Number Of Spontaneous Flinches Research Articles

Microglia Promote Inhibitory Synapse Phagocytosis in the Spinal Cord Dorsal Horn and Modulate Pain-Like Behaviors in a Murine Cancer-Induced Bone Pain Model.

The microglial activation has been implicated in cancer-induced bone pain. Recent studies have revealed that microglia mediate synaptic pruning in the central nervous system, where the cluster of differentiation 47-signal regulatory protein α (CD47-SIRPα) axis creates a "don't eat me" signal and elicits an antiphagocytic effect to protect synapses against elimination. To date, the synaptic phagocytosis in microglia has never been investigated in the murine cancer-induced bone pain model. The present experiments sought to explore whether microglia phagocytize synapses in mice with bone cancer pain as well as the possible mechanisms. Male C3H/HeN mice were used to induce bone cancer pain. Minocycline and S-ketamine were injected into D14. The number of spontaneous flinches (NSF) and paw withdrawal mechanical thresholds (PWMT) were measured on D0, D4, D7, D10, D14, D21, and D28. Hematoxylin and eosin staining presented bone lesions. Western blotting examined the Gephyrin, CD47, and SIRPα expression. Flow cytometry evaluated the proportion of SIRPα+ cells in the spine. Immunofluorescence and 3-dimensional reconstruction showed the Gephyrin puncta inside microglial lysosomes. Mice embedded with tumor cells induced persistent spontaneous pain and mechanical hyperalgesia. Hematoxylin and eosin staining revealed bone destruction and tumor infiltration in marrow cavities. Microglia underwent a responsive and proliferative burst (t = -16.831, P < .001). Western blotting manifested lowered Gephyrin expression in the tumor group (D4, D7, D10, D14, D21, and D28: P < .001). Immunofluorescence and 3-dimensional reconstruction showed larger volumes of Gephyrin puncta inside microglial lysosomes (t = -23.273, P < .001; t = -27.997, P < .001). Treatment with minocycline or S-ketamine exhibited pain relief and antiphagocytic effects (t = -6.191, P < .001, t = -7.083, P < .001; t = -20.767, P < .001, t = -17.080, P < .001; t = 11.789, P < .001, t = 16.777, P < .001; t = 8.868, P < .001, t = 21.319, P < .001). Last but not least, the levels of CD47 and SIRPα proteins were downregulated (D10: P = .004, D14, D21, and D28: P < .001; D10, D14, D21, and D28: P < .001). Flow cytometry and immunofluorescence substantiated reduced microglial SIRPα (t = 11.311, P < .001; t = 12.189, P < .001). Microglia-mediated GABAergic synapse pruning in the spinal cord dorsal horn in bone cancer pain mice, which might be associated with the declined CD47-SIRPα signal. Our research uncovered an innovative mechanism that highlighted microglia-mediated synaptic phagocytosis in a murine cancer-induced bone pain model.

MiR-199a-3p suppresses neuroinflammation by directly targeting MyD88 in a mouse model of bone cancer pain

AimsPain is a profoundly debilitating symptom in cancer patients, leading to disability, immobility, and a marked decline in their quality of life. This study aimed to investigate the potential roles of miR-199a-3p in a murine model of bone cancer pain induced by tumor cell implantation in the medullary cavity of the femur. Materials and methodsWe assessed pain-related behaviors, including the paw withdrawal mechanical threshold (PWMT) and the number of spontaneous flinches (NSF). To investigate miRNA expression and its targets in astrocytes, we employed a combination of RNA-seq analysis, qRT-PCR, Western blotting, EdU, TUNEL, ChIP, ELISA, and luciferase reporter assays in mice (C3H/HeJ) with bone cancer pain and control groups. Key findingsOn days 10, 14, 21, and 28 post-surgery, we observed significant differences in PWTL, PWMT, and NSF when compared to the sham group (P < 0.001). qRT-PCR assays and miRNA sequencing results confirmed reduced miR-199a-3p expression in astrocytes of mice with bone cancer pain. Gain- and loss-of-function experiments demonstrated that miR-199a-3p suppressed astrocyte activation and the expression of inflammatory cytokines. In vitro investigations revealed that miR-199a-3p mimics reduced the levels of inflammatory factors in astrocytes and MyD88/NF-κB proteins. Furthermore, treatment with a miR-199a-3p agonist resulted in reduced expression of MyD88, TAK1, p-p65, and inflammatory mediators, along with decreased astrocyte activation in the spinal cord. SignificanceCollectively, these findings demonstrate that upregulation of miR-199a-3p may offer a therapeutic avenue for mitigating bone cancer pain in mice by suppressing neuroinflammation and inhibiting the MyD88/NF-κB signaling pathway.

