p38βMAPK在大鼠骨癌痛痛觉过敏中的作用
摘要
研究背景和目的
骨癌痛是一种较严重的癌痛,通常由原发性骨肉瘤或继发于乳腺癌、前列腺癌、肺癌等肿瘤转移所致,其疼痛程度与骨组织破坏紧密相关。探寻其生物机理很大程度依赖于动物模型建立。根据Medhurst等报道方法,本实验用MADB-106大鼠乳腺癌细胞复制骨癌痛模型,以期对骨癌痛机制进行研究。
伤害刺激和组织炎症能导致外周(增加伤害初级传入神经末梢兴奋性)及中枢(增加后角神经元兴奋性)敏化,大量实验研究证明中枢神经系统中胶质细胞(星形胶质细胞和小胶质细胞)被炎症或外周神经损伤所激活,参与脊髓痛觉传递和中枢敏化。在炎性痛及神经病理痛中星形胶质细胞标记物GFAP和小胶质细胞标记物OX-42被激活,而且小胶质细胞激活早于星形胶质细胞。然而,有关胶质细胞在骨癌痛中的作用研究很少。
p38是丝裂原活化蛋白激酶(Mitogen Activated Protein Kinase,MAPK)家族成员之一,与细胞应激有关,已知有四种亚型即α,β,γ和δ。这些亚型分布在外周组织,而p38α与局部炎症级联效应有关。这些亚型在基质选择、活化方式、对抑制剂的反应性等方面各不相同。在哺乳动物中枢神经系统,仅有p38α和p38β表达,p38α和p38β表达在鼠脑神经元,p38β也在胶质细胞表达。实验研究证明外周炎症或神经损伤引起的疼痛以对传入刺激高反应为特征,痛觉高敏发生在脊髓水平对伤害传入易化。伤害传入导致脊髓上肽类物质和兴奋性氨基酸释放,它们通过各自受体、信号通路产生中枢敏化。如p38活化磷脂酶A2产生花生四烯酸,花生四烯酸经环加氧酶途径代谢产生能易化脊髓背角活性的前列腺素。组织及神经损伤或脊髓NK-1受体、NMDA受体直接激活脊髓小胶质细胞内的p38,这提示小胶质细胞内的p38MAPK在脊髓痛觉传递和敏化中起重要作用。有关p38α和p38β在骨癌痛大鼠脊髓中的细胞定位、表达及在痛敏中作用尚不清楚。当今治疗骨癌痛的方法有通过减小肿瘤体积,包括放疗,化疗,和/或手术;用非甾体抗炎药或鸦片类药物减轻炎性相关疼痛等,这些方法常伴有较多副作用。反义寡核苷酸技术由于其寡核苷酸分子能够特异性结合并阻断靶基因mRNA转录、翻译,从而抑制有害目的基因表达,而被引用至慢性疼痛研究领域。本研究旨在将反义寡核苷酸技术引入骨癌痛的实验研究,试图为临床癌痛治疗提供一条新途径。
研究方法与结果
1、大鼠骨癌痛模型建立
方法:选择雌性SD大鼠20只,随机分为对照组和模型组,每组10只。对照组:左侧胫骨上段骨髓腔注入3μl Hank液;模型组:按照Medhurst等的方法制作胫骨癌痛模型,大鼠麻醉后仰卧位捆绑,从左侧胫骨上部切开皮肤,分离肌肉,暴露胫骨,使用20ml注射器针头穿刺打孔,之后换上10μl注射器进入骨髓腔,缓慢注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl ),注射完毕后用骨蜡封住针孔,皮肤缝合。术前测定两组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～22d隔日测定术侧后爪机械痛及辐射热痛的缩爪阈值。术后第8、14、22 d,两组大鼠术侧后肢进行X射线拍片和苏木精-伊红染色,观察骨质破坏情况。
结果:(1)疼痛行为学观察:术后1～6d,模型组大鼠术侧后爪的机械痛缩爪阈值较对照组无统计学差异(P >0.05),而辐射热痛的缩爪阈值较对照组显著升高(P<0.05);术后14～22d,模型组大鼠术侧后爪的机械痛和辐射热痛缩爪阈值分别较对照组显著降低(P<0.05)。(2)影像学观察:X射线显示,模型组大鼠在术后第8d仅见注射部位松质骨有小的放射性缺省病灶;术后第14d松质骨放射性病灶增多,单侧骨皮质缺失;术后第22d双侧骨皮质缺失并移位性骨折。而对照组大鼠在术后第8、14、22d术侧后肢未发现骨质破坏。(3)组织学观察:术后第8、14、22d将术侧后肢石蜡切片、苏木精-伊红染色显示,对照组大鼠未见骨小梁、骨皮质破坏。而模型组大鼠在术后第8d可见骨髓腔有异型细胞,骨小梁未见广泛破坏;术后第14d可见肿瘤细胞充填骨髓腔,骨小梁广泛破坏;术后第22d可见肿瘤细胞穿破骨皮质,侵及周围肌肉及软组织。
2、骨癌痛大鼠术侧腰段脊髓星形胶质细胞和小胶质细胞活化的研究
方法:选择雌性SD大鼠20只,随机分为对照组和模型组,每组10只。对照组:左侧胫骨上段骨髓腔注入3μl Hank液;模型组:左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl)。术前测定两组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～22d隔日测定术侧后肢机械痛及辐射热痛的缩爪阈值。术后22d测痛后,使用4%多聚甲醛灌注大鼠,取大鼠脊髓L4-6节段,采用免疫组织化学方法观察骨癌痛大鼠腰段脊髓中星形胶质细胞胶质纤维酸性蛋白(GFAP)免疫反应阳性产物及OX42标记的小胶质细胞阳性产物分布及变化。
结果:(1)术后1～6d,模型组大鼠术侧后爪的机械痛缩爪阈值较对照组无统计学差异(P>0.05),而辐射热痛的缩爪阈值较对照组显著升高(P<0.05);术后14～22d,模型组大鼠术侧后爪的机械痛和辐射热痛缩爪阈值分别较对照组显著降低(P<0.05)。(2)免疫组化显示,模型组大鼠术侧腰脊髓背角和腹角可见大量GFAP阳性星形胶质细胞(染色加深,胞体增大,突起变粗变长),最明显在背角I-IV层和X层(中央管周围),而对照组术侧腰段脊髓背角仅见少量GFAP阳性星形胶质细胞。模型组大鼠术侧腰脊髓背角GFAP免疫反应光密度值明显高于对照组(P<0.05)。模型组大鼠术侧腰段脊髓背角可见大量OX42阳性的小胶质细胞(表现为分支减少且肥大),而对照组术侧腰段脊髓背角仅见少量OX42阳性的小胶质细胞。模型组大鼠术侧腰脊髓背角OX42免疫反应光密度值明显高于对照组(P<0.05)。
3、骨癌痛大鼠术侧腰段脊髓p38ɑ和p38β的细胞定位及表达
方法:选择雌性SD大鼠20只,随机分为对照组和模型组,每组10只。对照组:左侧胫骨上段骨髓腔注3μl Hank液;模型组:左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl)。术前测定两组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～14d隔日测定术侧后肢机械痛及辐射热痛的缩爪阈值。术后14d测痛结束,灌注固定取大鼠脊髓L4-6节段,采用免疫组织化学ABC法和荧光双标法观察大鼠术侧腰段脊髓背角p38α和p38β的分布和细胞定位。每组剩余7只大鼠断头处死后分离L4-6左右侧脊髓,提取大鼠脊髓的RNA,以RT-PCR技术检测模型组与对照组大鼠p38α和p38β表达的相对差异。
