| Cancer-induced bone pain(CIBP)is a common problem of cancer and treatments for CIBP are less effective with side effects. Involvement of spinal glial activation in cancer-induced bone pain was proved. Stimulation from tumour cells may induce the alteration of pain-related genes in spinal glial cells. An abnormal ubiquitin-proteasome system (UPS) may be one of the transcription factor-mediated epigenetic mechanisms underlying the nervous system diseases in the glial cells. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme which maintains the levels of mono-ubiquitin. In previous studies,UCHL1 invovled in neural and tumour tissues and played important roles in the nervous system diseases. Spinal UCHL1 and its mRNA levels were robustly increased in different rat models of chronic pain, which support the hypothesis that UPS is involved in nociceptive processing. Although the spinal UCHL1 involved in chronic pain modulation, the precise mechanisms remains unclear.Previoulsy, the UPS in the chronic pain diseases was mainly focused on neurons. While pain modulation of UPS in glial cells was still unknown. Glial cells secreted different kinds of cytokine and chemokine after noxious stimulation in the chronic pain models. Therefore, glial cells may interplay with disease-associated proteins and the UPS which may affect glial cells homeostasis and neuronal functions. Therefore, the changes of UPS (involving ubiquitin-ligases or ubiquitin C-terminal hydrolase in neurons or glial cells) in the nervous system diseases may be new therapeutic targets for CIBP.Presently, it is speculated that spinal UCHL1 increased in the CIBP rats, and inhibition of UCHL1 exhibits antinociceptive effects in a CIBP model. Thus, we asked the following questions:1) Does UCHL1 or ubiquitin co-localize with spinal glial cells and neurons in CIBP rats? 2) Is the glial cells activation and expression of spinal UCHL1/ubiquitin affected by CIBP? 3) How does the ketamine or inhibition of UCHL1 affect pain facilitation, UCHL1/ubiquitin and glial cells activation?Part 1:The pain hyperalgesia and glial activation change in a cancer-induced bone pain modelObjective To investigate the changes of glial activation and pain hyperalgesia in the spine of CIBP rats.Methods Female SD rats 160-180g were divided into the groups:naive group, sham group, CIBP group. The CIBP group then was divided into the following groups:1,3,5,7,9,11,14,21 days groups. The walker 256 rat mammary gland carcinoma cells were implanted into the right medullary cavity of the tibia in female Sprague Dawley rats (160-180 g) (tumour cell implantation, TCI). Equivolume heat killed cells were injected into sham animals. The 50% paw withdrawal mechanical threshold (50%PWMT) and Score of Ambulantory pain were observed on the 1,3, 5,7,9,11,14,21 days after tumour cell implantation. Furthermore, we observed that whether minocycline or fluorocitrate attenuate the established hypersensitivity in a CIBP model. The rats were sacrificed on postoperative day 14 after behaviors tested, and the spinal cord around lumbar enlargement were taken for detecting glial activation by immunofluorescence staining.ResultsBehavioral tests:In the sham group, the heat killed cells did not affect the pain thresholds (P>0.05). Compared to sham group, walker 256 rat mammary gland carcinoma cells decreased the pain threshold (the increase of Score of Ambulantory pain, and decrease of 50% PWMT) (P<0.05). The intrathecal injection of the minocycline (microglial inhibitor,5 μg/μl) or fluorocitrate (astrocyte inhibitor,1/3 nmol) alleviated the mechanical sensitivity in CIBP rats (P<0.05)The immunofluorescence staining:The average optical density mean optical density (MOD) value of Iba-1 (microglial cell specific marker) or GFAP (glial cell specific marker) increased significantly after tumor cells inoculation (P< 0.05). The numbers of glial cells increased and corpuscular volume of microglial and astrocytic cells became larger indicating activation of these cells. The microglia and astrocytes are mainly distributed in the II-IV layers of the spinal cord of CIBP rats.Conclusions(1) The walker 256 rat mammary gland carcinoma cells leaded to down-regulation of the pain thresholds suggesting that we successfully established the bone cancer pain model.(2) The spinal microglia and astrocytes were activated following inoculation. The intrathecal injection of the glial inhibitors attenuated the mechanical sensitivity in CIBP rats. The results above suggested that glial activation medicated the development of cancer-induced bone pain.Part 2:The expression and distribution of spinal UCHL1/ubiquitin in a cancer-induced bone pain modelObjective To investigate the changes of UCHL1/ubiquitin expression and distribution in the spine of CIBP rats.Methods Female SD rats 160-180g were divided into the groups:naive group, sham group, CIBP group. The walker 256 rat mammary gland carcinoma cells were implanted into the right medullary cavity of the tibia in female Sprague Dawley rats. Equivolume heat killed cells were injected into sham animals. The 50% PWMT and Score of Ambulantory pain were observed on the 1,3,7,14,21 days after tumour cell implantation. The rats were sacrificed after behavioral tests, and the spinal cord around lumbar enlargement were taken for detecting