Downregulated CAV-1 in mouse spinal cord may alleviate bone cancer pain by inhibiting the ERK/CREB pathway

BackgroundThis study aimed to assess the potential function of Caveolin-1 (CAV-1) in mice with bone cancer pain. Method: Using a mice bone cancer pain model we explored the contribution of CAV-1 expression to bone cancer pain on the 14th day after surgery, mice in the tumor group were randomized and treated with increasing doses of the CAV-1 inhibitor, methyl-beta-cyclodextrin. Pain was assessed by monitoring the number of spontaneous flinches (NSF) and paw withdrawal mechanical threshold (PMWT)mechanical withdrawal threshold (MWT). The localization and expression of CAV-1 in mouse neurons was also determined. Additionally, the protein levels of CAV-1, extracellular signal regulated kinase (ERK) 1/2, cAMP response element-binding protein (CREB) were monitored in mouse spinal cord tissues by western blotting. Results: CAV-1 was remarkably upregulated in the spinal cord of the tumor group on the 4th day after surgery, then downregulated on day 10, and upregulated again at day 14. Such CAV-1 levels were maintained until day 28. In the tumor group, the expression of p-ERK1/2 and p-CERB were upregulated at day 14 after surgery. Intrathecal injection of methyl-beta-cyclodextrin (MCD) downregulated p-ERK1/2 and p-CERB expression which correlated with alleviation of pain. Conclusion: Inhibition of CAV-1 in the spinal cord alleviates bone cancer pain in mice which correlates with inhibition of the ERK/CREB pathway.

MiR-135-5p Alleviates Bone Cancer Pain by Regulating Astrocyte-Mediated Neuroinflammation in Spinal Cord through JAK2/STAT3 Signaling Pathway

Bone cancer pain (BCP) was associated with microRNA dysregulation. In this study, we intended to clarify the potential role of miR-135-5p in a BCP mouse model, which was established by tumor cell implantation (TCI) in the medullary cavity of the mouse femur. The BCP-related behaviors were tested, including the paw withdrawal mechanical threshold (PWMT) and number of spontaneous flinches (NSF). The miRNA expression profiles in astrocytes of the sham and tumor groups were compared, and miRNA microarray and quantitative real-time PCR (qRT-PCR) assays confirmed that the amount of expression of miR-135-5p was significantly decreased in astrocytes of the tumor group. Gain- and loss-of-function studies showed that miR-135-5p could inhibit astrocyte activation and inflammation cytokine (TNF-α and IL-1β) expression. The relation between miR-135-5p and JAK2 was detected by bioinformatic analysis and dual luciferase reporter gene assay. By conducting in vitro experiments, it was shown that the miR-135-5P mimics lowered the level of JAK2/STAT3 proteins and inflammatory factors in astrocytes. Moreover, in vivo analysis on BCP mice model indicated that the miR-135-5p agonist could sufficiently increase PWMT and decrease NSF. Meanwhile, reduced activation of astrocytes in the spinal cord, as well as decreased expression of JAK2/STAT3 and inflammatory mediators, were found after miR-135-5p agonist treatment. Collectively, the results showed that miR-135-5p could potentially reduce BCP in mice through inhibiting astrocyte-mediated neuroinflammation and blocking of the JAK2/STAT3 signaling pathway, indicating that the upregulation of miR-135-5P could be a therapeutic focus in BCP treatment.

Lidocaine Alleviates Neuropathic Pain and Neuroinflammation by Inhibiting HMGB1 Expression to Mediate MIP-1α/CCR1 Pathway.