结果:术后第14d,模型组大鼠术侧后爪机械痛和辐射热痛的缩爪阈值较对照组显著降低(P<0.05)。模型组大鼠术侧腰段脊髓背角Ⅰ～Ⅳp38α和p38β的光密度值明显高于对照组(P<0.05)。荧光双标显示,模型组大鼠术侧腰段脊髓背角p38α与神经元特异性核蛋白标志物NeuN有共表达,p38β与小胶质细胞标志物OX-42有共表达。模型组大鼠手术侧p38α、p38β的mRNA表达较非手术侧显著升高(P<0.05),与对照组手术侧比较也明显升高(P<0.05)。
4、鞘内注射p38βMAPK反义寡核苷酸对骨癌痛大鼠痛觉过敏的影响
方法:选择雌性SD大鼠40只,随机分为四组,每组10只。A组(对照组):大鼠左侧胫骨上段骨髓腔注入3μl Hank液;B组(模型组):大鼠左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl);C组(p38β-SODN 20ug处理组)和D组(p38β-ASODN 20ug处理组)大鼠左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl),14d后鞘内分别注射p38β-SODN 20ug及p38β-ASODN 20ug,1次/d,连续6d ,而A、B组以同样的方法鞘内注射等量无菌生理盐水作对照。术前测定四组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～22d隔日测定术侧后肢机械痛及辐射热痛的缩爪阈值。术后第22d,取大鼠脊髓L4-6节段,采用Western blot法检测脊髓p38β蛋白的表达。
结果:术后14～22d p38β-ASODN 20ug组大鼠术侧后爪机械痛和辐射热痛的缩爪阈值较模型组和p38β-SODN 20ug组显著升高(P<0.05),而与对照组比较差异无统计学意义(P >0.05)。Western Blot结果显示,p38β-ASODN 20ug组p38β蛋白质相对表达量较模型组和p38β-SODN 20ug组显著降低(P<0.05),而与对照组比较,差异无统计学意义(P >0.05)。
5、统计学方法
应用SPSS11.0软件包进行统计分析,计量资料以均数±标准差(x±s)表示。测痛数据采用重复测量数据的方差分析,其余数据采用t检验,P<0.05为差异有统计学意义。
研究结论
1.采用MADB-106大鼠乳腺癌细胞系能成功复制大鼠胫骨癌痛模型。
2.骨癌痛大鼠术侧腰段脊髓背角星形胶质细胞和小胶质细胞均被广泛激活。
3.脊髓p38α和p38β参与了骨癌痛的发生和发展,p38α主要在脊髓神经元表达,而p38β则在脊髓小胶质细胞中表达。
4.鞘内注射p38β反义寡核苷酸能减轻骨癌痛大鼠机械性痛觉超敏及辐射热痛觉过敏,这种作用可能是通过下调脊髓p38β蛋白质的表达来发挥的。
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 Results
1. Establishment and Evaluation of Rat Model of Tibial Cancer Pain
Methods 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 Rats
Methods 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 Rats
Methods 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 Rats
Methods 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).
Conclusions
1. 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.
骨癌痛是一种较严重的癌痛,通常由原发性骨肉瘤或继发于乳腺癌、前列腺癌、肺癌等肿瘤转移所致,其疼痛程度与骨组织破坏紧密相关。探寻其生物机理很大程度依赖于动物模型建立。根据Medhurst等报道方法,本实验用MADB-106大鼠乳腺癌细胞复制骨癌痛模型,以期对骨癌痛机制进行研究。
伤害刺激和组织炎症能导致外周(增加伤害初级传入神经末梢兴奋性)及中枢(增加后角神经元兴奋性)敏化,大量实验研究证明中枢神经系统中胶质细胞(星形胶质细胞和小胶质细胞)被炎症或外周神经损伤所激活,参与脊髓痛觉传递和中枢敏化。在炎性痛及神经病理痛中星形胶质细胞标记物GFAP和小胶质细胞标记物OX-42被激活,而且小胶质细胞激活早于星形胶质细胞。然而,有关胶质细胞在骨癌痛中的作用研究很少。
p38是丝裂原活化蛋白激酶(Mitogen Activated Protein Kinase,MAPK)家族成员之一,与细胞应激有关,已知有四种亚型即α,β,γ和δ。这些亚型分布在外周组织,而p38α与局部炎症级联效应有关。这些亚型在基质选择、活化方式、对抑制剂的反应性等方面各不相同。在哺乳动物中枢神经系统,仅有p38α和p38β表达,p38α和p38β表达在鼠脑神经元,p38β也在胶质细胞表达。实验研究证明外周炎症或神经损伤引起的疼痛以对传入刺激高反应为特征,痛觉高敏发生在脊髓水平对伤害传入易化。伤害传入导致脊髓上肽类物质和兴奋性氨基酸释放,它们通过各自受体、信号通路产生中枢敏化。如p38活化磷脂酶A2产生花生四烯酸,花生四烯酸经环加氧酶途径代谢产生能易化脊髓背角活性的前列腺素。组织及神经损伤或脊髓NK-1受体、NMDA受体直接激活脊髓小胶质细胞内的p38,这提示小胶质细胞内的p38MAPK在脊髓痛觉传递和敏化中起重要作用。有关p38α和p38β在骨癌痛大鼠脊髓中的细胞定位、表达及在痛敏中作用尚不清楚。当今治疗骨癌痛的方法有通过减小肿瘤体积,包括放疗,化疗,和/或手术;用非甾体抗炎药或鸦片类药物减轻炎性相关疼痛等,这些方法常伴有较多副作用。反义寡核苷酸技术由于其寡核苷酸分子能够特异性结合并阻断靶基因mRNA转录、翻译,从而抑制有害目的基因表达,而被引用至慢性疼痛研究领域。本研究旨在将反义寡核苷酸技术引入骨癌痛的实验研究,试图为临床癌痛治疗提供一条新途径。
研究方法与结果
1、大鼠骨癌痛模型建立
方法:选择雌性SD大鼠20只,随机分为对照组和模型组,每组10只。对照组:左侧胫骨上段骨髓腔注入3μl Hank液;模型组:按照Medhurst等的方法制作胫骨癌痛模型,大鼠麻醉后仰卧位捆绑,从左侧胫骨上部切开皮肤,分离肌肉,暴露胫骨,使用20ml注射器针头穿刺打孔,之后换上10μl注射器进入骨髓腔,缓慢注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl ),注射完毕后用骨蜡封住针孔,皮肤缝合。术前测定两组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～22d隔日测定术侧后爪机械痛及辐射热痛的缩爪阈值。术后第8、14、22 d,两组大鼠术侧后肢进行X射线拍片和苏木精-伊红染色,观察骨质破坏情况。
