancer-induced bone diseases are common and can have a devastating impact at the end of life. One of the most difficult sequelae of cancer is metastases to the skeleton, an event that results in bone destruction and bone cancer pain. Bone cancer pain is usually progressive as the disease advances, and is particularly difficult to treat. Recently, experimental models of bone cancer pain have been developed and have provided seminal insight in understanding the pathophysiology of bone cancer pain. Animal models of bone cancer provided the finding that bone destruction (osteolysis) is associated with pain, and it has been determined that cancer-induced osteolysis is mediated by osteoclasts. Having established that RANK ligand contributed to cancer-induced osteoclastogenesis, it was determined that disruption of the RANKL-RANK axis with OPG inhibited tumor-induced osteoclastogenesis and decreased bone cancer pain.

Endothelin and the tumorigenic component of bone cancer pain.

Tumors including sarcomas and breast, prostate, and lung carcinomas frequently grow in or metastasize to the skeleton where they can induce significant bone remodeling and cancer pain. To define products that are released from tumors that are involved in the generation and maintenance of bone cancer pain, we focus here on endothelin-1 (ET-1) and endothelin receptors as several tumors including human prostate and breast have been shown to express high levels of ETs and the application of ETs to peripheral nerves can induce pain. Here we show that in a murine osteolytic 2472 sarcoma model of bone cancer pain, the 2472 sarcoma cells express high levels of ET-1, but express low or undetectable levels of endothelin A (ETAR) or B (ETBR) receptors whereas a subpopulation of sensory neurons express the ETAR and non-myelinating Schwann cells express the ETBR. Acute (10 mg/kg, i.p.) or chronic (10 mg/kg/day, p.o.) administration of the ETAR selective antagonist ABT-627 significantly attenuated ongoing and movement-evoked bone cancer pain and chronic administration of ABT-627 reduced several neurochemical indices of peripheral and central sensitization without influencing tumor growth or bone destruction. In contrast, acute treatment (30 mg/kg, i.p.) with the ETBR selective antagonist, A-192621 increased several measures of ongoing and movement evoked pain. As tumor expression and release of ET-1 has been shown to be regulated by the local environment, location specific expression and release of ET-1 by tumor cells may provide insight into the mechanisms that underlie the heterogeneity of bone cancer pain that is frequently observed in humans with multiple skeletal metastases.

Radiation treatment decreases bone cancer pain, osteolysis and tumor size.

Radiotherapy is the cornerstone of palliative treatment for primary bone cancer in animals and metastatic bone cancer in humans. However, the mechanism(s) responsible for pain relief after irradiation is unknown. To identify the mechanism through which radiation treatment decreases bone cancer pain, the effect of radiation on mice with painful bone cancer was studied. Analysis of the effects of a 20-Gy treatment on localized sites of painful bone cancers was performed through assessments of animal behavior, radiographs and histological analysis. The findings indicated that radiation treatment reduced bone pain and supported reduced cancer burden and reduced osteolysis as mechanisms through which radiation reduces bone cancer pain.

Un enfoque mecanístico del dolor oncológico / A mechanism based understanding of cancer pain

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Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur.

Although skeletal pain plays a major role in reducing the quality of life in patients suffering from osteoarthritis, Paget's disease, sickle cell anemia and bone cancer, little is known about the mechanisms that generate and maintain this pain. To define the peripheral fibers involved in transmitting and modulating skeletal pain, we used immunohistochemistry with antigen retrieval, confocal microscopy and three-dimensional image reconstruction of the bone to examine the sensory and sympathetic innervation of mineralized bone, bone marrow and periosteum of the normal mouse femur. Thinly myelinated and unmyelinated peptidergic sensory fibers were labeled with antibodies raised against calcitonin gene-related peptide (CGRP) and the unmyelinated, non-peptidergic sensory fibers were labeled with the isolectin B4 (Bandeira simplicifolia). Myelinated sensory fibers were labeled with an antibody raised against 200-kDa neurofilament H (clone RT-97). Sympathetic fibers were labeled with an antibody raised against tyrosine hydroxylase. CGRP, RT-97, and tyrosine hydroxylase immunoreactive fibers, but not isolectin B4 positive fibers, were present throughout the bone marrow, mineralized bone and the periosteum. While the periosteum is the most densely innervated tissue, when the total volume of each tissue is considered, the bone marrow receives the greatest total number of sensory and sympathetic fibers followed by mineralized bone and then periosteum. Understanding the sensory and sympathetic innervation of bone should provide a better understanding of the mechanisms that drive bone pain and aid in developing therapeutic strategies for treating skeletal pain.

Enhanced responses of spinal dorsal horn neurons to heat and cold stimuli following mild freeze injury to the skin.