UCHLl/ubiquitin changes and distributions by western blotting analysis and double immunofluorescence analyses.ResultsBehavioral tests:In the sham group, the heat killed cells did not affect the pain thresholds (P>0.05). Compared to sham group, walker 256 rat mammary gland carcinoma cells decreased the pain threshold (the increase of Score of Ambulantory pain and decrease of 50%PWMT (P<0.05)Western blotting analysis:The intensities of specic UCHL1 and ubiquitin bands increased following tumour cell implantation (P<0.05). The timecourse of the increased UCHL1 and ubiquitin expression closely corresponded with the development of pain facilitation after implantation.Immunofluorescence analysis:Ubiquitin-IR cell co-localized NeuN, Iba-1 or GFAP. UCHL1-IR cells co-expressed NeuN, but not co-localized with Iba-1 or GFAP in the L4-5 spine of CIBP rats. Double immunofluorescence demonstrated that UCHL1 co-expressing with ubiquitin in the L4-5 spine of CIBP rats.Conclusions(1) Spinal UCHL1 and ubiquitin expression increased in the CIBP model.(2) UCHL1 predominantly distributed in neurons, while ubiquitin existed in neurons, astrocytes and microglia in the CIBP rats.Part 3 Inhibition of UCHL1 alleviated cancer-induced bone pain by inhibiting spinal ubiquitin and glial activition.Objective To investigate whether inhibition of spinal UCHL1 affects pain facilitation, glial activation and UCHL1/ubiquitin expression in a cancer-induced bone pain model.Methods The CIBP model was established and pain facilitation was obviously on the 14 days following tumour cell implantation. LDN57444 or DMSO was injected intrathecally on each of 3 consecutive days from the 14th to the 16th day after implantation. The rats were sacrificed after behavioral tests, and the spinal cord around lumbar enlargement were taken for detecting UCHL1/ubiquitin changes by western blotting analysis. The rats were sacrificed after behaviors tested, and the spinal cord around lumbar enlargement were taken for detecting glial activation by immunofluorescence staining.Behavioral tests:In the sham group, the heat killed cells did not affect the pain thresholds (P>0.05). Compared to sham group, walker 256 rat mammary gland carcinoma cells decreased the pain threshold (P< 0.05). LDN57444 increased the pain thresholds in CIBP rats (P<0.05)Western blotting analysis:Repeated injection of UCHL1 inhibitor decreased ubiquitin expression in contrast to CIBP/DMSO rats (P<0.05). LDN57444 did not affect spinal UCHL1 expression in CIBP rats (P>0.05)Immunofluorescence analysis:The mean density of Iba-1 or GFAP was obviously increased in the CIBP animals in contrast to that of control animals (P<0.05) Pre-injection of DMSO did not affect the increased densities of Iba-1 or GFAP (P>0.05). However, injection of LDN57444 attenuated the upregulation of Iba-1 or GFAP densities following the tumor cells inoculation, indicating inhibition of spinal glial cells (P<0.05).Conclusions(1) UCHL1/ubiquitin distributed in the spines of CIBP rats, that spinal UCHL1, ubiquitin and glial activation increased in the CIBP model. (2) Inhibition of spinal UCHL1 may be an effective measure for alleviation of cancer-induced bone pain, which might react by down-regulation of ubiquitin expression and glial activation.Part 4 Ketamine alleviated cancer-induced bone pain by inhibiting spinal glial activition* but not by affecting the UCHL1/ubiquitin expression.Objective To investigate whether ketamine alleviated cancer-induced bone pain by inhibiting spinal UCHL1/ubiquitin and glial activition.Methods The CIBP model was established and pain facilitation was obviously on the 14 days following tumour cell implantation. Ketamine or saline was injected intrathecally. The Score of Ambulantory pain and 50% PWMT were observed on predrug,30,60,90,120,150,180min. The rats were sacrificed after behavioral tests, and the spinal cord around lumbar enlargement were taken for detecting UCHL1/ubiquitin changes by western blotting analysis. The rats were sacrificed after behaviors tested,and the spinal cord around lumbar enlargement were taken for detecting glial activation by immunofluorescence staining.Behavioral tests:In the sham group, the heat killed cells did not affect the pain thresholds (P>0.05). Compared to sham group, walker 256 rat mammary gland carcinoma cells decreased the pain threshold (P<0.05). Ketamine increased the pain thresholds in CIBP rats (P<0.05)Western blotting analysis:Repeated injection of ketamine did not affect UCHL1/ubiquitin expression in contrast to CIBP/saline rats (P<0.05)Immunofluorescence analysis:The mean density of Iba-1 or GFAP was obviously increased in the CIBP animals in contrast to that of control animals (P<0.05) Pre-injection of saline did not affect the increased densities of Iba-1 or GFAP (P>0.05). Ketamine (60,120 ug/d) attenuated the up-regulation of Iba-1 or GFAP densities following the tumor cells inoculation, indicating inhibition of spinal glial cells (P<0.05)Conclusions(1) UCHL1/ubiquitin distributed in the spines of CIBP rats, that spinal UCHL1, ubiquitin and glial activation increased in the CIBP model.(2) Ketamine may be an effective measure for alleviation of cancer-induced bone pain, which might inhibit glial activation, but not affect spinal UCHL1/ubiquitin. |
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