结果:(1)疼痛行为学观察:术后1～6d,模型组大鼠术侧后爪的机械痛缩爪阈值较对照组无统计学差异(P >0.05),而辐射热痛的缩爪阈值较对照组显著升高(P<0.05);术后14～22d,模型组大鼠术侧后爪的机械痛和辐射热痛缩爪阈值分别较对照组显著降低(P<0.05)。(2)影像学观察:X射线显示,模型组大鼠在术后第8d仅见注射部位松质骨有小的放射性缺省病灶;术后第14d松质骨放射性病灶增多,单侧骨皮质缺失;术后第22d双侧骨皮质缺失并移位性骨折。而对照组大鼠在术后第8、14、22d术侧后肢未发现骨质破坏。(3)组织学观察:术后第8、14、22d将术侧后肢石蜡切片、苏木精-伊红染色显示,对照组大鼠未见骨小梁、骨皮质破坏。而模型组大鼠在术后第8d可见骨髓腔有异型细胞,骨小梁未见广泛破坏;术后第14d可见肿瘤细胞充填骨髓腔,骨小梁广泛破坏;术后第22d可见肿瘤细胞穿破骨皮质,侵及周围肌肉及软组织。
2、骨癌痛大鼠术侧腰段脊髓星形胶质细胞和小胶质细胞活化的研究
方法:选择雌性SD大鼠20只,随机分为对照组和模型组,每组10只。对照组:左侧胫骨上段骨髓腔注入3μl Hank液;模型组:左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl)。术前测定两组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～22d隔日测定术侧后肢机械痛及辐射热痛的缩爪阈值。术后22d测痛后,使用4%多聚甲醛灌注大鼠,取大鼠脊髓L4-6节段,采用免疫组织化学方法观察骨癌痛大鼠腰段脊髓中星形胶质细胞胶质纤维酸性蛋白(GFAP)免疫反应阳性产物及OX42标记的小胶质细胞阳性产物分布及变化。
结果:(1)术后1～6d,模型组大鼠术侧后爪的机械痛缩爪阈值较对照组无统计学差异(P>0.05),而辐射热痛的缩爪阈值较对照组显著升高(P<0.05);术后14～22d,模型组大鼠术侧后爪的机械痛和辐射热痛缩爪阈值分别较对照组显著降低(P<0.05)。(2)免疫组化显示,模型组大鼠术侧腰脊髓背角和腹角可见大量GFAP阳性星形胶质细胞(染色加深,胞体增大,突起变粗变长),最明显在背角I-IV层和X层(中央管周围),而对照组术侧腰段脊髓背角仅见少量GFAP阳性星形胶质细胞。模型组大鼠术侧腰脊髓背角GFAP免疫反应光密度值明显高于对照组(P<0.05)。模型组大鼠术侧腰段脊髓背角可见大量OX42阳性的小胶质细胞(表现为分支减少且肥大),而对照组术侧腰段脊髓背角仅见少量OX42阳性的小胶质细胞。模型组大鼠术侧腰脊髓背角OX42免疫反应光密度值明显高于对照组(P<0.05)。
3、骨癌痛大鼠术侧腰段脊髓p38ɑ和p38β的细胞定位及表达
方法:选择雌性SD大鼠20只,随机分为对照组和模型组,每组10只。对照组:左侧胫骨上段骨髓腔注3μl Hank液;模型组:左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl)。术前测定两组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～14d隔日测定术侧后肢机械痛及辐射热痛的缩爪阈值。术后14d测痛结束,灌注固定取大鼠脊髓L4-6节段,采用免疫组织化学ABC法和荧光双标法观察大鼠术侧腰段脊髓背角p38α和p38β的分布和细胞定位。每组剩余7只大鼠断头处死后分离L4-6左右侧脊髓,提取大鼠脊髓的RNA,以RT-PCR技术检测模型组与对照组大鼠p38α和p38β表达的相对差异。
结果:术后第14d,模型组大鼠术侧后爪机械痛和辐射热痛的缩爪阈值较对照组显著降低(P<0.05)。模型组大鼠术侧腰段脊髓背角Ⅰ～Ⅳp38α和p38β的光密度值明显高于对照组(P<0.05)。荧光双标显示,模型组大鼠术侧腰段脊髓背角p38α与神经元特异性核蛋白标志物NeuN有共表达,p38β与小胶质细胞标志物OX-42有共表达。模型组大鼠手术侧p38α、p38β的mRNA表达较非手术侧显著升高(P<0.05),与对照组手术侧比较也明显升高(P<0.05)。
4、鞘内注射p38βMAPK反义寡核苷酸对骨癌痛大鼠痛觉过敏的影响
方法:选择雌性SD大鼠40只,随机分为四组,每组10只。A组(对照组):大鼠左侧胫骨上段骨髓腔注入3μl Hank液;B组(模型组):大鼠左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl);C组(p38β-SODN 20ug处理组)和D组(p38β-ASODN 20ug处理组)大鼠左侧胫骨上段骨髓腔注入3μl MADB-106大鼠乳腺癌细胞(4.8×103/μl),14d后鞘内分别注射p38β-SODN 20ug及p38β-ASODN 20ug,1次/d,连续6d ,而A、B组以同样的方法鞘内注射等量无菌生理盐水作对照。术前测定四组大鼠术侧后爪机械痛及辐射热痛的缩爪阈值,术后1～22d隔日测定术侧后肢机械痛及辐射热痛的缩爪阈值。术后第22d,取大鼠脊髓L4-6节段,采用Western blot法检测脊髓p38β蛋白的表达。
结果:术后14～22d p38β-ASODN 20ug组大鼠术侧后爪机械痛和辐射热痛的缩爪阈值较模型组和p38β-SODN 20ug组显著升高(P<0.05),而与对照组比较差异无统计学意义(P >0.05)。Western Blot结果显示,p38β-ASODN 20ug组p38β蛋白质相对表达量较模型组和p38β-SODN 20ug组显著降低(P<0.05),而与对照组比较,差异无统计学意义(P >0.05)。
5、统计学方法
应用SPSS11.0软件包进行统计分析,计量资料以均数±标准差(x±s)表示。测痛数据采用重复测量数据的方差分析,其余数据采用t检验,P<0.05为差异有统计学意义。
研究结论
1.采用MADB-106大鼠乳腺癌细胞系能成功复制大鼠胫骨癌痛模型。
2.骨癌痛大鼠术侧腰段脊髓背角星形胶质细胞和小胶质细胞均被广泛激活。
3.脊髓p38α和p38β参与了骨癌痛的发生和发展,p38α主要在脊髓神经元表达,而p38β则在脊髓小胶质细胞中表达。
4.鞘内注射p38β反义寡核苷酸能减轻骨癌痛大鼠机械性痛觉超敏及辐射热痛觉过敏,这种作用可能是通过下调脊髓p38β蛋白质的表达来发挥的。
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 Results
1. Establishment and Evaluation of Rat Model of Tibial Cancer Pain
Methods 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 Rats
Methods 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 Rats
Methods 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 Rats
Methods 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).
