The Effects Of P38βMAPK On Hyperalgesia Of Bone Cancer Pain In Rats

Bone cancer pain, one of the most serious cancer pain, is usually induced by primary bone cancer or secondary bone metastasis from breast, prostate, lung cancer, etc. The severity of the pain is closely correlated with the extent of bone destruction. Understanding biological mechanisms for bone cancer pain largely depends on the use of animal models. According to the rat bone cancer pain model described by Medhurst et al, here we establish a new model of tibial cancer pain with MADB-106 mammary gland carcinoma cell line of rats which would contribute to further study of the mechanisms underlying cancer pain.Noxious stimuli and tissue inflammation can produce pain hypersensitivity that may result both from peripheral sensitization (an increased excitability of the peripheral terminals of nociceptive primary sensory neurons) and central sensitization (an increased excitability of dorsal horn neurons). There are accumulating evidences that glia (astrocytes and microglia) in the central nervous system are activated by inflammation or peripheral nerve injury, and are both involved in spinal nociceptive transmission and central sensitization. For example, the use of markers for astrocytes (e.g., glial fibrillary acidic protein [GFAP]) and microglia (e.g., OX-42; Iba-1) reveals that astrocytes and microglia become activated in spinal models of both neuropathic and inflammatory pain, with the microglial activation usually preceding astrocyte activation. However, very little attention has been given to the involvement of glia in central mechanisms related to bone cancer pain.p38MAPK is a member of the MAPK family which is known to regulate events associated with cellular stress. To date, four different isoforms,α,β,γandδ, have been identified. These isoforms have been found in peripheral tissues and p38αin particular has been associated with local inflammatory cascades. The isoforms differ in their substrate preference, activation modes and response to inhibitors. In the mature CNS, only p38αand p38βare constitutively expressed and it has been shown that, in mouse brain, both p38αand p38βare present in neurons, while p38βis also expressed in glial cells. Accumulating evidences now suggest that pain states arising from tissue injury and inflammation are characterized by an enhanced response to subsequent afferent stimulation. This hyperalgesia arises in large part from a facilitated processing of noxious input at the spinal level. Thus, injury-evoked afferent input leads to spinal release of peptides and excitatory amino acids that activate, through their respective receptors, signaling pathways that generate spinal sensitization. For example, spinal p38 can activate phospholipase A2 that liberates arachidonic acid. Arachidonic acid is converted by spinal constitutive cyclooxygenase to prostaglandins which have been shown to facilitate dorsal horn activity. Afferent input generated by tissue and nerve injury or by the direct activation of spinal neurokinin-1 (NK-1) or NMDA receptors leads to phosphorylation (activation) of p38 in spinal microglia. These results suggest that spinal microglial p38MAPK is involved in pain and plays an important role in spinal nociceptive processing and sensitization.However, the anatomical and cellular distribution and location of p38α/βisoforms in rat spinal cord of bone cancer pain is not clear.The current treatments for bone cancer pain involve a variety of modalities. Therapies, including radiation, chemotherapy and/or surgery, targeted at decreasing tumor size are often less effective. Moreover, medications targeted at decreasing inflammation- associated pain, such as non-steroidal anti-inflammatory drugs or opiates have a number of unwanted side effects. Therefore, more and more attention is paid to searching for an efficient intervention at the gene levels to release that pain. Antisense oligonucleotide has been introduced to the chronic pain research just for its excellent characteristics; it can specifically disrupt the target gene mRNA transcription and translation, and then inhibit the noxious target gene expression. The purpose of this study is to provide a new view to treat bone cancer pain with the introduction of antisense oligonucleotide.Methods and Results1. Establishment and Evaluation of Rat Model of Tibial Cancer PainMethods Twenty female SD rats were randomly divided into control group and model group with 10 rats in each group. Models of tibial cancer pain were made according to the method described by Medhurst et al. Rats in the model group were injected with 3μl MADB-106 mammary gland carcinoma cells of rats (4.8×103/μl) into the top segment of left tibial cavitas medullaris, while rats in the control group were injected with the same volume of Hank balanced salt solution into the same site. Mechanical withdrawal thresholds and radiant heat threshold of rats'hind paws were measured before operation and every other day until 22 days of post-operation. The structural damage to the tibia was evaluated by radiological analysis and histological examination on the 8th, 14th and 22nd day post-operation.Results (1)Behavioral tests of pain: During the first 6 days of post-operation, the radiant heat threshold was significantly increased in the model group compared with the control group(P<0.05); However , no significant differences in mechanical withdrawal threshold were found at that time(P >0.05). On day 14 to day 22 after operation, mechanical withdrawal threshold and radiant heat threshold in the model group decreased remarkably compared with those in the control group(P<0.05). (2) Imageological observation: On the 8th, 14th and 22nd day after operation, no radiological change was found in ipsilateral hind limbs of rats in the control group. However, on the 8th day after operation, small radiolucent lesions in the proximal epiphysis were seen in cancellous bone close to sites of injection; On the 14th day after operation, X-rays showed that radiolucent lesions increased and unicortical bone lost; On the 22nd day after MADB-106 injection, bicortical bone loss and displaced fractures were observed. (3)Histological observation: Affected hind limbs were made into paraffin sections respectively on the 8th, 14th and 22nd day after operation, there were no damage in cortical bone and bone trabecula in the control group, whereas tumour cells were densely packed in cavitas medullaris of hind limbs in model group on the 8th day of post-operation