Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108
Performing Department
Veterinary Biomedical Sciences
Non Technical Summary
Justification: Pain is something that all animals and humans experience and untreated pain has important welfare consequences for domestic animals. Clinically relevant pain is the result of complex processes involving peripheral transduction and transmission as well as central modulation and processing that leads to the final conscious sensation of pain. Unfortunately despite the wealth of information regarding pain mechanisms, there is still much to be learned and such information is critical not only to our understanding of how pain is initiated and processed, but importantly to devising better treatments for alleviating pain and suffering particularly in food animals where welfare concerns arise when animals are not managed properly or when painful procedures are performed without pain medication. Our long-term goal is to better understand the mechanisms underlying bone cancer and arthritic pain and to determine if acupuncture treatment or inhibition of selected pain mediators is effective in alleviating this type of pain. We will examine whether 5 potential pain mediators (resistin, adiponectin, KC. CCL2 and osteopontin) are released from a bone tumor or from the inflammed knee joint and whether giving acupuncture or inhibitors of these mediators reduces either bone cancer pain or arthritic pain. Our findings will have important implications for treating arthritic and cancer pain in production animals as well as humans
Animal Health Component
10%
Research Effort Categories
Basic
75%
Applied
10%
Developmental
15%
Goals / Objectives
Our long-term goal is to better understand the mechanisms underlying bone cancer and arthritic pain and to determine the mechanisms by which acupuncture treatment alleviates this type of pain in order to develop better therapeutic approaches to alleviating pain in production animals. The central hypothesis is that cancer pain and joint pain are associated with inflammation and that acupuncture reduces the inflammatory response associated with these conditions. The overall objectives of our study are: 1. to determine the peripheral mechanisms by which inflammation induces pain in rodent models of cancer pain and arthritic pain and 2. to understand how acupuncture reduces these types of pain in order to discover key mediators that can be used to develop better therapeutic approaches to treating pain in poultry and other production animals. Joint and bone pain occur in several production animals particularly turkeys. Identifying underlying mechanisms and developing better treatment approaches will serve to help alleviate animal welfare concerns related to pain and will allow increased movement and less lameness particularly in turkeys, but this will also have an important impact on beef and other production animals. The overall objective of our study is to determine the peripheral mechanisms by which inflammation induces pain in rodent models of cancer pain and arthritic pain and to understand how acupuncture reduces these types of pain. Here we will investigate whether resistin, adiponectin, KC. CCL2 and osteopontin are associated with tumor-induced nociception and arthritic nociception and whether acupuncture reduces the levels of these chemokines and adipokines. The impact of our findings is that they will significantly advance our understanding of the role of inflammation in producing bone cancer and arthritic pain as well as the mechanisms by which acupuncture can relieve pain associated with these conditions. By examining the mechanisms of how acupuncture alleviates pain, we will identify pharmacological targets for the development of drugs that can have similar analgesic effects for use in production animals. This will lead to better therapeutic approaches to treating these conditions and impact animal welfare
Project Methods
Procedures:Animals: For our proposed studies we will use age matched C3H male mice. Adult mice will either be injected with 105 fibrosarcoma cells (controls: PBS) into the calcaneus bone of the hindpaw as previously described (9, 10, 11) or with 3% carrageenan into the knee joint (controls vehicle; 17).Behavioral Assays: All mice in both the tumor and knee arthritis groups and the corresponding control groups will be subjected to two or more of the following behavioral tests for nociception:1. Von Frey testing for mechanical hyperalgesia: Before any of the behavioral assays, mice will be habituated in the testing environments for at least 1 h. All behavioral assays will be performed blinded to injection of drug versus placebo. Mechanical sensitivity was tested using calibrated von Frey filaments (0.02-2.0 g, Stoelting, Wood Dale, IL) exerting bending forces between 0.02 and 2 g. Each filament will be gently applied for 2-3 s to the glabrous skin of the hindpaw of a mouse standing on a metal mesh. Each filament will be applied 8-10 times with at least 10 min between trials, and the frequency of withdrawal responses will be scored at each force. The 50% paw withdrawal threshold will be determined using the up-down method (9, 11).2. Thermal Sensory Response Assessment - Hot Plate Thermal Hypersensitivity:The hot plate is maintained at 53°C (Columbus Instrument, Columbus, OH). Once placed on the hot plate in an enclosed container (20×16×25cm3), mice are observed for licking or rapid shaking of one hind paw. The time transpired until the first avoidance response of either hind limb, such as licking or rapid shaking, is recorded as the latency of response. A cut-off value of 30 s is used to avoid tissue damage.3. Thermal Sensory Response Assessment - Cold Plate Hypersensitivity:The cold plate is maintained at 0 plus/minus 0.5°C . Once placed on the cold plate in an enclosed container (28×17×13cm3), mice are observed