Conclusions
1. 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.
引文
1.项红兵,安珂,杨辉,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5): 520-522
2. Sabino MAC, Mantyh PW. Clinically related behavioral models: bone cancer pain. Drug Discovery Today: Disease Models, 2004, 1 (2 ): 127-134
3. Petersa CM,Ghilardid JR, Keysera CP, et al .Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain. Exp Neurol 2005;193(1):85–100
4. Sasamura T,Nakamura S,Iida Y, et al . Morphine analgesia suppresses tumor growth and metastasis in a mouse model of cancer pain produced by orthotopic tumor inoculation. Eur J Pharmacol 2002;441(3): 185-191
5. Kehl LJ, Hamamoto DT, Wacnik PW, et al. A cannabinoid agonist differentially attenuates deep tissue hyperalgesia in animal models of cancer and inflammatory muscle pain. Pain 2003;103(1-2):175-186
6.刘凌云,张毅,吴志坚,等.低温对大鼠自然杀伤细胞活性和肿瘤转移的影响.华中科技大学学报(医学版),2003,32(3):263-265
7. Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain 2002;96(1-2):129-140
8. Luger NM., Mach DB, Sevcik MA et al. Bone Cancer Pain: From Model to Mechanism to Therapy. Journal of Pain and Symptom Management, 2005, 29 (5): 32-46
9.张瑛,韩济生,王韵.癌症痛的神经生物学机制研究进展[J].生理科学进展,2004,35(3):224-228
10. Manthyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Nature Rev (Cancer) 2002, 2(3): 201-209
11. Menendez L, Lastra A, Fresno MF,et al. Initial thermal heat hypoalgesia and delayed hyperalgesia in a murine model of bone cancer pain. Brain Res 2003;969(1-2):102-109
1. Schwei MJ, Honore P, Rogers SD, et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci,1999,19:10886- 10897.
2. Medhurst SJ,Walker K,Bowes M,et al. A rat model of bone cancer pain. Pain,2002,96:129-140.
3. Wacnik PW, Eikmeier LJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve : morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2001, 21(23):9355–9366.
4.项红兵,安珂,杨辉,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5):520-522.
5. Makoto Tsuda, Kazuhide Inoue, Michael W. Salter. Neuropathic pain and spinal microglia: a big problem from molecules in‘small’glia.TRENDS in Neurosciences, 2005, 28:101 -107.
6. Vasudeva Raghavendra, Joyce A. DeLeo. The role of astrocytes and microglia in persistent pain. Advances in Molecular and Cell Biology, 2004, 31:951–966.
7. Wieseler-Frank J,Maier SF ,Watkins LR,et al. Glial activation and pathological pain. Neurochemistry International,2004,45:389– 395.
8. Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain, 2005, 118(1-2):125 - 136.
9. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nat Rev Drug Discov, 2003, 2:973- 985.
10.陈敏,项红兵.胶质细胞参与疼痛中枢敏感化的机制.中国临床康复,2005,9(45):127- 129.
1. Tsuda M, Mizokoshi A, Shigemoto-mogami Y, et al. Activation of p38 mitogen- activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia, 2004, 89(1):89–95.
2. Jin SX. p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci, 2003, 23(10):4017–4022.
3. Svensson CI, Fitzsimmons B, Aziz S. Spinal p38βisoform mediates tissue injury- induced hyperalgesia and spinal sensitization. J Neurochem, 2005, 92(6):1508–1520.
4. Medhurst SJ,Walker K,Bowes M,et al. A rat model of bone cancer pain. Pain,2002,96(1-2):129-140.
5. Manthyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Nature Rev (Cancer) 2002;2(3):201-209.
6.项红兵,安珂,杨辉,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5):520-522.
7.董航,田玉科,项红兵.大鼠胫骨癌痛模型的制备及评价.中国临床康复,2006,10(20):77-79.
8. Stone LS, Vulchanova L. The pain of antisense: in vivo application of antisense oligonucleotides for functional genomics in pain and analgesia. Adv Drug Deliv Rev, 2003,55(8):1081–1112.
9. Hua XY, Moore A, Malkmus S, et al. Inhibition of spinal protein kinase C alpha expression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance. Neuroscience,2002,113(1):99–107.
10. Baba H, Kohno T, Moore KA, et al. Direct activation of rat spinal dorsal horn neurons by prostaglandin E2. J Neurosci,2001,21(5):1750–1756.
1. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science, 2000, 288: 1765–1769.
2. Wool CJ. Dissecting out mechanisms responsible for peripheral neuropathic pain: implications for diagnosis and therapy. Life Sci, 2004, 74: 2605–2610.