High mobility group box1 (HMGB1) released from sensory nerve tissues can induce neuropathic pain. Whether HMGB1 is implicated in the mechanism underlying the effect of lidocaine in pain management remains to be determined. This study aims to explore the effect of lidocaine in a rat model of spared nerve injury (SNI) and the underlying mechanism. An SNI model was established via nerve ligation. Two weeks after the SNI model was established, rats were intrathecally injected with lidocaine, an HMGB1 antibody (HMG Ab), an MIP-1α antibody (MIP-1α Ab), a CCR1 inhibitor (CCR1-RS) or a CCR5 antagonist (CCR5-Mar). Pain behaviors were assessed before and after model establishment to calculate the number of spontaneous flinches (NSF), paw withdrawal threshold (PWT), paw withdrawal thermal latency (PWL) and sciatic function index (SFI). Cell apoptosis and the inflammatory response in the cerebrospinal fluid (CSF) were detected by TUNEL staining and ELISA. The mRNA and protein expression levels of MIP-1α, CCR1 and CCR5 were determined by RT-PCR and Western blotting. The expression levels of HMGB1, MIP-1α, CCR1 and CCR5 were measured by Western blotting and immunofluorescence. Pain behavior testing in SNI rats showed that SNI rats exhibited an increased NSF and adecreased PWT, PWL and SFI. Cell apoptosis in the spinal dorsal horn and the generation of inflammatory cytokines were enhanced in SNI rats, and the expression levelsof HMGB1, MIP-1α, CCR1 and CCR5 were upregulated. HMGB1 cytoplasmic translocation, the coexpression of MIP-1α with NeuN, and the coexpression of CCR1 and CCR5 with OX42 were also observed in SNI rats. Neuropathic pain and neuroinflammation were suppressed by the intrathecal injection of lidocaine, HMG Ab, MIP-1α Ab, CCR1-RS or CCR5-Mar. Lidocaine inhibited the expression levels of HMGB1, MIP-1α, CCR1 and CCR5, and the HMGB1 antibody suppressed the expression of MIP-1α, CCR1 and CCR5. Lidocaine attenuates neuropathic pain and neuroinflammation by inhibiting HMGB1 to regulate the MIP-1α/CCR1/CCR5 pathway. Graphical Abstract.

Endoplasmic Reticulum Stress Contributes to Nociception via Neuroinflammation in a Murine Bone Cancer Pain Model.

Prolonged endoplasmic reticulum stress has been identified in various diseases. Inflammatory mediators, which have been shown to induce endoplasmic reticulum stress in several studies, have been suggested to serve as the important modulators in pain development. In this study, the authors hypothesized that the endoplasmic reticulum stress triggered by inflammatory mediators contributed to pain development. The authors used a male mouse model of bone cancer pain. The control mice were intrathecally injected with tumor necrosis factor-α (TNF-α) and lipopolysaccharide, the bone cancer pain mice were intrathecally injected with the endoplasmic reticulum stress inhibitors 4-PBA and GSK2606414. The nociceptive behaviors, endoplasmic reticulum stress markers, and inflammatory mediators were assessed. Increased expression of the p-RNA-dependent protein kinase-like endoplasmic reticulum kinase and p-eukaryotic initiation factor 2α were found in the spinal neurons during bone cancer pain, along with upregulation of inflammatory mediators (TNF-α, interleukin 1β, and interleukin 6). Intrathecal administration of TNF-α or lipopolysaccharide increased the expression of endoplasmic reticulum stress markers in control mice. Inhibition of endoplasmic reticulum stress by intrathecal administration of 4-PBA (baseline vs. 3 h: 0.34 ± 0.16 g vs. 1.65 ± 0.40 g in paw withdrawal mechanical threshold, 8.00 ± 1.20 times per 2 min vs. 0.88 ± 0.64 times per 2 min in number of spontaneous flinches, P < 0.001, n = 8) or GSK2606414 (baseline vs. 3 h: 0.37 ± 0.08 g vs. 1.38 ± 0.11 g in paw withdrawal mechanical threshold, 8.00 ± 0.93 times per 2 min vs. 3.25 ± 1.04 times per 2 min in number of spontaneous flinches, P < 0.001, n = 8) showed time- and dose-dependent antinociception. Meanwhile, decreased expression of inflammatory mediators (TNF-α, interleukin 1β, and interleukin 6), as well as decreased activation of astrocytes in the spinal cord, were found after 4-PBA or GSK2606414 treatment. Inhibition of inflammatory mediator-triggered endoplasmic reticulum stress in spinal neurons attenuates bone cancer pain via modulation of neuroinflammation, which suggests new approaches to pain relief.

