Lisa A. Majuta

Medullary pain facilitating neurons mediate allodynia in headache-related pain†

To develop and validate a model of cutaneous allodynia triggered by dural inflammation for pain associated with headaches. To explore neural mechanisms underlying cephalic and extracephalic allodynia.

Morphine treatment accelerates sarcoma-induced bone pain, bone loss, and spontaneous fracture in a murine model of bone cancer

Abstract Metastatic bone cancer causes severe pain that is primarily treated with opioids. A model of bone cancer pain in which the progression of cancer pain and bone destruction is tightly controlled was used to evaluate the effects of sustained morphine treatment. In cancer‐treated mice, morphine enhanced, rather than diminished, spontaneous, and evoked pain; these effects were dose‐dependent and naloxone‐sensitive. SP and CGRP positive DRG cells did not differ between sarcoma or control mice, but were increased following morphine in both groups. Morphine increased ATF‐3 expression only in DRG cells of sarcoma mice. Morphine did not alter tumor growth in vitro or tumor burden in vivo but accelerated sarcoma‐induced bone destruction and doubled the incidence of spontaneous fracture in a dose‐ and naloxone‐sensitive manner. Morphine increased osteoclast activity and upregulated IL‐1β within the femurs of sarcoma‐treated mice suggesting enhancement of sarcoma‐induced osteolysis. These results indicate that sustained morphine increases pain, osteolysis, bone loss, and spontaneous fracture, as well as markers of neuronal damage in DRG cells and expression of pro‐inflammatory cytokines. Morphine treatment may result in “add‐on” mechanisms of pain beyond those engaged by sarcoma alone. While it is not known whether the present findings in this model of osteolytic sarcoma will generalize to other cancers or opioids, the data suggest a need for increased understanding of neurobiological consequences of prolonged opioid exposure which may allow improvements in the use of opiates in the effective management of cancer pain.

The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model.

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.

NGF Blockade at Early Times during Bone Cancer Development Attenuates Bone Destruction and Increases Limb Use

Studies in animals and humans show that blockade of nerve growth factor (NGF) attenuates both malignant and nonmalignant skeletal pain. While reduction of pain is important, a largely unanswered question is what other benefits NGF blockade might confer in patients with bone cancer. Using a mouse graft model of bone sarcoma, we demonstrate that early treatment with an NGF antibody reduced tumor-induced bone destruction, delayed time to bone fracture, and increased the use of the tumor-bearing limb. Consistent with animal studies in osteoarthritis and head and neck cancer, early blockade of NGF reduced weight loss in mice with bone sarcoma. In terms of the extent and time course of pain relief, NGF blockade also reduced pain 40% to 70%, depending on the metric assessed. Importantly, this analgesic effect was maintained even in animals with late-stage disease. Our results suggest that NGF blockade immediately upon detection of tumor metastasis to bone may help preserve the integrity and use, delay the time to tumor-induced bone fracture, and maintain body weight.

3,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase (nNOS) inhibitors.

A series of 1,6-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). By varying the basic amine side chain at the 1-position of the indole ring, several potent and selective inhibitors of human neuronal NOS were identified. In general compounds with bulkier side chains displayed increased selectivity for nNOS over eNOS and iNOS isoforms. One of the compounds, (R)-8 was shown to reduce tactile hyperesthesia (allodynia) after oral administration (30 mg/kg) in an in vivo rat model of dural inflammation relevant to migraine pain.

Exuberant sprouting of sensory and sympathetic nerve fibers in nonhealed bone fractures and the generation and maintenance of chronic skeletal pain.

