| Cancer-induced pain, one kind of common clinical chronic pathological pain syndrome, decreased the patients' quality of life greatly. Approximately 30-50% of all cancer patients will experience moderate to severe pain, and 75-95% of patients with advanced-stage or metastatic cancer will experience substantial, life-altering cancer-induced pain. According to the guidelines of the World Health Organization's 'analgesic ladder', treatment with non-steroidal anti-inflammatory drugs and/or opioids, great progress has been made in cancer pain relieving. However, it has been reported that 45% of cancer patients have inadequate and undermanaged pain control because of the relative ineffectiveness and the side effects of current treatments. Therefore, the focus of current research was to establish the proper animal model of cancer pain, which would provide a new impetus for the study of the mechanisms of cancer pain and development of new approaches for cancer pain management. This was one of the research hotspots in chronic pain condition.Previous researches showed inflammation, neuropathy and cancer each generated unique neurochemical changes in the spinal cord. The greatest change in response to cancer pain in the spinal cord was the massive astrocyte hypertrophy, which was significantly greater than peripheral neuropathic and inflammatory pain. More and more evidence supported that glial cells was one of the key players in chronic pathological pain. Glial cells were involved in metabolism, uptake, synthesization and removal of the glutamate, one of the key substances in pain sensation and transmission. Furthermore, upon activation, a variety of algesic substances are released by glia that may potentiate pain transmission by neurons. Of these glial products, proinflammatory cytokines, such as interleukin-1β(IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α), have been demonstrated to be common mediators of allodynia and hyperalgesia. Thus glial cells played an important role in the development and maintainance of the chronic pathological pain. Therefore, another part of our studies was to explore the changes of glial cells in spinal dorsal horn in animal model of bone cancer pain.Acupuncture is an ancient form of medicine that originated in China, which has been practiced for over 2000 years. It is one of the most effective alternative medical treatments with the advantages of simple application, low cost and minimal side effects in pain management. In particular, previous clinical and basic reports showed that acupuncture displayed notable analgesic effect in many kinds of chronic pain conditions, such as peripheral inflammatory and neuropathic pain. Further more, previous reports also showed that acupuncture was one of the most important alternatively medical treatments used in cancer patients for pain relieving and immune modulation. It is beneficial for cancer patients to improve their quality of life. However, very little literature had been reported on its related basic experimental studies due to the lack of proper animal model of cancer pain in a long period. It has been hampered to research on cancer pain mechanisms and to develop new effective therapies for cancer pain relieving. Until 1998, Wacnik et al described a mouse model of cancer pain. Then several models of bone cancer pain were described in mice and rats. It provided a new impetus for the study of cancer pain.According to the reported animal model of cancer pain, the presented study was aimed to:I. Establishment of murine models of cancer painAccording to the rat model of bone cancer pain described by Medhurst, establishing the modified rat model of tibia bone cancer pain and evaluating the animal by dectecting the body weight, bone destruction, ambulatory pain, thermal hyperalgesia, mechanical allodynia and weight bearing on hind limbs; Establishing the rat model of calcaneus cancer pain and evaluating the animal by detecting the thermal hyperalgesia and mechanical allodynia; According the mice model described by Sasamura et al, establishing the mouse model of cutaneous cancer pain and evaluating the animal model by detecting the varicose perimeter of cancer growth and thermal hyperalgesia. II. Changes of glial activity in spinal cord of bone cancer pain ratsExamining expression of glial fibrillary acid protein (GFAP, a markerof astrocyte), CD11b (a marker of microglia), myelin/oligodendrocyte specific protein (MOSP, a marker of oligodendrocyte), proinflammatory cytokines (IL—1β, TNF-α, IL-6) and Toll like receptor (TLR) in spinal dorsal horn in rat model of bone caner pain, by using immunohistochemistry, Western Blot, RT-PCR and ELISA.III. Effects of electroacupuncture (EA) and/or celebrex on cancer pain relievingExamining the effects of EA on mouse model of cutaneous cancer pain and rat model of tibia or calcutaneous bone cancer pain, effects of different dose of celebrex or EA combined with low dose of celebrex on rat model of tibia bone cancer pain.The results are as follows:I. Establishment of murine models of cancer painAccording to the rat model of tibia bone cancer pain, the modified rat model of tibia bone cancer pain was established by inoculating the Walker 256 rat mammary gland carcinoma cells into tibia bone cavity directly, using microsyringe. X-ray and HE staining showed that the rats inoculated with live tumor cells showed significant tibia bone destruction as compared to Normal, PBS and Heat-Killed group rats. The tumor bearing rats displayed significant body weight loss, ambulatory pain, mechanical allodynia and weight bearing differences. The pain related behavior was exaggerated as time and tumor growth. These results suggested that the rat model of bone cancer pain we have developed in this study shares many key features with human bone cancer pain. Our rat model of bone cancer pain was induced by ascitic cancer cells and avoided the boiling cell culture in vitro. The model showed the advantages of simple surgical procedure, stability quality, minimal surgical wound and high successful possibility. Notably, we first reported the bilateral mechanical allodynia induced by unilateral cancer inoculation. The present study provided a valuable animal model for cancer pain research.By inoculating the Walker 256 carcinoma cells into calcaneus bone, the rat model