Expression of spinal PD-L1 in bone cancer pain model mice and its effect on pain behaviors

Objective To evaluate the role of programmed cell death ligand-1(PD-L1) in a mouse model of bone cancer pain. Methods Ninety-six male C3H/HeN mice (20-25 g, 4-6 weeks old), which inoculated with osteolytic NCTC 2472 cells, were used to build the model of bone cancer pain. Part one: sixty-four male C3H/HeJ mice were randomly divided into sham group (group Sham, n=32) and tumor group (group Tumor, n=32). Part two: Twenty-four male C3H/HeJ mice which were inoculated with osteolytic NCTC 2472 cells were randomly divided into group T (tumor, n=8), group PD-L1 (intrathecal injection with PLX3397, 1μg /5μl, n=8) and group NS (intrathecal injection with normal saline, n=8). Also, there were eight male C3H/HeJ mice in group S which were intra-femur inoculated with α-MEM. The pain behaviors of Sham group and Tumor group were observed and the expression of PD-L1 was detected before inoculation and on 4 , 7 , 10 , 14 and 21 days after inoculation, including paw withdrawal mechanical threshold (PWMT) and the number of spontaneous flinches (NSF). On 14 d after inoculation, the mice of group PD-L1 and group NS were intrathecal injected with drugs respectively. Pain behaviors were observed before injection and 2, 4, 6, 24h after injection. Results Compared with group Sham, PWMT was significantly decreased and NSF was increased on 7~21 d after inoculation in group Tumor (P<0.05). Compared with baseline and group S (baseline (0.38±0.06), group Sham (0.35±0.08), (0.38±0.08), (0.36±0.07)), the expression of PD-L1 was up-regulated on 10-21 d after inoculation in group Tumor ((0.77±0.06), (1.21±0.04), (1.18±0.06)) (P<0.05). Compared with group NS, PWMT was significantly increased (group NS (0.25±0.12), (0.25±0.12), (0.31±0.12), group PD-L1 (1.43±0.49), (1.35±0.44), (0.95±0.26)), and NSF was decreased on 2-6 h after injection in group PD-L1 (group NS(11.74±1.31), (13.78±0.0.91), (13.63±1.06), group PD-L1(4.90±0.82), (4.15±0.71), (7.65±0.56)) (P<0.05). Conclusion Expression of PD-L1 in spinal cord was up-regulated in the mouse model of bone cancer pain. Intrathecal injection of recombinant PD-L1 has an analgesic effect on mice with bone cancer. Key words: Bone cancer pain; Spinal cord; Programmed cell death ligand-1; Mice

Blockage of spinal endothelin A receptors attenuates bone cancer pain via regulation of the Akt/ERK signaling pathway in mice

Bone cancer pain (BCP) is a common source of pain in patients with advanced stage and metastatic cancer; however, existing treatment for this kind of pain remains deficient. Being closely related to sensory change and inflammatory pain in both the central and peripheral nervous systems, endothelin A receptor (ETAR) plays an essential role in pain processing. As a result, ETAR antagonist has been reported to alleviate both neuropathic and inflammatory pain. Thus far, the role of ETAR in the process of BCP is still ambiguous. In this study, by using a BCP mouse model, the analgesic effect and molecular mechanism of the ETAR antagonist BQ-123 was investigated. Pain sensation in the BCP mouse model was investigated by the number of spontaneous flinches (NSF) and pain withdrawal threshold (PWT), and the mechanism of BCP was assessed by measuring p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 levels in L4–6 segments of the spinal cord. Our results demonstrated that BCP mice showed a higher NSF and a lower PWT score than Sham mice. In addition to the development of nociceptive sensitization, p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 were up-regulated correspondingly in L4–6 segments of the spinal cord in BCP mice. BQ-123 treatment showed a promising analgesic effect, and the effect was correlated to the down-regulation of p-Akt, p-Akt/t-Akt, p-ERK-1/2 and p-ERK-1/2/t-ERK-1/2 in spinal cord cells. The results suggested that intrathecal administration of BQ-123 was able to relieve BCP in mice as a consequence of suppressing the Akt and ERK signalling pathways.

