The Mechanism Of Spinal Glia Cell And The Anti-hyperalgesic Effect Of Intrathecal Propentofylline On Bone Cancer Pain In Rats

Part I Study of A SD Rat Model of Bone Cancer Pain Created by Using Walker 256 Cultured in Vitro or in VivoObjective To investigate the possibility and verify the reliability of a SD rat model of bone cancer pain using Walker 256 cultivate in Vitro or in Vivo. Methods Bone cancer pain model was created by receiving left superior extremity intra-tibial inoculations of Walker 256 syngeneic SD rat carcinoma cells which culture and proliferation in vitro(group V1) or in vivo to produce ascites (group A1) in female SD rats weighing 150-180g,n=8. Rat of group N1 or group K1 were inoculated of Hank's or heat-killed cells. The development of bone tumour and pain were observated by digital radiology, ECT, MRI, histology, pain behavioural signs indicative of pain and analgesic pharmacodynamics. Results Rats of four groups did not show changes in body weight during 18 days. Rats of group A and group V displayed decrease of left hint limb activity and minute defect of bone trabecula in the proximal epiphysis by radiological analysis by day 6; Further deterioration was detected at day 12 post-injection with full thickness unicortical bone loss by radiological analysis and bone formation active in region by SPECT; Rats of group A and group V displayed signs of weight loss by day 15. At the final time point, 18 days after the cells injection, full thickness bicortical bone loss and form soft tissue tumor were observed by X ray. Bones inoculated with live cells showed infiltration of bone marrow spaces by malignant tumor on day 18 after inoculation in histology. Rats of group A and group V displayed the gradual development reduced of PWT, von Frey threshold and weight bearing of left hind limb between day 6 and day 18(P <0.01) .Morphine (2-16 mg/kg, S.C.) produced a dose-dependent reduction and Naloxone antagonist manner in the response frequency of hind paw withdrawal to Mechanical stimulation on 15 day following intra-tibia inoculation. Conclusions These results suggested that SD rat model of bone cancer pain can be established by using Walker 256 Culture in Vitro or in Vivo , and the latter was more convenient; the gradual development of mechanical allodynia and mechanical hyperalgesia and reduced of weight bearing on the affected limb, beginning on day 6 following inoculation, whereas the thermal hyperalgesia was not significant; Behavioral data suggest a reasonable time window for study the mechanism of bone cancer pain and evaluation of antinociceptive agents between day 6 and 18 after walker 256 inoculations.Part II Spinal Glia Cell Activation in The SD Rat Model of Bone Cancer Pain Produced by Walker 256 Cancer Cell Inoculation in TibiaInvestigations demonstrated that glial activation in the spinal cord are involved in the development and persistent of pain induced by nerve injury and tissue inflammation. However, the alteration and role of glial cells in spinal cord of bone cancer pain is not well understood. Objective This study explored the spinal glial alteration in a rat model of bone cancer pain produced by injecting syngeneic Walker 256 rat mammary gland Cancer cells into the unilateral tibia of female SD rats. Thereby desire to study the mechanism of development and persistent of bone cancer pain. Methods Bone cancer pain model was created by receiving left superior extremity intra-tibial inoculations of Walker 256 syngeneic SD rat carcinoma cells which culture and proliferation in vivo to produce ascites (A2) in 25 female SD rats weighing 150-180g. The rats were divided by 5 groups randomly which included group N2(control), group K2 (inoculated of heat-killed cells) and group A2, and group A2 was consisted of A2(d6), A2d(12) and A2d(18) groups depended on the day of post inoculation. Results The results showed that astrocytes and microglia were significantly enhanced and activated in the L4-L6 superficial spinal cord bilateralis in rats of inoculation of Walker cancer cells, characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker, P <0.05, P <0.01) and OX-42 (microglial marker,P <0.05, P <0.01). Spinal GFAP staining progression increase on day 6 to day 18 post cell inoculation, and it was prominent on day of 18 postoperation (P <0.05, P <0.01). While Spinal OX-42 staining progression decrease on day 6 to day 18 post cell inoculation, and it was distinguished on day 6 post inoculation (P <0.05, P <0.01). Conclusions These results demonstrate that injection of Walker 256 cancer cells into the tibia activates the spinal glial cells, which may contribute to hyperalgesia and allodynia. Furthermore, the astrocytes and microglia activated in ipsi- and Contralateral superficial dorsal horns highlights the possible involvement in the mirror imaging pain phenomenon in neuroethology of this model of bone cancer pain.Part III Anti-nociceptive and Anti-hyperalgesic Effect of Intrathecal Propentofylline on A Rat Model of Bone Cancer Pain and Its Mechanism in Spinal CordObjective The present study was undertaken to determine whether intrathecal injection propentofylline, a glial modulating agent, could generate anti-nociceptive, anti-hyperalgesic and anti-allodynic effect on a rat model of bone cancer pain. Moreover, investigate its effective mechanism through the alteration of spinal glial and p-ERK in spinal cord. Methods 40 SD rats were divided by 4 groups(n=10) randomly which included group N3(control), group P3 (intrathecal injection PPF 10μg), group A3(model of bone cancer pain and 0.9%NS intrathecal injection), and group PA3 (model of bone cancer pain and PPF 10μg intrathecal injection). Propentofylline or 0.9% was administered daily intrathecally during 9-12 day postoperation. Determination of von Frey threshold before and after intrathecal administered. Spinal cords (L4-L6 segments) were removed for immunohistochemical stainning and Western blot analysis 4h after intrathecal injection on day 12 post operation. Results The von Frey threshold in group PA3 was increased obviously during 3-5 h post PPF intrathecal injection compare to that in group A3 (P <0.01) , and the pain threshold was increased after PPF i.t. daily during 9-12d post inoculation. GFAP stained astrocyte were atrophied and become less ramified in group PA3 compared to those in group A3. OX-42 stained microglia were atrophied and become more ramified in group PA3 compared to those in group A3. Otherwise, p-ERK stained increased in group PA3 compared to those in group A3. These results indicate spianl microglial, astrocytic and ERK activation was decreased by intrathecal daily administration of propentofylline in existing antihyperalgia and allodynia paradigms (P<0.05 , P<0.01 ) .Conclusions Propentofylline intrathecal injection could generate antinociceptive, antihyperalgia and antiallodynic effect on a rat model of bone cancer pain. Moreover, the antinociceptive, antihyperalgic and antiallodynic effect of PPF intrathecal were generated by restrainting the activation of spinal astrocytes ,microglia and p-ERK.