3. Tsuda M, Mizokoshi A, Shigemoto-mogami Y, et al. Activation of p38 mitogen- activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia, 2004, 89: 89–95.
4. Tsuda1 M, Inoue1 K, Salter MW. Neuropathic pain and spinal microglia: a big problem from molecules in‘small’glia. Trends in Neurosciences, 2005, 28 (2):101- 107.
5. Hamish UK. Microglia as a source and target of cytokines. Glia, 2002, 40: 140–155.
6. Decosterd I and Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87: 149–158.
7. Tanga FY. Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochem Int, 2004, 45: 397–407.
8. Raghavendra V. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J. Pharmacol. Exp. Ther, 2003, 306: 624–630.
9. Sweitzer SM. The differential role of spinal MHC class II and cellular adhesion molecules in peripheral inflammatory versus neuropathic pain in rodents. J. Neuroimmunol, 2002, 125: 82–93.
10. Sweitzer SM, DeLeo JA. The active metabolite of leflunomide, an immunosuppressive agent, reduces mechanical sensitivity in a rat mononeuropathy model. J. Pain, 2002, 3: 360–368.
11. Jin SX. p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J. Neurosci, 2003, 23: 4017–4022.
12. Svensson CI, Fitzsimmons B, Aziz S.Spinal p38βisoform mediates tissue injury- induced hyperalgesia and spinal sensitization. Journal of Neurochemistry, 2005, 92: 1508– 1520.
13. Inoue K, Schuichi KC. Signaling of ATP receptors in glia-neuron interaction and pain. Life Sciences 2003,74:189–197.
14. Tsuda M, Shigemoto-Mogami Y. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature, 2003,424:778-783.
15. Inoue K. Microglial activation by purines and pyrimidines. Glia, 2002, 40(2):156-63.
16. Chaban VV, Mayer EA. Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons. Neuroscience,2003, 118: 941-948.
17. Eric Boue-Grabot, Vincent Archambault. A protein kinase C site highly conserved in P2X subunits controls the desensitization Kinetics of P2X2 ATP-gated Channels. The Journal of Biological Chemistry,2000,275: 10190–10195.
18. Xiao-Ying H, Ping C. Inhibition of spinal protein kinase C reduces nerve injury-induced tactile allodynia in neuropathic rats. Neuroscience Letters, 1999,276: 99-102.
19. Visentin S. Two different ionotropic receptors areactivated by ATP in rat microglia. J Physiol,1999,519: 723–736.
20. Ambrosini E,Aloisi F. Chemokines and glial cells: a complex network in the central nervous system. Neurochem Res, 2004, 29: 1017–1038.
21. Sweitzer SM. Focal peripheral nerve injury induces leukocyte trafficking into the central nervous system: potential relationship to neuropathic pain. Pain,2002,100: 163–170.
22. Kerschensteiner M. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med,1999,189: 865–870.
23. Moalem G. Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J Autoimmun,2000,15:331–345.
24. Aldskogius H, Kozlova EN. Central neuron–glial and glial–glial interactions following axon injury. Prog Neurobiol, 1998, 55:1–26.
25. Watkins LR, Maier SF. Targeting glia to control clinical pain:an idea whose time has come. Drug discovery today,2004,1: 83-89.
1. Vermeirsch H, Nuydens RM, Salmon PL, et al TF. Bone cancer pain model in mice: evaluation of pain behavior, bone destruction and morphine sensitivity. Pharmacol Biochem Behav. 2004, 79(2):243-251.
2.项红兵,杨辉,安珂,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5):520-522.
3. Mach DB, Rogers SD, Sabino MC, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience , 2002,113(1):155-166.
4.张瑛,韩济生,王韵.癌症痛的神经生物学机制研究进展.生理科学进展,2004,35(3):224-228.
5. Wacnik PW, Eikmeier LJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2001, 21(23): 9355–9366.
6. Sevcik MA, Luger NM, Mach DB, et al. Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain. 2004 ,111 (1-2) :169-180.
7. Schwei MJ, Honore P, Rogers SD, et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci, 1999, 19: 10886–10897.
8. Wacnik PW,Kehl LJ,Trempe TM, et al. Tumor implantation in mouse humerus evokes movement2related hyperalgesia exceeding that evoked by intramuscular carrageenan. Pain,2003,101:175～186.
9. Sarlak A, Gundes H, Ozkurkcugil C, et al. Solitary calcaneal metastasis in superficial bladder carcinoma. Int J Clin Pract,2000,54:681–682.
10. Medhurst SJ,Walker K,Bowes M,et al. A rat model of bone cancer pain. Pain,2002,96:129-140.
11.董航,田玉科,项红兵.大鼠胫骨癌痛模型的制备及评价.中国临床康复,2006,10(20):77-79.
12. Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain, 2005,123:1–12.
13. Schwei MJ, Honore P, Rogers SD,et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bonecancer pain. J Neurosci, 1999, 19: 10886–10897.
14. Urch CE, Donovan-Rodriguez T, Dickenson AH. Alterations in dorsal horn neurones in a rat model of cancer-induced bone pain. Pain,2003,106(3):347-356.
15. Sevcik MA, Ghilardi JR, Halvorson KG,et al. Analgesic efficacy of bradykinin B1 antagonists in a murine bone cancer pain model. J Pain. 2005,6(11):771-775.
16. Halvorson KG, Kubota K, Sevcik MA, et al. A blocking antibody to nerve growth factor attenuates skeletal pain induced by prostate tumor cells growing in bone. Cancer Res. 2005,65(20):9426-9435.
17. El Mouedden M, Meert TF. Evaluation of pain-related behavior, bone destruction and effectiveness of fentanyl, sufentanil, and morphine in a murine model of cancer pain. Pharmacol Biochem Behav. 2005,82(1):109-119.
18. Saito O, Aoe T, Yamamoto T. Analgesic effects of nonsteroidal antiinflammatory drugs, acetaminophen, and morphine in a mouse model of bone cancer pain. J Anesth,2005,19(3): 218-224.
19. Lee BH, Seong J, Kim UJ, et al. Behavioral characteristics of a mouse model of cancer pain. Yonsei Med J. 2005,46 (2):252-259.
20. Ghilardi JR, Rohrich H, Lindsay TH, et al. Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci. 2005,25(12):3126-3131.
21. Vit JP, Ohara PT, Tien DA, et al. The analgesic effect of low dose focal irradiation ina mouse model of bone cancer is associated with spinal changes in neuro-mediators of nociception. Pain. 2006,120(1-2):188-201.