Expression of IGF-1 in serum and phosphorylated IGF-1 receptor in spinal cord in mouse model of bone cancer pain

Objective To investigate the expression of insulin-like growth factors -1(IGF-1) in serum and phosphorylated IGF-1 receptor in spinal cord in mouse model of bone cancer pain. Methods Sixty male C3H/HeJ mice weighed 18-22 g were randomly divided into Sham group(n=30) and Tumor group(n=30). The mice in Tumor group were inoculated with NCTC fibrosarcoma cells in the right femur bone marrow cavity. Paw withdrawl mechanical threshold(PWMT) and the number of spontaneous flinches(NSF) were measured on 1d before inoculation and on 4 d, 7 d, 10 d, 14 d, 21 d after inoculation(n=8). At each time point, the mice of each group were taken blood by removal eyeball and the samples of blood were obtained to detect the expression of IGF-1 by enzyme-linked immunosorbent assay(n=4). The mice after taken blood on 14 d after inoculation were perfused and the samples of spinal cord lumber(L3~5) segment were obtained to detect the expression of phosphorylated IGF-1 receptor by immunofluorescence assay(n=6). Results Compared with Sham group, PWMT was significantly decreased(P<0.05) and NSF was significantly increased(P<0.05) on 7~21 d after inoculation. Compared with baseline value and Sham group(baseline value(27.33±0.52)pg/ml, Sham group(29.11±1.86)pg/ml, (24.51±3.61)pg/ml, (23.33±4.59)pg/ml, (25.29±2.99)pg/ml), the expression of IGF-1 in serum was significantly increased on 7~21 d after inoculation in Tumor group((39.76±3.92)pg/ml, (36.93±2.18)pg/ml, (38.85±2.40)pg/ml, (39.70±2.62)pg/ml). The mean fluorescence intensity of phosphorylated IGF-1 receptor was significantly higher on 14d after inoculation in Tumor group(2.40±0.11) compared with Sham group(0.05±0.01). Conclusion Expression of IGF-1 in serum and phosphorylated IGF-1 receptor in spinal cord were significantly increased in mice with bone cancer pain, and this change may be involved in the development and maintenance of bone cancer pain. Key words: Bone cancer pain; Insulin like growth factor-1; Phosphorylation insulin like growth factor-1 receptor

Changes in expression of spinal endothelin-1 and its receptors in a mouse model of bone cancer pain

Objective To evaluate the changes in the expression of spinal endothelin-1(ET-1)and its receptors in a mouse model of bone cancer pain(BCP). Methods Ninety-six healthy male SPF C3H/HeN mice, aged 4-6 weeks, weighing 20-25 g, were divided into 2 groups(n=48 each)using a random number table: sham operation group(group S)and BCP group.BCP was produced by injecting α-MEM 20 μl containing 1×104 cells/μl NCTC 2472 osteosarcoma cells into the distal medullary cavity of the right femur bone.In group S, α-MEM 20 μl was injected into the distal medullary cavity of the right femur bone.Mechanical paw withdrawal threshold(MWT)and the number of spontaneous flinches(NSF)were measured on 1 day before inoculation(T0)and 4, 7, 10, 14 and 21 days after inoculation(T1-5). Twelve mice of each group were randomly sacrificed at T0, 2, 4, 5, and the lumbar enlargement segments of the spinal cord were harvested to detect the expression of ET-1, endothelin type A receptor and endothelin type B receptor protein and mRNA(using Western blot or real-time polymerase chain reaction). Results The MWT was significantly lower and the NSF was higher at T1 in group S and at T1-5 in group BCP than at T0(P<0.05). Compared with group S, the MWT was significantly decreased and the NSF was increased at T2-5, and the expression of ET-1, endothelin type A receptor and endothelin type B receptor protein and mRNA was down-regulated at T2, 4, 5 in group BCP(P<0.05). Conclusion The pathophysiological process of BCP is associated with down-regulating the expression of spinal ET-1 and its receptors in mice. Key words: Receptor, endothelin type A; Receptor, endothelin type B; Receptors, G protein-coupled; Bone neoplasms; Pain; Spinal cord

Role of spinal neuronal Mas-related gene receptor C in maintenance of bone cancer pain in mice

