| BackgroundCancer may show a variety of physiological and psychological symptoms, but the pain caused by cancer is one of the key symptoms. Untreated pain can seriously affect the patients' quality of life, and can influence the prognosis of cancer patients through affecting their tolerance to the treatments. With the technical progress of cancer treatment, cancer patients' survival time was significantly prolonged; to ease the pain of cancer patients and improve their quality of life becomes a prominent problem. It is estimated that cancer pain prevalence is 25% in newly diagnosed cancer patients and 33% in cancer patients with active treatment. In patients with advanced cancer, the prevalence of cancer pain is over 75%. Due to the limitations of existing clinical treatments of cancer pain, about 45% of patients with cancer pain have not been effectively treated. Thus, a comprehensive in-depth study of cancer pain mechanisms and analgesia means are of great importance.Cancer induced bone pain (CIBP) is the most common type of pain in cancer patients and one of the most common symptoms caused by tumor metastasis to bone. About one-third of bone metastasis happened in patients with advanced, while more than 70% bone metastasis occurred in patients with advanced breast and prostate cancer. Bone metastasis is the most common cause of cancer pain. CIBP is a typical representative of cancer pain, its main manifestations are:ongoing pain, breakthrough pain, hyperalgesia and allodynia. Currently, patients are still lack of effective cancer pain treatments without adverse effects; inadequately-treated pain not only seriously affects the patients' quality of life but also significantly shortens their survival period. This is because of lack of understanding of the pathogenesis of CIBP. The mechanism of CIBP is complex. For example, various pro-hyperalgesic mediators released by local-infiltration tumor cells, inflammatory cells, osteoclasts and osteoblasts induce sensitization of peripheral and central nervous systems and jointly participate in the development of CIBP. And different types, different stages of tumors and different tumor growth sites can affect the development of CIBP.Recent studies have shown that activation of descending facilitation in the central nervous system play an important role in the maintenance of chronic pain. The rostral ventromedial medulla (RVM) is a key component of descending pain modulation system. RVM is mainly consistent of nucleus raphe magnus, nucleus reticularis gigantocellularis pars alpha and ventral nucleus reticularis gigantocellularis. RVM receives and integrates pain modulation signals from supraspinal areas and relays to the spinal cord. About 50% of neurons within RVM are tryptophan hydroxylase (TPH) containing serotonergic neurons, which can synthsize 5-hydroxytryptamine (5-HT) and project it to the spinal dorsal horn. 5-HT is an important neurotransmitter involved in the influence of descending facilitation system on spinal cord pain processing. Descending 5-HT from RVM plays an important role in spinal cord pain processing. Besides, it was reported that RVM glial activation played an important role in the development of chronic pain. However, the specific role of descending 5-HT from the RVM projected to spinal dorsal horn on spinal cord pain processing and whether RVM glial cells involved in descending pain modulation are still unclear in CIBP. Cancer pain treatments targeted RVM are free from the restrictions of tumor growth and metastatic sites and can effectively control multiple neuropathic pain caused by cancer treatments. Based on mentioned above, this study aimed to explore the role of descending serotonergic pathway projected from RVM to spinal cord and RVM glial cells in a rat model of cancer induced bone pain and find new therapeutic targets and strategies for the treatment of cancer pain.Methods and Results1. Significance of changes in 5-HT content in spinal dorsal horn in a rat model of cancer induced bone painMethods:Experiment 1 (correlation analysis of 5-HT content in the spinal dorsal horn of CIBP rat and the pain threshold):Fifty-seven female SD rats were randomly divided into 3 groups (n=19).1) normal control group (Naive):no treatment; 2) sham operation group (Sham):D-hank's solution (10μl) was injected into the right tibia upper marrow cavity; 3) cancer induced bone pain group (CIBP):Walker 256 rat breast cancer cell suspension 10μl (about 3×104) was injected into the right tibia upper bone marrow cavity for preparation of CIBP model. Experiment 2 (influence of intrathecal injection of selective serotonergic neurotoxin 5,7-DHT on the pain threshold of CIBP rat):Fifty-six female SD rats were randomly divided into 4 groups (n=14).1) Sham-saline group:intrathecal catheter was placed ten days after sham operation, normal saline (20μl) was injected intrathecally on the 14th day; 2) Sham-5,7-DHT group:intrathecal catheter was placed ten days after sham operation,5,7-DHT (60μg) was injected intrathecally on the 14th day;3) CIBP-saline group: intrathecal catheter was placed ten days after modeling, normal saline (20μl) was injected intrathecally on the 14th day; 4) CIBP-5,7-DHT group:intrathecal catheter was placed ten days after modeling,5,7-DHT (60μg) was injected intrathecally on the 14th day. Pain behavior tests and histopathology method were used to identify successful model. Spinal dorsal horn tissues of rats from different group were collected on 7th,14th and 21st after surgery.5-HT content in the spinal dorsal horn was determined using high performance liquid chromatography (HPLC) and was correlated with corresponding mechanical paw withdrawal threshold and paw withdrawal duration. The effect of intrathecal 5,7-DHT on rat pain behavior was assayed. HPLC and immunofluorescence staining were used to determine ablation effect of 5,7-DHT on 5-HT containing nerve in spinal dorsal horn.Results:Mechanical withdrawal thresholds significantly reduced, paw withdrawal duration prolonged and ambulatory pain score increased (P<0.05) from 7th to 21st day in CIBP rats after inoculation of tumor cells. Under light microscope, lots of tumor cells infiltration and severe bone structure damage at the injection site were observed, indicating that the CIBP model was successfully constructed. Compared with the control group, mechanical pain threshold decreased from 7th to 21st day after modeling.7,14 and 21 days after inoculation, 5-HT levels in spinal dorsal horn increased.5-HT content and mechanical pain threshold were significantly correlated. Intrathecal administration of 5,7-DHT significantly decreased 5-HT content in spinal dorsal horn and attenuated pain behaviors of CIBP rats.2. Analgesic effect of targeted inhibition of the descending serotonergic facilitation from RVM in a rat model of cancer induced bone painMethods:One hundred and eight female SD rats were randomly divided into four groups (n=27).1) Sham-Saline group:14 days after sham operation,0.5μl saline was injected to RVM; 2) Sham-PCPA group:14 days after sham operation, PCPA (50μg) was injected to RVM; 3) CIBP-Saline group:14 days after modeling,0.5μl saline was injected to RVM; 4) CIBP-PCPA group:14 days after modeling, PCPA (50μg) was injected to RVM. Mechanical paw withdrawal threshold, paw withdrawal duration and ambulatory pain score were assayed in all groups at different time-points.5-HT content in spinal dorsal horn was determined at different time points after RVM administration using HPLC. Expression of 5-HT in RVM and spinal dorsal horn was detected by immunofluorescence staining.Results:Selective tryptophan hydroxylase inhibitor PCPA downregulated the 5-HT expression in RVM and reduced 5-HT content in spinal dorsal horn in the injected rat (P <0.05). Behavioral tests showed increased mechanical paw withdrawal threshold, decreased withdrawal duration and reduced ambulatory pain score (P<0.05). The immunofluorescence staining results showed that RVM injection of PCPA reduced 5-HT content in spinal dorsal horn.3. The mechanisms of descending serotonergic facilitation from RVM participating in cancer induced bone pain in ratsMethods:Experiment 1:Forty-eight female SD rats were randomly divided into 4 groups (n=12).1) Sham-DMSO group:DMSO (1μl) was injected to RVM14 days after sham operation; 2) Sham-SB203580 group:SB203580 (50μg) was injected to RVM14 days after sham operation; 3) CIBP-DMSO group:DMSO (1μl) was injected to RVM 14 days after modeling; 4) CIBP-SB203580 group:SB203580 (50μg) was injected to RVM 14 days after modeling. Pain thresholds of rats in each group were determined at different time points. TPH expression in RVM was assayed by western blot after drug injection.5-HT content in spinal dorsal horn was determined using HPLC. Immunofluorescence staining was used to detect the expression of TPH in RVM and 5-HT in spinal dorsal horn respectively. Experiment 2:Twelve female SD rats were randomly divided into 2 groups. Normal control group (Naive group, n=3) and cancer induced bone pain group (CIBP group, n=9). The rats were sacrificed on the 7th,14th and 21st day after modeling, the spinal