&NA; Characterizing the ectopic sensory and sympathetic nerve growth that occurs in nonhealed fractures may provide insight into the mechanisms that drive chronic skeletal pain. &NA; Skeletal injury is a leading cause of chronic pain and long‐term disability worldwide. While most acute skeletal pain can be effectively managed with nonsteroidal anti‐inflammatory drugs and opiates, chronic skeletal pain is more difficult to control using these same therapy regimens. One possibility as to why chronic skeletal pain is more difficult to manage over time is that there may be nerve sprouting in nonhealed areas of the skeleton that normally receive little (mineralized bone) to no (articular cartilage) innervation. If such ectopic sprouting did occur, it could result in normally nonnoxious loading of the skeleton being perceived as noxious and/or the generation of a neuropathic pain state. To explore this possibility, a mouse model of skeletal pain was generated by inducing a closed fracture of the femur. Examined animals had comminuted fractures and did not fully heal even at 90+ days post fracture. In all mice with nonhealed fractures, exuberant sensory and sympathetic nerve sprouting, an increase in the density of nerve fibers, and the formation of neuroma‐like structures near the fracture site were observed. Additionally, all of these animals exhibited significant pain behaviors upon palpation of the nonhealed fracture site. In contrast, sprouting of sensory and sympathetic nerve fibers or significant palpation‐induced pain behaviors was never observed in naïve animals. Understanding what drives this ectopic nerve sprouting and the role it plays in skeletal pain may allow a better understanding and treatment of this currently difficult‐to‐control pain state.

Orthopedic surgery and bone fracture pain are both significantly attenuated by sustained blockade of nerve growth factor.

Abstract The number of patients suffering from postoperative pain due to orthopedic surgery and bone fracture is projected to dramatically increase because the human life span, weight, and involvement in high-activity sports continue to rise worldwide. Joint replacement or bone fracture frequently results in skeletal pain that needs to be adequately controlled for the patient to fully participate in needed physical rehabilitation. Currently, the 2 major therapies used to control skeletal pain are nonsteroidal anti-inflammatory drugs and opiates, both of which have significant unwanted side effects. To assess the efficacy of novel therapies, mouse models of orthopedic and fracture pain were developed and evaluated here. These models, orthopedic surgery pain and bone fracture pain, resulted in skeletal pain–related behaviors that lasted 3 weeks and 8 to 10 weeks, respectively. These skeletal pain behaviors included spontaneous and palpation-induced nocifensive behaviors, dynamic weight bearing, limb use, and voluntary mechanical loading of the injured hind limb. Administration of anti–nerve growth factor before orthopedic surgery or after bone fracture attenuated skeletal pain behaviors by 40% to 70% depending on the end point being assessed. These data suggest that nerve growth factor is involved in driving pain due to orthopedic surgery or bone fracture. These animal models may be useful in developing an understanding of the mechanisms that drive postoperative orthopedic and bone fracture pain and the development of novel therapies to treat these skeletal pains.

Dissociation between the relief of skeletal pain behaviors and skin hypersensitivity in a model of bone cancer pain

Abstract Recent studies have suggested that in humans and animals with significant skeletal pain, changes in the mechanical hypersensitivity of the skin can be detected. However, whether measuring changes in skin hypersensitivity can be a reliable surrogate for measuring skeletal pain itself remains unclear. To explore this question, we generated skeletal pain by injecting and confining GFP-transfected NCTC 2472 osteosarcoma cells unilaterally to the femur of C3H male mice. Beginning at day 7 post-tumor injection, animals were administered vehicle, an antibody to the P2X3 receptor (anti-P2X3) or anti-NGF antibody. Pain and analgesic efficacy were then measured on days 21, 28, and 35 post-tumor injection using a battery of skeletal pain-related behaviors and von Frey assessment of mechanical hypersensitivity on the plantar surface of the hind paw. Animals with bone cancer pain treated with anti-P2X3 showed a reduction in skin hypersensitivity but no attenuation of skeletal pain behaviors, whereas animals with bone cancer pain treated with anti-NGF showed a reduction in both skin hypersensitivity and skeletal pain behaviors. These results suggest that although bone cancer can induce significant skeletal pain-related behaviors and hypersensitivity of the skin, relief of hypersensitivity of the skin is not always accompanied by attenuation of skeletal pain. Understanding the relationship between skeletal and skin pain may provide insight into how pain is processed and integrated and help define the preclinical measures of skeletal pain that are predictive end points for clinical trials.

Anti-nerve growth factor therapy increases spontaneous day/night activity in mice with orthopedic surgery-induced pain.