Objective To evaluate the role of spinal neuronal Mas-related gene receptor C (MrgC) in the maintenance of bone cancer pain (BCP) in mice. Methods A total of 132 SPF male C3H/HeJ mice, aged 8-10 weeks, weighing 18-22 g, were randomly divided into 4 groups (n=33 each) using a random number table: sham operation group (S group), BCP group, bovine adrenal medulla peptide 8-22 (BAM8-22, a highly selective MrgC agonist) group (group BAM), and MrgC antibody group (group MA). BCP was produced by injecting α-MEM 20 μl containing 2×105 NCTC2472 cells into the distal medullary cavity of right femur bone.While α-MEM 20 μl was injected only in group S. The artificial cerebrospinal fluid 5 μl was injected intrathecally in S and BCP groups, and BAM 8-22 8 nmol/5 μl and MrgC antibody 5 μl were injected intrathecally in BAM and MA groups, respectively, once a day for 7 consecutive days starting from the day 14 after inoculation of the tumor cells.At 1 day before inoculation (T0), before administration (T1), and at 14, 16 19 and 21 days after inoculation (T2-5, at 0.5 h before the initial administration and 2 h after each administration), the number of spontaneous flinches (NSF) and mechanical paw withdrawal threshold (MWT) were measured.Five animals selected from each group at each time point were sacrificed, and the lumbar enlargement segments of the spinal cord were removed for determination of MrgC expression in the spinal neurons (by immunofluorescence). Results Compared with group S, NSF was significantly increased, MWT was decreased, and the expression of MrgC was up-regulated at T1-5 in BCP, BAM and MA groups.Compared with group BCP, NSF was significantly decreased, MWT was increased, and the expression of MrgC was up-regulated at T2-5 in group BAM, and NSF was significantly increased, MWT was decreased, and the expression of MrgC was down-regulated at T2-5 in group MA. Conclusion Spinal neuronal MrgC is involved in the maintenance of BCP in mice. Key words: Receptors, G-protein-coupled; Bone neoplasms; Pain; Neurons; Spinal cord

Role of spinal neuronal microRNA-132 in a mouse model of bone cancer pain

Objective To evaluate the role of spinal neuronal microRNA-132 (miR-132) in a mouse model of bone cancer pain (BCP).Methods Male C3H/HeJ mice,aged 4-6 weeks,weighing 20-25 g,were used in the study.The experiment was performed in 2 parts.In experiment Ⅰ,fifty-two male C3H/HeJ mice were randomly divided into 2 groups (n =26 each) using a random number table:sham operation group (S group) and BCP group.In experiment Ⅱ,thirty-two male C3H/HeJ mice were randomly divided into 4 groups (n =8 each) using a random number table:sham operation + intrathecal DNAse/RNAse-free water group (S + Veh group),BCP + intrathecal DNAse/RNAse-free water group (BCP + Veh group),BCP + intrathecal negative control locked nucleic acid (LNA) group (BCP + C group),and BCP + intrathecal miR-132 antisense LNA group (BCP + AS group).BCP was induced by injecting 2 x 105 NCTC 2472 cells into the distal medullary cavity of the right tibia.In BCP+ C and BCP + AS groups,negative control LNA and miR-132 antisense LNA 12 pmol/5 μl were intrathecally injected,respectively,once a day for 7 consecutive days starting from day 14 after inoculation.In S + Veh and BCP + Veh groups,DNAse/RNAse-free water 5 μl was intrathecally injected once a day for 7 consecutive days starting frowm day 14 after inoculation.In S and BCP groups,the paw withdrawal threshold to mechanical stimuli (MWT) and the number of spontaneous flinches (NSF) were measured,and the expression of miR-132 in spinal cord was detected before inoculation and on 4,7,10,14 and 21 days after inoculation.In S + Veh,BCP + Veh,BCP + C and BCP + AS groups,the parameters mentioned above were measured at 14 days before inoculation and 15,16,17,18,19,20 and 21 days after inoculation.Results Compared with S group,MWT was significantly decreased,NSF was increased and the expression of miR-132 was up-regulated on 7-21 days after inoculation in BCP group.Compared with S + Veh group,MWT was significantly decreased,and NSF was increased at each time point in the other three groups.Compared with BCP + Veh group,MWT was significantly increased,and NSF was decreased on 16-21 days after inoculation in BCP + AS group.Conclusion Spinal neuronal miR-132 is involved in the development and maintenance of BCP in mice. Key words: MicroRNAs ; Spinal cord ; Bone neoplasms ; Pain

Changes in the expression of CREB-regulated transcription coactivator 1 in spinal cord in mouse model of bone cancer pain