after hematoxylin and eosin-stain and bone trabecula were not widely damaged; Cavitas medullaris were full of tumor cells, which induced wide damage of bone trabecula on the 14th day after operation; Tumor cells perforated the cortical bone, and invaded peripheral muscles and soft tissues on the 22nd day after operation .2. Glial Activation in the Lumbar Spinal Cord of the Bone Cancer Pain RatsMethods Twenty-four female SD rats weighing 180～220g were randomly divided into 2 groups(n=10 each):ⅠControl group: intra-tibial injection of 3μl Hank's solution;ⅡModel group: intra-tibial injection of 3μl MADB-106 mammary gland carcinoma cells of rats (4.8×103/μl). Mechanical withdrawal thresholds and radiant heat threshold of rats'hind paws were measured before operation and every other day until 22 days of post-operation. The lumbar 4–6 spinal cord was removed on the 22nd day. The changes of the spinal GFAP and OX42 expression were detected by immunohistochemistry SABC method.Results (1)During the first 6 days of post-operation the radiant heat threshold was significantly increased in the model group compared with the control group(P<0.05); However , no significant differences in mechanical withdrawal threshold were found at that time(P >0.05). On day 14 to day 22 after operation, mechanical withdrawal threshold and radiant heat threshold in the model group decreased remarkably compared with those in the control group(P<0.05).(2)Activation of astrocytes in the ipsilateral spinal cord increased on the 22nd day after MADB-106 mammary gland carcinoma cells inoculation of the tibia by GFAP staining.This enhanced staining appeared in all areas of the spinal grey matter, with the most prominent increase being in dorsal horn laminaeⅠ-Ⅳand lamina X (central canal area). GFAP stained astrocytes were hypertrophied. The optical density on the inoculated side of the spinal grey matter showed stronger staining than that in the control group (P<0.05). (3)Activation of microglia OX-42 staining was greater in the ipsilateral spinal cord on the 22nd day after MADB-106 mammary gland carcinoma cells inoculation of the tibia. This enhanced staining appeared mainly in the dorsal horn of the spinal grey matter. OX-42 stained microglia on the ipsilateral side became less ramified and was hypertrophied compared to those on the contralateral side. Optical immunostaining density on the ipsilateral spinal dorsal horn showed stronger staining than that in the control group (P<0.05).3. The Cellular Location and Expression of p38ɑ/βIsoforms in the Lumbar Spinal Cord of the Bone Cancer Pain RatsMethods Twenty female SD rats weighing 180～220g were randomly divided into 2 groups(n=10 each):ⅠControl group:intra-tibial injection of 3μl Hank's solution;ⅡModel group: intra-tibial injection of 3μl MADB-106 mammary gland carcinoma cells of rats (4.8×103/μl) . Mechanical withdrawal threshold and radiant heat threshold of rats'hind paws were measured before operation and every other day until 14 days of post-operation. The lumbar 4-6 spinal cord was removed on the 14th day. The cellular distribution and location of the spinal p38ɑ/βimmunoreactivity were detected by immunohistochemistry SABC and double immunofluorescence methods. The operative side of spinal cord segment in control group, and bilateral spinal cord segment in model group were taken for RT-PCR. Results On the 14th day after operation, mechanical pain threshold and radiant heat threshold in model group were significantly decreased in the model group compared with the control group (P<0.05). The p38α/βimmunoreactivity of model group in laminaeⅠ～Ⅳof dorsal horn showed stronger staining than that in the control group(P<0.05). Double immunofluorescence confocal micrographs showed that spinal p38αand the neuronal marker neuronal N (NeuN) were colocalized in the dorsal horn, indicating that p38αexpressed in neurons. Double immunofluorescence micrographs demonstrated that antibodies against p38βand the microglia marker OX42 labeled the same cell, indicating that p38βexpressed in microglia. The expression of p38αand p38βin the operative side of model group were increased significantly compared to the operative side of control group and the non-operative side of model group (P<0.05).4. The Inhibitory Effects of Hyperalgesia by Intrathecal p38βAntisense Oligonucleotide in the Bone Cancer Pain RatsMethods Twenty-four female SD rats weighing 180～220g were randomly divided into 4 groups(n=10 each): group A ( control group): intra-tibial injection of 3μl Hank's solution; group B ( model group): intra-tibial injection of 3μl MADB-106 mammary gland carcinoma cells of rats (4.8×103/μl) ; group C (p38β-SODN 20μg) ; group D (p38β-ASODN 20μg). The rats in group C and D were the same bone cancer models as that in the group B. From the 14th day after operation, p38β-SODN 20ug and p38β-ASODN 20ug were injected intrathecally into group C and D respectively and normal saline was injected into group A and B per day for 6 days. Mechanical withdrawal threshold and radiant heat threshold of rats'hind paws were measured before operation and every other day until 22 days of post-operation. The lumbar 4–6 spinal cord was removed on the 22nd day. The expression of p38βprotein in the spinal cord was determined using Western blot. Results No significant differences in mechanical withdrawal threshold and radiant heat threshold were found at all time points in group A. During 14-22 days after operation, mechanical pain threshold and radiant heat threshold were significantly increased in group D compared with those in the group B and C (P<0.05), but the differences were not remarkable between group A and group D(P >0.05). The expression of p38βprotein in lumbar spinal cord was significantly higher in group B and C than that in group A (P<0.05). There was no significant difference in p38βprotein expression between group D and group A (P >0.05).Conclusions1. The model of tibial cancer pain was successfully established with MADB-106 mammary gland carcinoma cell line of rats.2. Both astrocytes and microglia were activated ipsilaterally in the spinal cord of rats with our rodent model of bone cancer pain.3. Our studies indicate that p38ɑand p38βare invovled in the generation and maintenance of bone cancer pain states. P38ɑpredominantly expressed in neurons, while p38βmainly expressed in microglia.4. The hyperalgesia induced by bone cancer can be inhibited by intrathecal administration of p38βantisense oligonucleotide, which is achieved by reducing expression of p38βprotein.