| BACKGROUND AND OBJECTIVEBone cancer pain is a common symptom of malignant tumour patients with bone metastases, which may seriously affect the quality of life of patients. Often accompanied with breakthrough pain, bone cancer pain remains one of the most challenging of cancer pains to fully control. Current treatments of bone cancer pain are single or multi agents combination treatments, including non-steroidal anti-inflammatory drugs, cox-2inhibitors, chemotherapy, radiotherapy, nitrogen-containing bisphosphonates and opioids. When patients who received these treatments still have pain, even the pain aggressive, there is need to identify the cause of under control pain, which is due to tumor progression or non-response to the treatments or the doses of the drugs are not enough. The main diagnose methods of lesions and extent of bone metastases are bone scan and some imagining methods, such as X-ray, CT and MRI. However, these methods can not accurately quantify the amount and the speed of bone lesions, and can detect lesions only as them progress to a certain extent. Therefore, there is need to find a marker, which can simply, fast and sensitively find the bone changes, monitor the progress of disease and assess the treatments’ responses.Recently, the bone collagen metabolites-type I collagen cross-linked telopeptide (NTx) and type I collagen cross-linked C-terminal peptide (CTx) are gradually focused on. The type I collagen is the only collagen in bone tissue, accounting for90%of the bone matrix. Previous study shows that the NTx separately origin from type I collagen, and form a new epitope in the stage of bone resorption, which has high specificity, is a direct product of the osteoclasts and cannot further metabolism. The NTx levels can be measured in serum or urine samples, and the urinary samples are non-invasive and easy to collect, which have favour for monitoring the bone lesions progress and the responses to pain treatments.Therefore, the aim of the present study was to evaluate correlations between the rat urine NTx or the growth rate of NTx and bone cancer pain, to further explore the relationship between urine NTx and the pain response to chemotherapy and bisphosphonates therapy of bone metastases, and to evaluate that whether urine NTx can be a marker of early detection of bone lesion and assessment of response to analgesic therapy.Before starting this study, we first summarized the various animal models of bone cancer pain, which were previously used in molecular and pathological researches and the basic treatments studies. And we further investigated the clinical conditions of patients of bone cancer pain, to explore if the currently commonly used animal models (mice and rats) can well mimic the clinic condition, including the type of disease, bone metastasis sites, sites of bone pain and the characteristics of pain. And we try to find a animal model which is most close to clinical characteristics to do our experimental research. Furthermore, we try to improve the screened animal model to be easier to operate and less damage.METHODS1. Characterization of cancer-induced bone pain of patients based on animal modelsThe animal model research literatures including bone cancer pain animal studies from the pubmed database from1999to May2012. A retrospective study was performed, targeting hospitalized bone tumor patients from May2010to March2011and primary bone tumors patients from May2010to May2012in Tongji Hospital of Huazhong University of Science and Technology. The database included patient demographic information, including age, gender, primary cancer site, and information on bone cancer pain-related factors.2. A new rat model of bone cancer pain produced by rat breast cancer cell implantation of the shaft of femur at the third trochanter levelIn our model of Walker256cell implantation of the shaft of femur at the third trochanter level, the anatomical structure is relatively simple and the drilled hole was vertical in the cortical bone only1-2mm in depth without injury of the distal femur. Pain behaviors and tumor growth were observed for21days. And neurochemical changes were further investigated in this model.3. The correlation between bone metabolic marker NTx and bone cancer painIn this randomized controlled study, we implanted intra-femur different concentrations of Walker256mammary gland carcinoma cells (104,105or106/10μl) to make different tumor progression, and recorded rats’paw thresholds of mechanical allodynia and measured urine NTx values before and7days,14days and21days after injection operations. Furthermore, we gave the rats, which were implanted the same concentration of tumor cells normal saline, zoledronic acid or paclitaxel intervention on the10th day post-operation, recorded paw thresholds and measured urine NTx values on the same days as above mentioned. And we stained bone sections and osteoclasts on the14th days post-operation (4days after treatments), to observe tumor invasion and osteoclasts activity. And the mRNA expressions of c-fos and TRPV1in spinal cord and ASCI3in dorsal root ganglion were analyzed, to explore the potential mechanism underlying the association between the bone cancer pain and urine NTx.RESULTS1. Characterization of cancer-induced bone pain of patients based on animal modelsThis study included seven different types bone cancer pain animal models from26articles. The metastatic bone cancers consist of95.3%of bone cancers. Several significant differences between bone metastases pain and primary bone cancer pain were shown, including age at referral, whether pain occurred before the diagnosis of bone cancer and pain types. There is more common of proximal limb bone metastasis than distal bone. A better bone cancer pain model that can mimic clinical situation should include the injection of metastatic tumor cells, at the femur, measurement methods containing the tests of breakthrough pain.2. A new rat model of bone cancer pain produced by rat breast cancer cell implantation of the shaft of femur at the third trochanter levelCancer-bearing rats demonstrated a decreased limb use score from day14, a increased spontaneous flinching and guarding times from day7and a decreased withdrawal threshold from day6. The tumour infiltration of bone were monitored by MRI and further verified by histological examination. C-Fos and the capsaicin receptor (TRPV1) positive neurons were more expressed in cancer-bearing rats, and the substance P expression has no difference, suggesting that neurons were activated in the model.3. The correlation between bone metabolic marker NTx and bone cancer painThe bone cancer pain of rats has a correlation with urine NTx (r=0.873, p<0.001) instead of the growth rate of NTx(r=0.265,p=0.181), and there is a significant correlation between rat urine NTx and the pain response to zoledronic acid or paclitaxel treatments of bone cancer pain. The H&E stainings of bone sections showed that there is no difference of tumor invasion lesions among each treatment groups and tumor control group, but it was noted that the remaining trabeculars in zoledronic acid group were obviously more than other groups, and the TRAP stainings showed that the osteoclasts activity in zoledronic acid group was significantly reduced compared with tumor control group. The mRNA expression of c-fos in spinal cord and ASCI3in dorsal root ganglion of zoledronic acid group was significantly lower than tumor control group early after treatment (4days). And in the paclitaxel group, this difference occurred later (11days after treatment). In the contrast, the mRNA expressions of TRPV1had no difference in each treatment groups and tumor control group.CONCLUSIONS1. A better bone cancer pain model that can mimic clinical situation should include the injection of metastatic tumor cells, at the femur, measurement methods containing the tests of breakthrough pain. And its findings can provide more effective reference for clinical research and treatment.2. Our animal model demonstrated time-dependent tumor growth and pain behaviors, and will be a novel animal model of bone cancer pain in the future.3. Urine NTx can be a marker of bone cancer pain, early detection of bone lesions and pain evaluation of treatment response. |
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