The effects of a mild freeze injury to the skin on responses of nociceptive dorsal horn neurons to cold and heat stimuli were examined in anesthetized rats. Electrophysiological recordings were obtained from 72 nociceptive spinal neurons located in the superficial and deep dorsal horn. All neurons had receptive fields (RFs) on the glabrous skin of the hindpaw, and neurons were functionally divided into wide dynamic range (WDR) and high-threshold (HT) neurons. Forty-four neurons (61%) were classified as WDR and responded to both innocuous and noxious mechanical stimuli (mean mechanical threshold of 12.8 +/- 1.6 mN). Twenty-eight neurons (39%) were classified as HT and were excited only by noxious mechanical stimuli (mean mechanical threshold of 154.2 +/- 18.3 mN). Neurons were characterized for their sensitivity heat (35 to 51 degrees C) and cold (28 to -12 degrees C) stimuli applied to their RF. Among WDR neurons, 86% were excited by both noxious heat and cold stimuli, while 14% responded only to heat. For HT neurons, 61% responded to heat and cold stimuli, 32% responded only to noxious heat, and 7% responded only to noxious cold. Effects of a mild freeze injury (-15 degrees C applied to the RF for 20 s) on responses to heat and cold stimuli were examined in 30 WDR and 22 HT neurons. Skin freezing was verified as an abrupt increase in skin temperature at the site of injury due to the exothermic reaction associated with crystallization. Freezing produced a decrease in response thresholds to heat and cold stimuli in most WDR and HT neurons. WDR and HT neurons exhibited a mean decrease in response threshold for cold of 9.0 +/- 1.3 degrees C and 10.0 +/- 1.6 degrees C, respectively. Mean response thresholds for heat decreased 4.0 +/- 0.4 degrees C and 4.3 +/- 1.3 degrees C in WDR and HT neurons, respectively. In addition, responses to suprathreshold cold and heat stimuli increased. WDR and HT neurons exhibited an 89% and a 192% increase in response across all cold stimuli, and a 93 and 92% increase in responses evoked across all heat stimuli, respectively. Our results demonstrate that many spinal neurons encode intensity of noxious cold as well as noxious heat over a broad range of stimulus temperatures. Enhanced responses of WDR and HT neurons to cold and heat stimuli after a mild freeze injury is likely to contribute to thermal hyperalgesia following a similar freeze injury in humans.

Osteoprotegerin diminishes advanced bone cancer pain.

Bone cancer pain most commonly occurs when tumors originating in breast, prostate, or lung metastasize to long bones, spinal vertebrae, and/or pelvis. Primary and metastatic cancers involving bone account for approximately 400,000 new cancer cases per year in the United States alone, and >70% of patients with advanced breast or prostate cancer have skeletal metastases. Whereas pain resulting from bone cancer can dramatically impact an individual's quality of life, very little is known about the mechanisms that generate and maintain this pain. To begin to define the mechanisms that give rise to advanced bone cancer pain, osteolytic 2472 sarcoma cells or media were injected into the intramedullary space of the femur of C3H/HeJ mice, and the injection hole was sealed using dental amalgam, confining the tumor cells to the bone. Twelve days after injection of 2472 tumor cells, animals showed advanced tumor-induced bone destruction of the injected femur, bone cancer pain, and a stereotypic set of neurochemical changes in the spinal cord dorsal horn that receives sensory inputs from the affected femur. Administration of osteoprotegerin, a naturally secreted decoy receptor that inhibits osteoclast maturation and activity and induces osteoclast apoptosis, or vehicle was begun at 12 days, when significant bone destruction had already occurred, and administration was continued daily until day 21. Ongoing pain behaviors, movement-evoked pain behaviors, and bone destruction were assessed on days 10, 12, 14, 17, and 21. The neurochemistry of the spinal cord was evaluated at days 12 and 21. Results indicated that osteoprotegerin treatment halted further bone destruction, reduced ongoing and movement-evoked pain, and reversed several aspects of the neurochemical reorganization of the spinal cord. Thus, even in advanced stages of bone cancer, ongoing osteoclast activity appears to be involved in the generation and maintenance of ongoing and movement-evoked pain. Blockade of ongoing osteoclast activity appears to have the potential to reduce bone cancer pain in patients with advanced tumor-induced bone destruction.

The molecular dynamics of pain control.

Pain is necessary for survival, but persistent pain can result in anxiety, depression and a reduction in the quality of life. The discriminative and affective qualities of pain are both thought to be regulated in an activity-dependent fashion. Recent studies have identified cells and molecules that regulate pain sensitivity and the parallel pathways that distribute nociceptive information to limbic or sensory areas of the forebrain. Here, we emphasize the cellular and neurobiological consequences of pain, especially those that are involved in the generation and maintenance of chronic pain. These new insights into pain processing will significantly alter our approach to pain control and the development of new analgesics.