Abstract Total knee arthroplasty (TKA) and total hip arthroplasty (THA) are 2 of the most common and successful surgical interventions to relieve osteoarthritis pain. Control of postoperative pain is critical for patients to fully participate in the required physical therapy which is the most influential factor in effective postoperative knee rehabilitation. Currently, opiates are a mainstay for managing postoperative orthopedic surgery pain including TKA or THA pain. Recently, issues including efficacy, dependence, overdose, and death from opiates have made clinicians and researchers more critical of use of opioids for treating nonmalignant skeletal pain. In the present report, a nonopiate therapy using a monoclonal antibody raised against nerve growth factor (anti-NGF) was assessed for its ability to increase the spontaneous activity of the operated knee joint in a mouse model of orthopedic surgery pain–induced by drilling and coring the trochlear groove of the mouse femur. Horizontal activity and velocity and vertical rearing were continually assessed over a 20 hours day/night period using automated activity boxes in an effort to reduce observer bias and capture night activity when the mice are most active. At days 1 and 3, after orthopedic surgery, there was a marked reduction in spontaneous activity and vertical rearing; anti-NGF significantly attenuated this decline. The present data suggest that anti-NGF improves limb use in a rodent model of joint/orthopedic surgery and as such anti-NGF may be useful in controlling pain after orthopedic surgeries such as TKA or THA.Total knee arthroplasty (TKA) and total hip arthroplasty (THA) are 2 of the most common and successful surgical interventions to relieve osteoarthritis pain. Control of postoperative pain is critical for patients to fully participate in the required physical therapy which is the most influential factor in effective postoperative knee rehabilitation. Currently, opiates are a mainstay for managing postoperative orthopedic surgery pain including TKA or THA pain. Recently, issues including efficacy, dependence, overdose, and death from opiates have made clinicians and researchers more critical of use of opioids for treating nonmalignant skeletal pain. In the present report, a nonopiate therapy using a monoclonal antibody raised against nerve growth factor (anti-NGF) was assessed for its ability to increase the spontaneous activity of the operated knee joint in a mouse model of orthopedic surgery pain-induced by drilling and coring the trochlear groove of the mouse femur. Horizontal activity and velocity and vertical rearing were continually assessed over a 20 hours day/night period using automated activity boxes in an effort to reduce observer bias and capture night activity when the mice are most active. At days 1 and 3, after orthopedic surgery, there was a marked reduction in spontaneous activity and vertical rearing; anti-NGF significantly attenuated this decline. The present data suggest that anti-NGF improves limb use in a rodent model of joint/orthopedic surgery and as such anti-NGF may be useful in controlling pain after orthopedic surgeries such as TKA or THA.

Anti–nerve growth factor does not change physical activity in normal young or aging mice but does increase activity in mice with skeletal pain

Abstract Anti–nerve growth factor (anti-NGF) therapy has shown significant promise in attenuating several types of skeletal pain. However, whether anti-NGF therapy changes the level of physical activity in individuals with or without skeletal pain is largely unknown. Here, automated day/night activity boxes monitored the effects of anti-NGF treatment on physical activity in normal young (3 months old) and aging (18-23 months old) mice and mice with bone fracture pain. Although aging mice were clearly less active and showed loss of bone mass compared with young mice, anti-NGF treatment had no effect on any measure of day/night activity in either the young or aging mice. By contrast, in mice with femoral fracture pain, anti-NGF treatment produced a clear increase (10%-27%) in horizontal activity, vertical rearing, and velocity of travel compared with the Fracture + Vehicle group. These results suggest, just as in humans, mice titrate their level of physical activity to their level of skeletal pain. The level of skeletal pain may in part be determined by the level of free NGF that seems to rise after injury but not normal aging of the skeleton. In terms of bone healing, animals that received anti-NGF showed an increase in the size of calcified callus but no increase in the number of displaced fractures or time to cortical union. As physical activity is the best nondrug treatment for many patients with skeletal pain, anti-NGF may be useful in reducing pain and promoting activity in these patients.