cord lumbar enlargements were collected for gene microarray analysis of 5-HT receptor expression.Results:CIBP rats received RVM injection of SB203580 showed increased mechanical paw withdrawal threshold, decreased paw withdrawal duration and reduced ambulatory pain score (P<0.05). Western blot result showed RVM injection of SB203580 significantly inhibited TPH expression in RVM and produced analgesic effect (P<0.05). Immunofluorescence staining showed TPH positive neurons decreased in RVM after injection of SB203580. HPLC showed reduced 5-HT level in the spinal dorsal horn (P <0.05). The gene microarray result showed that both facilitory and inhibitory 5-HT receptors were up-regulated in the spinal cord of CIBP rat, however, the expression of facilitory 5-HT receptors was much higher than that of inhibitory 5-HT receptors.4. The mechanisms of RVM glial activation involved in cancer induced bone pain in ratsMethods:Ninety-nine female SD rats were randomly divided into 7 groups:1) Naive group (n= 6):no intervention;2) Sham group (n= 18):10μl D-hank's solution was injected into right tibia; 3) CIBP group (n=18):10μl Walker 256 breast cancer cell suspension (3×104) was injected into right tibia; 4) sham/NS (n= 15):0.5μl normal saline was injected into RVM 10 days after sham operation; 5) CIBP/NS (n= 15):0.5μl normal saline was injected into RVM 10 days after CIBP modeling; 6) CIBP/Minocycline (n=15): 0.5μl (25μg) microglial inhibitor minocycline was injected into RVM 10 days after CIBP modeling; 7) CIBP/Fluorocitrate (n= 12):0.5μl (1μg) astrocyte inhibitor fluorocitrate was injected into RVM 10 days after CIBP modeling. Tibial destruction was detected using radiograph. Expression of the RVM glial cell activation markers mRNA was detected by quantitative PCR. Western-blot and immunofluorescence staining were used to determine the effect of drug treatments on the activation of RVM glial cells. Pain thresholds were assayed. The expression of pro-inflammatory mediators IL-1β,IL-6, TNF-αand brain-derived neurotrophic factor was assayed using quantitative PCR after RVM injection of microglial inhibitor.Results:CIBP rats showed significant activation of microglia and astrocytes, and also up-regulation of proinflammatory mediators released by glial cells (IL-1β,IL-6,TNF-αand BDNF) in the RVM. Stereotaxic microinjection of the glial inhibitors (minocycline and fluorocitrate) into CIBP rats'RVM could reverse the glial activation and significantly attenuate mechanical allodynia in a time-related manner. RVM injection of microglial inhibitor reversed the associated up-regulation of proinflammatory mediators and significantly attenuated mechanical allodynia.5. Statistical analysisAll of the analyses were performed by SPSS13.0 software package. Data were expressed as the mean±standard deviation (SD). Comparisons between two groups were made using r test, comparisons between multiple groups were made using ANOVA followed by post-hoc test (Student-Newman-Keuls test), a bivariate correlation analysis was made between 5-HT in spinal dorsal horn and mechanical pain threshold. P<0.05 was considered statistically significant.Conclusion1. Descending serotonergic facilitation from RVM was involved in the maintenance of cancer induced bone pain in rats, the mechanism included increased 5-HT projection from RVM to spinal dorsal horn and the 5-HT receptors balance of pain inhibition and facilitation switch to pain facilitation.2. RVM glial activation contributed to the development of CIBP through promoting descending facilitation. Activation of microglia was involved in the modulation of the expression of RVM pro-inflammatory mediators, which might be a part of effects of RVM microglial activation in CIBP.SignificanceAccumulating evidence suggests that inappropriate tonic-descending facilitation arising from the RVM has been established to underlie some chronic pathologic pain. Our research selectively inhibited TPH expressing neurons in the RVM, presumed to be the source of descending serotonergic projection to spinal cord, by a single intra-RVM microinjection with PCPA, and subsequently attenuated the experimental CIBP effectively. On the other hand, data of this research established RVM glia might serve as a novel target for the treatment of intractable CIBP. The targeted inhibition of descending facilitation is a safe and reliable approach to treat pathologic pain. Thus, this research is a novel exploration of mechanism-based therapy for CIBP. providing us some experimental evidence for future clinical application.
|
PDF Full Text Request