22. Sevcik MA, Luger NM, Mach DB, et al. Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain. 2004 ,111 (1-2) :169-180.
23. Honore P, Mantyh PW. Bone cancer pain: from mechanism to model to therapy. Pain Med. 2000,1 (4):303-309.
24. Baamonde A, Lastra A, Juarez L, et al. Effects of the local administration of selective mu-, delta-and kappa-opioid receptor agonists on osteosarcoma-induced hyperalgesia. Naunyn Schmiedebergs Arch Pharmacol. 2005,372(3):213-219.
25. Donovan-Rodriguez T, Urch CE, Dickenson AH. Evidence of a role for descending serotonergic facilitation in a rat model of cancer-induced bone pain. Neurosci Lett. 2006,393(2-3):237-242.
26. Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain,2005,118(1-2):125-136.
27. Donovan-Rodriguez T, Dickenson AH, Urch CE. Gabapentin normalizes spinal neuronal responses that correlate with behavior in a rat model of cancer-induced bone pain. Anesthesiology. 2005,102(1):132-140.
28. Fox A, Medhurst S, Courade JP, et al. Anti-hyperalgesic activity of the cox-2 inhibitor lumiracoxib in a model of bone cancer pain in the rat. Pain,2004,107(1-2):33-40.
29. Urch CE, Donovan-Rodriguez T, Dickenson AH. Alterations in dorsal horn neurones in a rat model of cancer-induced bone pain. Pain,2003,106(3):347-356.
30. Weber KL, Doucet M, Price JE. Renal cell carcinoma bone metastasis: epidermal growth factor receptor targeting. Clin Orthop Relat Res. 2003,(415 Suppl):S86-94.
31.张伟倍.动物模型与实验外科.中华实验外科杂志,2003,20:293-295.
1. Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain 2002;96(1-2):129-140
2. Kjonniksen I, Winderen M, Bruland O, et al. Validity and usefulness of human tumor models established by intratibial cell inoculation in nude rats. Cancer Res,1994,54:1715
3. Schwei MJ, Honore P, Rogers SD,et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bonecancer pain. J Neurosci, 1999, 19: 10886–10897.
4. Wacnik PW, Eikmeier LJ, Ruggles T R, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2001, 21: 9355–9366
5. Sarlak A, Gundes H, Ozkurkcugil C, et al. Solitary calcaneal metastasis in superficial bladder carcinoma. Int J Clin Pract,2000,54:681–682.
6. O’Connell JX, Nanthakumar SS, Nielsen GP, et al. Osteoid osteoma:the uniquely innervated bone tumor. Modern Pathol ,1998,11:175–180.
7. Olson TH, Rield MS, Vulchanova XR, et al. An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats. NeuroReport ,1998,9:1109–1113.
8. Watkins LR, Wiertelak EP, Goehler LE, et al. Characterization of cytokine- induced hyperalgesia. Brain Res,1994,654:15–26.
9. Webb DJ. Endothelin: from molecule to man. Br J Clin Pharmacol,1997,44:9–20.
10. Davar G, Hans G, Fareed MU, et al. Behavioral signs of acute pain produced by application of endothelin-1 to rat sciatic nerve. NeuroReport,1998,9:2279–2283.
11. Pomonis JD, Rogers SD, Peters CM, et al. Expression and localization of endothelinreceptors: implications for the involvement of peripheral glia in nociception. J Neurosci,2001,21: 999– 1006.
12. Lehre KP, Danbolt NC. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci,1998,18:8751– 8757.
13. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nature reviews, 2003, 2: 974.
14. Honore P, Rogers SD, Schwei MJ, et al. Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience,2000,98:585–598
15. Colburn RW, DeLeo JA, Rickman AJ, et al. Dissociation of microglial activation and neuropathic pain behavior following peripheral nerve injury in the rat. J Neuroimmunol,1997,79:163–175.
16. Petrenko AB, Yamakura T, Baba H, et al. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg. 2003, 97: 1108-1116
2. Sabino MAC, Mantyh PW. Clinically related behavioral models: bone cancer pain. Drug Discovery Today: Disease Models, 2004, 1 (2 ): 127-134
3. Petersa CM,Ghilardid JR, Keysera CP, et al .Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain. Exp Neurol 2005;193(1):85–100
4. Sasamura T,Nakamura S,Iida Y, et al . Morphine analgesia suppresses tumor growth and metastasis in a mouse model of cancer pain produced by orthotopic tumor inoculation. Eur J Pharmacol 2002;441(3): 185-191
5. Kehl LJ, Hamamoto DT, Wacnik PW, et al. A cannabinoid agonist differentially attenuates deep tissue hyperalgesia in animal models of cancer and inflammatory muscle pain. Pain 2003;103(1-2):175-186
6.刘凌云,张毅,吴志坚,等.低温对大鼠自然杀伤细胞活性和肿瘤转移的影响.华中科技大学学报(医学版),2003,32(3):263-265
7. Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain 2002;96(1-2):129-140
8. Luger NM., Mach DB, Sevcik MA et al. Bone Cancer Pain: From Model to Mechanism to Therapy. Journal of Pain and Symptom Management, 2005, 29 (5): 32-46
9.张瑛,韩济生,王韵.癌症痛的神经生物学机制研究进展[J].生理科学进展,2004,35(3):224-228
10. Manthyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Nature Rev (Cancer) 2002, 2(3): 201-209
11. Menendez L, Lastra A, Fresno MF,et al. Initial thermal heat hypoalgesia and delayed hyperalgesia in a murine model of bone cancer pain. Brain Res 2003;969(1-2):102-109
1. Schwei MJ, Honore P, Rogers SD, et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci,1999,19:10886- 10897.
2. Medhurst SJ,Walker K,Bowes M,et al. A rat model of bone cancer pain. Pain,2002,96:129-140.
3. Wacnik PW, Eikmeier LJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve : morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2001, 21(23):9355–9366.
4.项红兵,安珂,杨辉,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5):520-522.
5. Makoto Tsuda, Kazuhide Inoue, Michael W. Salter. Neuropathic pain and spinal microglia: a big problem from molecules in‘small’glia.TRENDS in Neurosciences, 2005, 28:101 -107.
6. Vasudeva Raghavendra, Joyce A. DeLeo. The role of astrocytes and microglia in persistent pain. Advances in Molecular and Cell Biology, 2004, 31:951–966.
7. Wieseler-Frank J,Maier SF ,Watkins LR,et al. Glial activation and pathological pain. Neurochemistry International,2004,45:389– 395.
8. Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain, 2005, 118(1-2):125 - 136.
9. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nat Rev Drug Discov, 2003, 2:973- 985.
10.陈敏,项红兵.胶质细胞参与疼痛中枢敏感化的机制.中国临床康复,2005,9(45):127- 129.
1. Tsuda M, Mizokoshi A, Shigemoto-mogami Y, et al. Activation of p38 mitogen- activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia, 2004, 89(1):89–95.
2. Jin SX. p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci, 2003, 23(10):4017–4022.
3. Svensson CI, Fitzsimmons B, Aziz S. Spinal p38βisoform mediates tissue injury- induced hyperalgesia and spinal sensitization. J Neurochem, 2005, 92(6):1508–1520.
4. Medhurst SJ,Walker K,Bowes M,et al. A rat model of bone cancer pain. Pain,2002,96(1-2):129-140.
5. Manthyh PW, Clohisy DR, Koltzenburg M, et al. Molecular mechanisms of cancer pain. Nature Rev (Cancer) 2002;2(3):201-209.
6.项红兵,安珂,杨辉,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5):520-522.
7.董航,田玉科,项红兵.大鼠胫骨癌痛模型的制备及评价.中国临床康复,2006,10(20):77-79.
8. Stone LS, Vulchanova L. The pain of antisense: in vivo application of antisense oligonucleotides for functional genomics in pain and analgesia. Adv Drug Deliv Rev, 2003,55(8):1081–1112.
9. Hua XY, Moore A, Malkmus S, et al. Inhibition of spinal protein kinase C alpha expression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance. Neuroscience,2002,113(1):99–107.
10. Baba H, Kohno T, Moore KA, et al. Direct activation of rat spinal dorsal horn neurons by prostaglandin E2. J Neurosci,2001,21(5):1750–1756.
1. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science, 2000, 288: 1765–1769.
2. Wool CJ. Dissecting out mechanisms responsible for peripheral neuropathic pain: implications for diagnosis and therapy. Life Sci, 2004, 74: 2605–2610.
3. Tsuda M, Mizokoshi A, Shigemoto-mogami Y, et al. Activation of p38 mitogen- activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia, 2004, 89: 89–95.
4. Tsuda1 M, Inoue1 K, Salter MW. Neuropathic pain and spinal microglia: a big problem from molecules in‘small’glia. Trends in Neurosciences, 2005, 28 (2):101- 107.
5. Hamish UK. Microglia as a source and target of cytokines. Glia, 2002, 40: 140–155.
6. Decosterd I and Woolf CJ. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain, 2000, 87: 149–158.
7. Tanga FY. Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochem Int, 2004, 45: 397–407.
8. Raghavendra V. Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J. Pharmacol. Exp. Ther, 2003, 306: 624–630.
9. Sweitzer SM. The differential role of spinal MHC class II and cellular adhesion molecules in peripheral inflammatory versus neuropathic pain in rodents. J. Neuroimmunol, 2002, 125: 82–93.
10. Sweitzer SM, DeLeo JA. The active metabolite of leflunomide, an immunosuppressive agent, reduces mechanical sensitivity in a rat mononeuropathy model. J. Pain, 2002, 3: 360–368.
11. Jin SX. p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J. Neurosci, 2003, 23: 4017–4022.
12. Svensson CI, Fitzsimmons B, Aziz S.Spinal p38βisoform mediates tissue injury- induced hyperalgesia and spinal sensitization. Journal of Neurochemistry, 2005, 92: 1508– 1520.
13. Inoue K, Schuichi KC. Signaling of ATP receptors in glia-neuron interaction and pain. Life Sciences 2003,74:189–197.
14. Tsuda M, Shigemoto-Mogami Y. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature, 2003,424:778-783.
15. Inoue K. Microglial activation by purines and pyrimidines. Glia, 2002, 40(2):156-63.
16. Chaban VV, Mayer EA. Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons. Neuroscience,2003, 118: 941-948.
17. Eric Boue-Grabot, Vincent Archambault. A protein kinase C site highly conserved in P2X subunits controls the desensitization Kinetics of P2X2 ATP-gated Channels. The Journal of Biological Chemistry,2000,275: 10190–10195.
18. Xiao-Ying H, Ping C. Inhibition of spinal protein kinase C reduces nerve injury-induced tactile allodynia in neuropathic rats. Neuroscience Letters, 1999,276: 99-102.
19. Visentin S. Two different ionotropic receptors areactivated by ATP in rat microglia. J Physiol,1999,519: 723–736.
20. Ambrosini E,Aloisi F. Chemokines and glial cells: a complex network in the central nervous system. Neurochem Res, 2004, 29: 1017–1038.
21. Sweitzer SM. Focal peripheral nerve injury induces leukocyte trafficking into the central nervous system: potential relationship to neuropathic pain. Pain,2002,100: 163–170.
22. Kerschensteiner M. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med,1999,189: 865–870.
23. Moalem G. Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity. J Autoimmun,2000,15:331–345.
24. Aldskogius H, Kozlova EN. Central neuron–glial and glial–glial interactions following axon injury. Prog Neurobiol, 1998, 55:1–26.
25. Watkins LR, Maier SF. Targeting glia to control clinical pain:an idea whose time has come. Drug discovery today,2004,1: 83-89.
1. Vermeirsch H, Nuydens RM, Salmon PL, et al TF. Bone cancer pain model in mice: evaluation of pain behavior, bone destruction and morphine sensitivity. Pharmacol Biochem Behav. 2004, 79(2):243-251.
2.项红兵,杨辉,安珂,等.骨癌痛模型和相关疼痛病理机制研究进展.临床麻醉学杂志,2005,21(5):520-522.
3. Mach DB, Rogers SD, Sabino MC, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience , 2002,113(1):155-166.
4.张瑛,韩济生,王韵.癌症痛的神经生物学机制研究进展.生理科学进展,2004,35(3):224-228.
5. Wacnik PW, Eikmeier LJ, Ruggles TR, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2001, 21(23): 9355–9366.
6. Sevcik MA, Luger NM, Mach DB, et al. Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain. 2004 ,111 (1-2) :169-180.
7. Schwei MJ, Honore P, Rogers SD, et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci, 1999, 19: 10886–10897.
8. Wacnik PW,Kehl LJ,Trempe TM, et al. Tumor implantation in mouse humerus evokes movement2related hyperalgesia exceeding that evoked by intramuscular carrageenan. Pain,2003,101:175～186.