of calcaneus bone cancer pain was established in this study. Wistar rats displayed significant mechanical allodynia on both side of hind limbs after unilateral inoculation. However, there was no significant thermal hyperalgesia on both sides of hind limbs during the whole experiment.By orthotopic inoculating of B16-BL6 melanoam cells into hind paw, the mouse model of cutaneous cancer pain was established according to Sasamura's description. The varicose perimeter of inoculated paw in situ was increased markedly in C57BL/6 mice after B16-BL6 melanoma cells inoculation. The B16-BL6 bearing mice showed significant thermal hyperalgesia after melanoma inoculation.II. Changes of glial activity in spinal cord of bone cancer pain ratsWistar rats were divided randomly into four groups: Normal, PBS, Heat-Killed, Walker 256 (4×10~5) groups. Immunohistochemistry, Western Blot and RT-PCR were used to determine the expression of GFAP, CD11b and MOSP in both sides of spinal dorsal horn. The results showed that there were significant astrocyte and microglia hypertrophy in both sides of spinal dorsal horn induced by unilateral bone cancer while the oligodendrocyte showed no significant changes in this model of bone cancer. Notably, the Western Blot results showed that the level of GFAP and CD11b were also significant increased in both sides of spinal dorsal horn after unilateral Heat-Killed tumor cells inoculation.To examine the level of proinflammatory cytokines (IL-1β, TNF-α, IL-6) in both sides of spinal dorsal horn of tumor bearing rats, RT-PCR and ELISA were used in the study. RT-PCR analysis showed that the mRNA level of proinflammatory cytokines (IL-1β, TNF-α, IL-6) were significant increased in both sides of spinal dorsal horn of tumor bearing rats but not in Heat-Killed rats on 8, 16 and 22 days after unilateral inoculation. ELISA results showed that the level of proinflammatory cytokines (IL-1β, TNF-α, IL-6) were decreased significantly in both sides of spinal dorsal horn on 8 days after unilateral tumor inoculation, and then increased to normal level on 16 days after inoculation.To investigate the changes of Toll like receptor (TLR) in spinal dorsal horn of bone cancer pain rats, RT-PCR analysis was used in the study. The results showed that the mRNA level of TLR(4, 2) were significant increased in both sides of spinal dorsal horn of tumor bearing rats on 8, 16, 22 days after unilateral tumor cells inoculation.All these indicated that the spinal astrocytes and microglia were activated after intra tibia tumor inoculation. It suggested that spinal glial cells and proinflammatory cytokines might be involved in development and maintainance of bone cancer pain.III. Effects of electroacupuncture (EA) and/or celebrex on cancer pain relievingIn order to investigate the analgesic effects of EA on cancer pain, EA treatment was applied to mouse model of cutaneous cancer pain and rat model of tibia/calcaneus bone cancer pain every other days. Zu-San-Li and Kun-Lun acupoints were selected in the study. The results showed that EA treatment displayed significant analgesia effect on mouse model of cutaneous cancer pain at early stage of pain while displayed no significant analgesia effect at late stage of pain. However, EA treatment showed no significant analgesia on rat model of tibia/calcaneus bone cancer pain at the whole stage of cancer pain. It suggested that EA analgesia correlated with the type and the time window of cancer pain.To examine the analgesia effect of celebrex, a kind of selective COX-2 inhibitor, on rat model of tibia bone cancer pain, different dose of celebrex (5, 10, 30 mg/kg/day, p. o.) was administered to bone cancer pain rats twice a day. The results showed that only celebrex (10 mg/kg/day) displayed significant analgesic effect on bone cancer pain. Celebrex (30 mg/kg/day) might produce strong gastrointestinal reaction and toxic side effect while the concentration of celebrex (5 mg/kg/day) might not enough to produce significant analgesic effect, so we could not observe the analgesic effect of these two dose of celebrex on bone cancer pain.To investigate of the Synergistic effect of EA combined with low dose of celebrex (5mg/kg/day) on rat model of bone cancer pain, celebrex (5mg/kg/day) was administered twice a day while EA treatment every other day. The results showed that EA combined with celebrex displayed significant analgesic effect on rat bone cancer pain, indicating Synergistic analgesic effect of EA and celebrex on bone cancer pain. The combined therapy of acupuncture and medication might provide a new analgesic strategy for cancer pain relieving.The present study suggested:i. The rats or mice showed a series of pain related behavior after unilateral inoculation of tumor cells indicating the success in establishment of animal model of cancer pain.ii. Massive astrocytes and microglia hypertrophy, significantly increased expression of proinflammatory cytokines (IL-1β, TNF-α, IL-6) mRNA were observed in both side of spinal dorsal horn on rat model of tibia bone cancer pain. This suggested that the spinal astrocytes and microglia activation might be involved in cancer pain.iii. Bilateral activation of astrocytes and microglia might be involved in the development and maintainance of bilateral mechanical allodynia induced by unilateral tibia inoculation of tumor cells, indicating the involvement of 'mirror image' pain in bone cancer pain.iv. Increased expression of TLR(4, 2) mRNA in bilateral side of spinal dorsal horn was observed in rat tibia bone cancer pain, indicating the TLR-microglia-proinflammatory cytokines pathway might play important role in bone cancer pain.v. EA treatment displayed significant analgesic effect at early stage but no analgesic effect at late stage of mouse cutaneous cancer pain. EA showed no significant analgesic effect on rat bone cancer pain. These suggested EA analgesia correlated with the type and the time window of cancer pain.vi. Celebrex (10mg/kg/day) has significant analgesic effect while celebrex (5mg/kg/day) has no analgesic effect on rat tibia bone cancer pain.vii. EA combined with celebrex (5 mg/kg/day) showed significant analgesic effect on rat tibia bone cancer pain, indicating a Synergistic analgesic effect of EA and celebrex on bone cancer pain...
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