Expression and localization of endothelin receptors: implications for the involvement of peripheral glia in nociception.

The endothelins (ETs) are peptides that have a diverse array of functions mediated by two receptor subtypes, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). Pharmacological studies have suggested that in peripheral tissues, ET(A)R expression may play a role in signaling acute or neuropathic pain, whereas ET(B)R expression may be involved in the transmission of chronic inflammatory pain. To begin to define the mechanisms by which ET can drive nociceptive signaling, autoradiography and immunohistochemistry were used to examine the distribution of ET(A)R and ET(B)R in dorsal root ganglia (DRG) and peripheral nerve of the rat, rabbit, and monkey. In DRG and peripheral nerve, ET(A)R-immunoreactivity was present in a subset of small-sized peptidergic and nonpeptidergic sensory neurons and their axons and to a lesser extent in a subset of medium-sized sensory neurons. However, ET(B)R-immunoreactivity was not seen in DRG neurons or axons but rather in DRG satellite cells and nonmyelinating ensheathing Schwann cells. Thus, when ETs are released in peripheral tissues, they could act directly on ET(A)R-expressing sensory neurons and on ET(B)R-expressing DRG satellite cells or nonmyelinating Schwann cells. These data indicate that ETs can have direct, nociceptive effects on the peripheral sensory nervous system and that peripheral glia may be directly involved in signaling nociceptive events in peripheral tissues.

Activation barriers to structural transition determine deposition rates of Alzheimer's disease a beta amyloid.

Brain amyloid composed of the approximately 40-amino-acid human beta-amyloid peptide A beta is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Abeta during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of A beta congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Abeta congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type A beta. The hyperactive peptide, which is linked to a heritable A beta amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type A beta. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.

Alzheimer's disease amyloid propagation by a template-dependent dock-lock mechanism.

Amyloid plaques composed of the peptide Abeta are an integral part of Alzheimer's disease (AD) pathogenesis. We have modeled the process of amyloid plaque growth by monitoring the deposition of soluble Abeta onto amyloid in AD brain tissue or synthetic amyloid fibrils and show that it is mediated by two distinct kinetic processes. In the first phase, "dock", Abeta addition to the amyloid template is fully reversible (dissociation t(1/2) approximately 10 min), while in the second phase, "lock", the deposited peptide becomes irreversibly associated (dissociation t(1/2) >> 1000 min) with the template in a time-dependent manner. The most recently deposited peptide dissociates first while Abeta previously deposited becomes irreversibly "locked" onto the template. Thus, the transition from monomer to neurotoxic amyloid is mediated by interaction with the template, a mechanism that has also been proposed for the prion diseases. Interestingly, two Abeta peptides bearing primary sequence alterations implicated in heritable Abeta amyloidoses displayed faster lock-phase kinetics than wild-type Abeta. Inhibiting the initial weak docking interaction between depositing Abeta and the template is a viable therapeutic target to prevent the critical conformational transition in the conversion of Abeta((solution)) to Abeta((amyloid)) and thus prevent stable amyloid accumulation. While thermodynamics suggest that inhibiting amyloid assembly would be difficult, the present study illustrates that the protein misfolding diseases are kinetically vulnerable to intervention.

Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons.

The aim of this investigation was to determine whether murine models of inflammatory, neuropathic and cancer pain are each characterized by a unique set of neurochemical changes in the spinal cord and sensory neurons. All models were generated in C3H/HeJ mice and hyperalgesia and allodynia behaviorally characterized. A variety of neurochemical markers that have been implicated in the generation and maintenance of chronic pain were then examined in spinal cord and primary afferent neurons.Three days after injection of complete Freund's adjuvant into the hindpaw (a model of persistent inflammatory pain) increases in substance P, calcitonin gene-related peptide, protein kinase C gamma, and substance P receptor were observed in the spinal cord. Following sciatic nerve transection or L5 spinal nerve ligation (a model of persistent neuropathic pain) significant decreases in substance P and calcitonin gene-related peptide and increases in galanin and neuropeptide Y were observed in both primary afferent neurons and the spinal cord. In contrast, in a model of cancer pain induced by injection of osteolytic sarcoma cells into the femur, there were no detectable changes in any of these markers in either primary afferent neurons or the spinal cord. However, in this cancer-pain model, changes including massive astrocyte hypertrophy without neuronal loss, increase in the neuronal expression of c-Fos, and increase in the number of dynorphin-immunoreactive neurons were observed in the spinal cord, ipsilateral to the limb with cancer. These results indicate that a unique set of neurochemical changes occur with inflammatory, neuropathic and cancer pain in C3H/HeJ mice and further suggest that cancer induces a unique persistent pain state. Determining whether these neurochemical changes are involved in the generation and maintenance of each type of persistent pain may provide insight into the mechanisms that underlie each of these pain states.