Objective To investigate the changes in the expression of CREB-regulated transcription coactivator 1 (CRTC1) in spinal cord during the development and maintenance of bone cancer pain in mice. Methods Forty six male C3H/HeJ mice were randomly divided into Sham group (n=23) and Tumor group (n=23). The mouse model of bone cancer pain was developed by intra-femur inoculation of α-minimal essence media (α-MEM) with osteolytic NCTC 2472 cells. The mice in sham group were inoculated with α-MEM without NCTC 2472 cells.Pain behaviors such as the paw withdrawal mechanical threshold (PWMT) and the number of spontaneous flinches (NSF) were assessed on 1 d before inoculation and on 4 d, 7 d , 10 d , 14 d , 21 d after inoculation. At each corresponding time point, three mice of each group were killed just after the pain behaviors assessment and the samples of spinal cord lumbar segment were obtained to detect the expression of CRTC1 using Western Blot. Results Compared with Sham group, PWMT((1.35±0.14) g, (1.10±0.28) g, (0.78±0.25) g, (0.47±0.16) g, (0.34±0.16)g) was significantly decreased (P<0.05) and NSF((3.12±0.74), (6.02±0.67), (7.42±1.22), (10.82±0.98), (12.48±1.06)) was significantly increased (P<0.05) on 7 d, 10 d, 14 d and 21 d after inoculation in Tumor group. Compared with baseline level, significant increase in the expression of CRTC1 in spinal cord was observed on 4 d in both Sham group and Tumor group (P<0.05). Compared with baseline level and Sham group, significant increase in the expression of CRTC1 in spinal cord was observed on 7 d, 10 d, 14 d and 21 d in Tumor group (P<0.05). Conclusion Expression of CRTC1 in spinal cord is up-regulated in mice with bone cancer pain, and this change may be involved in the development and maintenance of bone cancer pain. Key words: Bone neoplasms; Pain; CREB-regulated transcription coactivator 1; Spinal cord

Effect of agmatine on intrathecal morphine analgesia

Objective To observe the effect of intrathecal agmatine( AG) at the spinal cord level on morphine tolerance.Methods( 1) Twenty-four SD rats were divided into four groups: control group,pure intrathecal injection( i. t.) of saline( 15 μL);morphine group,i. t. morphine( 15 μg /15 μL); AG group,i. t. AG( 12. 5 μg /15 μL); and morphine + AG group,i. t. morphine( 15 μg/5 μg) and AG( 12.5 μg/10 μL). Rats of the four groups were injected with 0.2 mg bee venom subcutaneously in the plantar5 min after i. t,and the numbers of spontaneous paw withdrawal reflex were recorded within 1 hour.( 2) Twenty-four SD rats were divided into three groups: control group,pure intrathecal saline( 15 μL); morphine tolerance group,i. t. morphine( 15 μg /5 μL)twice a day for 4 consecutive days; and morphine tolerance + AG group,i. t. morphine( 15 μg /5 μL) twice a day for 4 consecutive days,on the fourth day the rats also received AG( 12. 5 μg /10 μL). Half of the rats were examined for thermal paw withdrawal latency and mechanical withdrawal threshold after i. t,and the other half was injected with 0. 2 mg bee venom subcutaneously in the plantar 10 min after last dose; the number of spontaneous paw withdrawal reflex were recorded within 1 hour. Results( 1) Compared with the control group,the numbers of flinches of intrathecal morphine and AG groups were significantly reduced( P 0. 05).( 2) In intrathecal morphine tolerance model,the thermal paw withdrawal latency and mechanical withdrawal threshold were significantly increased in the morphine tolerance + AG group compared with the control group( P 0. 05); morphine tolerance + AG group hadsignificantly stronger inhibitory effect against spontaneous s. c. BV-induced pain,with the number of spontaneous flinches within 1 hour decreased significantly in the morphine tolerance + AG group( P 0. 05). Conclusion Intrathecal AG has synergistic effect with morphine in pain relieving,and it can also reverse the morphine tolerance induced by repeated i. t. injection.