9. Sarlak A, Gundes H, Ozkurkcugil C, et al. Solitary calcaneal metastasis in superficial bladder carcinoma. Int J Clin Pract,2000,54:681–682.
10. Medhurst SJ,Walker K,Bowes M,et al. A rat model of bone cancer pain. Pain,2002,96:129-140.
11.董航,田玉科,项红兵.大鼠胫骨癌痛模型的制备及评价.中国临床康复,2006,10(20):77-79.
12. Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain, 2005,123:1–12.
13. Schwei MJ, Honore P, Rogers SD,et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bonecancer pain. J Neurosci, 1999, 19: 10886–10897.
14. Urch CE, Donovan-Rodriguez T, Dickenson AH. Alterations in dorsal horn neurones in a rat model of cancer-induced bone pain. Pain,2003,106(3):347-356.
15. Sevcik MA, Ghilardi JR, Halvorson KG,et al. Analgesic efficacy of bradykinin B1 antagonists in a murine bone cancer pain model. J Pain. 2005,6(11):771-775.
16. Halvorson KG, Kubota K, Sevcik MA, et al. A blocking antibody to nerve growth factor attenuates skeletal pain induced by prostate tumor cells growing in bone. Cancer Res. 2005,65(20):9426-9435.
17. El Mouedden M, Meert TF. Evaluation of pain-related behavior, bone destruction and effectiveness of fentanyl, sufentanil, and morphine in a murine model of cancer pain. Pharmacol Biochem Behav. 2005,82(1):109-119.
18. Saito O, Aoe T, Yamamoto T. Analgesic effects of nonsteroidal antiinflammatory drugs, acetaminophen, and morphine in a mouse model of bone cancer pain. J Anesth,2005,19(3): 218-224.
19. Lee BH, Seong J, Kim UJ, et al. Behavioral characteristics of a mouse model of cancer pain. Yonsei Med J. 2005,46 (2):252-259.
20. Ghilardi JR, Rohrich H, Lindsay TH, et al. Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci. 2005,25(12):3126-3131.
21. Vit JP, Ohara PT, Tien DA, et al. The analgesic effect of low dose focal irradiation ina mouse model of bone cancer is associated with spinal changes in neuro-mediators of nociception. Pain. 2006,120(1-2):188-201.
22. Sevcik MA, Luger NM, Mach DB, et al. Bone cancer pain: the effects of the bisphosphonate alendronate on pain, skeletal remodeling, tumor growth and tumor necrosis. Pain. 2004 ,111 (1-2) :169-180.
23. Honore P, Mantyh PW. Bone cancer pain: from mechanism to model to therapy. Pain Med. 2000,1 (4):303-309.
24. Baamonde A, Lastra A, Juarez L, et al. Effects of the local administration of selective mu-, delta-and kappa-opioid receptor agonists on osteosarcoma-induced hyperalgesia. Naunyn Schmiedebergs Arch Pharmacol. 2005,372(3):213-219.
25. Donovan-Rodriguez T, Urch CE, Dickenson AH. Evidence of a role for descending serotonergic facilitation in a rat model of cancer-induced bone pain. Neurosci Lett. 2006,393(2-3):237-242.
26. Zhang RX, Liu B, Wang L, et al. Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia. Pain,2005,118(1-2):125-136.
27. Donovan-Rodriguez T, Dickenson AH, Urch CE. Gabapentin normalizes spinal neuronal responses that correlate with behavior in a rat model of cancer-induced bone pain. Anesthesiology. 2005,102(1):132-140.
28. Fox A, Medhurst S, Courade JP, et al. Anti-hyperalgesic activity of the cox-2 inhibitor lumiracoxib in a model of bone cancer pain in the rat. Pain,2004,107(1-2):33-40.
29. Urch CE, Donovan-Rodriguez T, Dickenson AH. Alterations in dorsal horn neurones in a rat model of cancer-induced bone pain. Pain,2003,106(3):347-356.
30. Weber KL, Doucet M, Price JE. Renal cell carcinoma bone metastasis: epidermal growth factor receptor targeting. Clin Orthop Relat Res. 2003,(415 Suppl):S86-94.
31.张伟倍.动物模型与实验外科.中华实验外科杂志,2003,20:293-295.
1. Medhurst SJ, Walker K, Bowes M, et al. A rat model of bone cancer pain. Pain 2002;96(1-2):129-140
2. Kjonniksen I, Winderen M, Bruland O, et al. Validity and usefulness of human tumor models established by intratibial cell inoculation in nude rats. Cancer Res,1994,54:1715
3. Schwei MJ, Honore P, Rogers SD,et al. Neurochemical and cellular reorganization of the spinal cord in a murine model of bonecancer pain. J Neurosci, 1999, 19: 10886–10897.
4. Wacnik PW, Eikmeier LJ, Ruggles T R, et al. Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain. J Neurosci, 2001, 21: 9355–9366
5. Sarlak A, Gundes H, Ozkurkcugil C, et al. Solitary calcaneal metastasis in superficial bladder carcinoma. Int J Clin Pract,2000,54:681–682.
6. O’Connell JX, Nanthakumar SS, Nielsen GP, et al. Osteoid osteoma:the uniquely innervated bone tumor. Modern Pathol ,1998,11:175–180.
7. Olson TH, Rield MS, Vulchanova XR, et al. An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats. NeuroReport ,1998,9:1109–1113.
8. Watkins LR, Wiertelak EP, Goehler LE, et al. Characterization of cytokine- induced hyperalgesia. Brain Res,1994,654:15–26.
9. Webb DJ. Endothelin: from molecule to man. Br J Clin Pharmacol,1997,44:9–20.
10. Davar G, Hans G, Fareed MU, et al. Behavioral signs of acute pain produced by application of endothelin-1 to rat sciatic nerve. NeuroReport,1998,9:2279–2283.
11. Pomonis JD, Rogers SD, Peters CM, et al. Expression and localization of endothelinreceptors: implications for the involvement of peripheral glia in nociception. J Neurosci,2001,21: 999– 1006.
12. Lehre KP, Danbolt NC. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci,1998,18:8751– 8757.
13. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nature reviews, 2003, 2: 974.
14. Honore P, Rogers SD, Schwei MJ, et al. Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience,2000,98:585–598
15. Colburn RW, DeLeo JA, Rickman AJ, et al. Dissociation of microglial activation and neuropathic pain behavior following peripheral nerve injury in the rat. J Neuroimmunol,1997,79:163–175.
16. Petrenko AB, Yamakura T, Baba H, et al. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg. 2003, 97: 1108-1116