for licking or rapid shaking of one hind paw. The time transpired until the first avoidance response of the hind limb, such as licking or rapid shaking, is recorded as the latency of response. The total number of spontaneous behaviors as indicative of the same type of responses as latency are totaled for the length of the testing window. A cut-off value of 2 minutes is used to avoid tissue damage.4. Activity Box Assay: Each mouse is placed in a 43 X 43 X 21 cm Plexiglas activity box (Columbus Instruments) surrounded by movement recording sensors as previously reported (see references 1 and 2 below). Each box is surrounded by 32 XY emitter/detector sensor pairs and 16 Z emitter/detector sensor pairs. Bedding, food, and water are placed in locations that will not interfere with the ability of the sensors to record movements of the mouse. The total numbers of X, Y (horizontal), and Z (vertical, i.e. rearing) movements are recorded every 5 minutes for up to 72 hours depending on experimental design and treatment. The numbers of horizontal and vertical movements of tumor, arthritic and control animals will be compared. Upon completion of the study, mice will be removed from the activity boxes and returned to their normal conventional housing. Recent experiments performed in our laboratory have shown that in a model of inflammation and a bone tumor model that mice display marked reductions in both locomotor and spontaneous activity (similar to what is seen with turkeys experiencing leg/joint pain) as measured using the Columbus Activity Boxes. These preliminary results suggest that in the proposed models of joint pain and tumor pain, activity measurements can provide additional real-time nociceptive data as the knee inflammation or tumor develops and progresses.Other Experimental Procedures:Microperfusion studies: Implantation of a microperfusion probe into the tumor has been performed in our laboratory for the past 15 years. Dr. Beitz developed this procedure in his laboratory (see reference 9) and has extensive experience with the procedure. As part of our investigation of arthritic joint and tumor released mediators and their potential role in tumor- and arthritis-induced pain, we will measure the relative levels of resistin, adiponectin, KC, OPG and CCL2 secreted at the tumor site (At day 10 postimplantation) or from an inflamed joint (24 hours post-injection) in vivo. A microperfusion probe is implanted in the hindpaw bone tumor or into the joint as previously described (9). Briefly, following induction of anesthesia the microprobe will be inserted through the center of the tumor site or into the knee joint and secured to the skin with super glue. Two lengths of polyethylene tubing (PE-10, prefilled with heparin-nPBR; 30 U/ml) connect the microprobe to the fluid swivel and two peristaltic pumps. The inlet perfusion pump will push modified Ringer's through the microprobe at a rate of 10 ul/min; while the outlet pump will be set at a rate of 20 ul/min. Following a 45 min equilibration period, samples will be collected for a period of 3-4 hr and the above chemokines and adipokines are measured using either commercially available ELISA assays or protein arrays.Immunocytochemistry: To determine which cells are producing the above chemokines and adipokines we will perform immunohistochemistry on the hindpaw tumor and on the knee joint in both experimental and control animals as previously described (10, 11). The animals will be perfused transcardially with 10% neutral-buffered formalin, postfixed overnight at 4? C, 8 µm thick sections will be cut on a cryostat and processed for immunocytochemistry with antibodies against resistin, adiponectin, KC, OPG and CCL2. Antibodies will be obtained from Abcam (Resistin, adiponectin, Osteopontin) or R&D Systems (CCL2 and CXCL1).Electroacupuncture: We will test whether electroacupuncture (EA) given at ST-36 reduces the release of resistin, adiponectin, CXCL1 and osteopontin from the tumor site or from the knee joint. EA will be applied to ST-36 as previously described (11, 18). For tumor animals, EA will be applied to the ST-36 acupoint twice weekly beginning at postimplantation day 3 (PID 3). For arthritic animals EA will be given starting 2 days prior to carrageenan injection and will be given on days -2, 0 and 1 post-carrageenan. Microperfusion will be performed on PID 10 in tumor animals and 24 h post-injection in knee arthritic animals. Substances in the microperfusate samples will be analyzed using ELISA assays or protein arrays.Inhibition of Tumor and Arthritic Joint Nociceptive Mediators: In order to determine if resistin, adiponectin, or CCL2 contribute to bone tumor or joint pain we will administer antagonists to these three mediators (in separate experiments) and perform behavioral tests to assess the level of inhibition of pain behaviors. We will administer the CCL2 antagonist, RS504393 (purchased from Tocris, Bristol, UK, 60 µg/mouse), the resistin antagonist, CYT-23 (purchased from Ansh Labs, Webster, TX), the adiponectin antagonist, MAPP (1S,2R-D-erythro-2-N-myristoylamino)-1-phenyl-1-propanol, purchased from Cayman Chemical Company, Ann Arbor, MI; 5 mg/kg). Drugs will be administered at doses recommended in the literature.Statistical Analysis: Data will be presented as the mean ± SEM. Statistical analyses will be performed with unpaired Student's t-tests (two-tailed), Mann-Whitney U tests, or ANOVA. The number of animals indicated in this application are based on sample size calculations to generate statistical power for the detection of significant differences (n = sample size = [σ/δ]2 x [Z alpha + Z beta]2 where σ = standard deviation and δ = difference in SD units). Consequently, and according to the anticipated treatment effects and on previously observed experimental variance, n = 10 at 90% power with an one-sided p-value < 0.05 and Z alpha = 1.65.