Role of u-opioid receptor in attenuation of bone cancer pain by anti-nerve growth factor in rats

Objective To evaluate the role of μ-opioid receptor （MOR） in attenuation of bone cancer pain by anti-nerve growth factor （anti-NGF） in rats. Methods Part Ⅰ Sixty female SD rats weighing 200-220 g were randomly divided into 4 groups （n = 15 each）： sham operation group （group S）, sham operation ＋ anti-NGF group （group SN）, bone cancer pain group （group P） and bone cancer pain＋ anti-NGF group （group PN） . Bonecancer was induced by intra-tibial inoculation of 1 × 105 Walker 256 breast cancer cells in group P and PN. Group S and SN received injection of PBS 10 μl. APE 10 catheter was inserted at L2,3 interspace into the epidural space 13 days after cancer cell inoculation. Three days after the catheter was successfully placed, group SN and PN received intrachecal （IT） injection of anti-NGF 10 μg （in normal saline （NS） 10 μl） and group S and P IT injection of NS 10 μl twice a day for 5 consecutive days. The number of spontaneous flinches （NSF）, paw withdrawal latency （PWL） and paw withdrawal threshold （PWT） were measured before and 13, 16, 18, 21 day after cancer cell inoculation. The animals were sacrificed at 21 day after cancer cell inoculation and the spinal cord dorsal horn and dorsal root ganglion were removed for determination of MOR and MOR mRNA expression. Part Ⅱ Thirty female SD rats weighing 200-220 g were randomly divided into 2 groups （n = 15 each）： bone cancer pain ＋ anti-NGF group （group PN） and bone cancer pain ＋ naloxone ＋ anti-NGF group （group PNN）. Bone cancer was induced by intratibial inoculation of 1 × 105 Walker 256 breast cancer cells. APE 10 catheter was inserted at L2-3 interspace into the epidural space 13 days after cancer cell inoculation. Three days after the catheter was successfully placed,group PN received IT injection of anti-NGF 10 μg （in NS 10 μl） and group PNN IT injection of naloxone 10μg （in NS 25 μl） and 0.5 h later IT injection of anti-NGF 10 μg （in NS 25μl） twice a day for 5 consecutive days. NSF,PWL and PWT were measured before and 13, 16, 18, 21 days after cancer cell inoculation. Results Part ⅠCompared with group S, no significant change was found in NSF, PWL and PWT in group SN, and in MOR and MOR mRNA expression in group SN and PN （P 〉 0.05）, NSF was significantly increased, PWL shortened, PWT decreased at 13-21 days after inoculation in group P and PN, and MOR and MOR mRNA expression was down-regulated in group P （P 〈 0.05 or 0.01）. Compared with group P, NSF was significantly decreased, PWL prolonged, PWT increased, MOR and MOR mRNA expression was up-regulated in group PN at 18-21 days after inoculation （P 〈 0.05 or 0.01）. Part Ⅱ Compared with group PN, NSF was significantly increased, PWL shortened, PWT decreased at 18-21 days after inoculation in group PNN （P 〈 0.05 or 0. 01）. Conclusion The mechanism by which anti-NGF attenuates bone cancer pain in rats is related to the activation of MOR. Key words: Receptors,opioid; Nerve growth factor; Antibodies; Bone neoplasms; Pain

Effects of spinal naloxone and naltrindole on the antinociceptive action of intrathecally administered dexmedetomidine

Intrathecally administered alpha-2 adrenoceptor and opioid agonists are well known to exert antinociceptive effects in humans and various animals. To examine the interaction of these two groups of agents in the spinal cord, we tested the effect of the opioid antagonists naloxone or naltrindole on the antinociceptive action of an intrathecally administered alpha-2 agonist, dexmedetomidine, using a formalin test in rats. 19 groups of Sprague-Dawley rats (250-300 g) were prepared with chronic intrathecal catheters and examined for the effects of agents on the formalin test. Each group contained 6 animals. 50 μl of 5% formalin was injected subcutaneously in the plantar surface of one hind paw. For each animal, the number of spontaneous flinches, characterized by rapid and brief withdrawal of the injected paw, were counted separately at 1-2 min, 5-6 min (phase 1), and at 5-min intervals thereafter up to 60 min (phase 2). Intrathecal dexmedetomidine (1 μg) maximally depressed the behavioral changes in both phase 1 and phase 2 of the formalin test, which was antagonized by the alpha-2 adrenoceptor selective antagonist atipamezole (0.3 μg). Naloxone (0.1-10 μg) or naltrindole (1-10 μg), when coadministered with dexmedetomidine, showed a dose-dependent antagonism to the effect of dexmedetomidine, whereas naloxone, naltrindole, or atipamezole alone showed no effect on the nocieptive behavior due to formalin injection. These results indicate that the antinociceptive effect of intrathecally administered alpha-2 adrenoceptor agonists may involve opioid receptors in the spinal cord.