The Role Of P2X₃ Receptor On The Peripheral Sensitization In A Rat Model Of Bone Cancer Pain

Pain is one of the most common symptoms reported by patients with cancer. 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.Although bone cancer pain is one of the most severe and common of the chronic pains that accompany breast,prostate,and lung cancers,relatively little is known about the mechanisms that generate and maintain this pain.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.Metastasis of tumor cells to bone is particularly common in patients with lung,breast,and prostate cancer and patients with bone metastasis are more likely to experience severe pain.Thus, pain associated with tumor cells that have metastasized to bone is a frequent and debilitating complication of cancer.Understanding the neurobiological mechanisms underlying cancer pain is critical for improved management.The first rat model of bone cancer pain had been described by Medhurst et.al.in 2002.But the MRMT-1 tumor cell used in this model was difficult to be got domesticly,yet many other research was still due to develop the new rat models of bone cancer pain using new tumor cells.The recently published animal models of bone cancer pain have allowed greater insight into the plasticity of peripheral and central nervous system,and the further future studies on these models are expected to lead to new approaches for cancer pain management.In our previous study,we developed a modified rat model of bone cancer pain induced by inoculating another mammary gland carcinoma cells derived from Wistar rats—Walker 256 cells into the tibia cavity of rats.Bone destruction and the time course of pain-related behavioral changes were evaluated.In these models,inoculation of cancer cells into the intramedullary cavity of femur or tibia of syngeneic animals produced a series of behavioral,cellular,and neurochemical changes correlated with cancer growth and bone destruction,including the development of mechanical hyperalgesia and mechanical allodynia,the changes of weight bearing,and astrocyte hypertrophy.These clues suggested that bone cancer pain was a special type of chronic pain and pain sensitization mechanisms were involed in. P2X receptors are a family of ligand-gated ion channels,activated by extracellular ATP. The seven subunits cloned(P2X_1-7) can assemble to form homomeric and heteromeric receptors.Immunocytochemical studies revealed that P2X₃ receptor is almost exclusively localized in small and medium DRG sensory neurons and was likely to participate in the pain signal transmission.Interestingly,ATP is contained in tumor cells in exceptionally high concentration(Maehara et al.) and Gilchrist's studies demonstrated that increased expression of P2X₃ receptors on CGRP-ir epidermal nerve fibers during tumor growth in a murine model of cancer painand suggest a role for ATP and P2X₃ receptors in cancer-related pain.Among peripheral neurons,sensory neurons of the dorsal root ganglia (DRG) are the fisrt station of nociceptive transmission,so our study began from this.In the present study,we plained to determine whether ATP and P2X₃ receptors contribute to bone cancer pain using several experement techniques such as Immunocytochemistory,western blot,RT-PCR and pacth clamp.The experement started with this four steps:(1) establish a rat model of bone cancer pain by inoculating Walker 256 cells into the tibia cavity of femal wistar rats and determine hyperalgesia or allodynia by using behavial test;(2) observe the immunoreactive changes of P2X receptor of rat DRG neurons and determin the P2X₃ mRNA level using realtime RT-PCR techniques;(3) observe the electrophysiological changes of P2X₃ receptor using whole-cell patch clamp; (4) test whether intracheal administration of P2X₃ antagonist suramin 50μg could relieve bone cancer pain.The results were as follow.1.the condition and results of modelingSyngeneic Walker 256 mammary gland carcinoma cells(2×10⁵) were injected into the tibia medullary cavity of female Wistar rats,the rate of useful inoculation is 70%as be calculated PID 21d.The spontaneous ambulatory pain score(SPAS) was going higher on the injected hind limb from day 10.Rats showed significant limp from day 14 to 21 and some with the paw autophagia or decreased foodintake.Rats inoculated with live cells displayed a profound decrease in paw withdrawal threshold to von Frey hair stimulation but there was no significant change in paw withdrawal latency to radiant heat stimulation on the hind paws during the whole experiment which was according with the document report.No radiological change was found in PID 8d.By day 21d the tibia bone showed signs of radiolucent lesion in the proximal epiphysis,medullary bone loss and cortical bone loss.Sections obtained from the proximal end of tibia 21 days after the intra-tibia injection were stained with hematoxylin and eosin,tumor growth and various degrees of bone destruction were observed in the animals received live Walker 256 carcinoma cells. Remodeling of the bone was also observed in the affected bone.In a few cases of severe bone destruction,the tumor destroyed bone matrix and periosteum and grew outside of the bone.GFAP staining of the spinal cord showed increased staining in the ipsilateral spinal cord.In conclusion,the rat model of bone cancer pain has been succesfuly established.2.the change of the expression level of the P2X₃ receptor in DRGImmunohistochemistry showed that 30.71%,32.3%,48.78%and 46.27%of DRG neurons were P2X₃ receptor-positive neurons in control,sham,cancer pain 14d,and cancer pain 21d group,respectively.The results were also suportted by western blot which showed the related level(P2X₃/β-actin)were 0.58±0.16,0.56±0.17,1.26±0.25,1.30±0.43 in control,sham,cancer pain 14d,and cancer pain 21d group,respectively.It was significantly different from control and cancer pain group(p＜0.05).Realtime RT-PCR showed that P2X3 mRNA level increased PID 14d and 21d in cancer pain goup.These result sugessted that the hyperalgesia in the rats with bone cancer pain on PID 14d to 21d may be related to the increased expression of P2X₃ receptors in DRG which due to the P2X3 mRNA increase.3.electrophysiological changes of P2X₃ receptorThe morphology,resting membrane potential and membrane capacitance of DRG neurons showed no significant differecne between the cancer pain and control group.Passive membrane electrophysiological characters of DRG neurons were not markedly changed after tumor inoculation(p＞0.05).But the rate of neurons responed to aβ-meATP(100μM) and the inward current induced by application of aβ-meATP was increased in bone cancer pain group.Three type of inward currents induced by aβ-meATP had been recorded, the trainsient inward current,the biphasic current and the sustained current and the current could be fully inhibitted by suramin.Percentage of the three type currents were not markedly different after tumor inoculation.4.Effects of spinally administered P2X₃ receptor antagonists suramin on bone cancer painIntrathecal administration of 50μg/d of suramin to animals 14～16d after the inoculation of Walker256 cell produced a significant inhibition of the mechanical hyperalgesia,the paw withdrawal theshold to von Frey hair stimulation increased,while suramin did not alter the pain related behavier in normal animals.ConclusionFrom the results above we can get these conclusion, 1.The rat model of bone cancer pain has been succesfuly established.The main behavier characters of this model are spontaneous ambulatory pain and mechanical hyperalgesia.2.P2X₃ receptor might participate in the mechanism of the pain sensitization in bone cancer pain(1) The hyperalgesia in the rats with bone cancer pain may be related to the increased expression of P2X₃ receptors in DRG which due to the P2X3 mRNA increase.(2) The rate of neurons responed to aβ-meATP(100μM) and the inward current induced by application of aβ-meATP was increased in bone cancer pain group.(3) Intrathecal administration of suramin to animals with bone cancer pain produced a significant inhibition of the mechanical hyperalgesia...