The number of CB1 IR-positive neurons was also significantly decreased in stress rats (57

The number of CB1 IR-positive neurons was also significantly decreased in stress rats (57.5 5.8 %) compared with the control (84.3 4.2 %) (Fig. of WA stress rats with WIN or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the up-regulation of TRPV1. Conclusions These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia. test was used to ABT-639 examine the data for the expression of TRPV1 and CB1 obtained from Western blot and immunohistochemistry, for corticosterone and fecal pellet output measurements. Results were expressed as means SEM. P 0.05 was considered statistically significant. Results Chronic WA stress resulted in increased visceral nociception The serum corticosterone level was significantly higher in chronic WA rats compared with the controls ( 0.05). In control rats, ABT-639 the serum corticosterone level was 97.8 23.2 ng/ml, while it was 310.8 30.9 ng/ml in stressed rats (Fig. 1A). The VMR to grades intensities of CRD was recorded on day 0 before the start of WA stress and sham stress as the baseline level and recorded again on day 11 after chronic stress. In response to CRD, the VMR mean change, expressed as AUC percentage, was significantly higher in the WA stress rats at day 11 compared with the baseline level for the pressures of 20 and 40 mmHg (Fig 1B; 0.05). The AUC in the WA stress rats increased 109.7 37.2% compared with the controls for the pressure 60 mmHg ( 0.01). The VMR in rats following sham stress did not change significantly at day 11 compared with the baseline level. All 8 rats after repeated WA stress showed increased VMR to CRD, which is usually consistent with visceral hyperalgesia observed in the rats following repeated WA stress.12 Moreover, fecal pellet outputs were significantly increased in rats on day 1, day 5 and day 10 of WA stress compared with the controls (Fig 1C, 0.05), indicating the altered colonic motor function following repeated WA stress. Open in a separate window Physique 1 Effect of chronic water avoidance (WA) stress on serum corticosterone level, visceromotor response (VMR) to colorectal distension (CRD), and colonic motor function( 0.05; **, 0.01. Chronic WA stress increased the level of the endocannabinoid anandamide in DRGs Representative chromatographs depicting the content of anandamide in DRG tissue extracts are shown in Fig. 2A. The value from control L6-S2 DRGs was 111.3 ng/g tissue weight for anandamide, whereas it was 160.3 ng/g tissue weight in chronic WA stress rats, corresponding to 44% higher anandamide content than that of the controls (Fig. 2B). This percentage increase in anandamide content was similar to the significant increase in anandamide observed in lumbar DRG in a model of neuropathic pain.21 We also measured the content of 2-arachidonylglycerol (2-AG). However, the levels of 2-AG in DRG extracts from both control and WA stressed rats were too low to be detected, possibly due to the natural significant lower content of 2-AG as compared with anandamide in DRGs.21 Open in a separate window Figure 2 The level of the endocannabinoid anandamide was increased in L6-S2 DRGs from WA ratsRepresentative chromatograph from DRG extracts from control and WA stress rats depicting the relative abundance of endocannabinoid anandamide. The bar graph illustrated the increase in anandamide content in L6-S2 DRGs in rats following chronic WA stress compared to controls. L6-S2 DRGs from 5 rats in each group were combined for sample extraction to reach the adequate weight for LC-APCI/MS-MS analysis. Chronic WA Stress decreased CB1 Expression in DRG neurons To determine the expression of CB1 receptors, the colonic DRGs (L6-S2) corresponding to the distension area for VMR measurement were identified by.Scale bar: 80 m. changes in CB1 and TRPV1 were reproduced by treatment of ABT-639 control DRGs with anandamide with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats with WIN or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the up-regulation of TRPV1. Conclusions These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia. test was used to examine the data for the expression of TRPV1 and CB1 obtained from Western blot and immunohistochemistry, for corticosterone and ABT-639 fecal pellet output measurements. Results were expressed as means SEM. P 0.05 was considered statistically significant. Results Chronic WA stress resulted in increased visceral nociception The serum corticosterone level was significantly higher in chronic WA rats compared with the controls ( 0.05). In control rats, the serum corticosterone level was 97.8 23.2 ng/ml, while it was 310.8 30.9 ng/ml in stressed rats (Fig. 1A). The VMR to grades intensities of CRD was recorded on day 0 before the start of WA stress and sham stress as the baseline level and recorded again on day 11 after chronic stress. In response to CRD, the VMR mean change, expressed as AUC percentage, was significantly higher in the WA stress rats at day 11 compared with the baseline level for the pressures of 20 and 40 mmHg (Fig 1B; 0.05). The AUC in the WA stress rats increased 109.7 37.2% compared with the controls for the pressure 60 mmHg ( 0.01). The VMR in rats following sham stress did not change significantly at day 11 compared with the baseline level. All 8 rats after repeated WA stress showed increased VMR to CRD, which is consistent with visceral hyperalgesia observed in the rats following repeated WA stress.12 Moreover, fecal pellet outputs were significantly increased in rats on day 1, day 5 and day 10 of WA stress compared with the controls (Fig 1C, 0.05), indicating the altered colonic motor function following repeated WA stress. Open in a separate window Figure 1 Effect of chronic water avoidance (WA) stress on serum corticosterone level, visceromotor response (VMR) to colorectal distension (CRD), and colonic motor function( 0.05; **, 0.01. Chronic WA stress increased the level of the endocannabinoid anandamide in DRGs Representative chromatographs depicting the content of anandamide in DRG tissue extracts are shown in Fig. 2A. The value from control L6-S2 DRGs was 111.3 ng/g tissue weight for anandamide, whereas it was 160.3 ng/g tissue weight in chronic WA stress rats, corresponding to 44% higher anandamide content than that of the controls (Fig. 2B). This percentage increase in anandamide content was similar to the significant increase in anandamide observed in lumbar BSG DRG in a model of neuropathic pain.21 We also measured the content of 2-arachidonylglycerol (2-AG). However, the levels of 2-AG in DRG extracts from both control and WA stressed rats were too low to be detected, possibly due to the natural significant lower content of 2-AG as compared with anandamide in DRGs.21 Open in a separate window Figure 2 The level of the endocannabinoid anandamide was increased in L6-S2 DRGs from WA ratsRepresentative chromatograph from DRG extracts from control and WA stress rats depicting the relative abundance of endocannabinoid anandamide. The bar graph illustrated the increase in anandamide content in L6-S2 DRGs in rats following chronic WA stress compared to controls. L6-S2 DRGs from 5 rats in each group were combined for sample extraction to reach the adequate weight for LC-APCI/MS-MS analysis. Chronic WA Stress decreased CB1 Expression in DRG neurons To determine the expression of CB1 receptors, the colonic DRGs (L6-S2) corresponding to the distension area for VMR measurement were identified by retrograde labeling (data not shown). As shown in Fig 3ACB, The CB1 immunoreactivity.In response to CRD, the VMR mean change, expressed as AUC percentage, was significantly higher in the WA stress rats at day 11 compared with the baseline level for the pressures of 20 and 40 mmHg (Fig 1B; 0.05). increased significantly in DRGs from stressed rats. Immunofluorescence and Western blot analysis showed a significant decrease in CB1 and reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats with WIN or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the up-regulation of TRPV1. Conclusions These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia. test was used to examine the data for the expression of TRPV1 and CB1 obtained from Western blot and immunohistochemistry, for corticosterone and fecal pellet output measurements. Results were expressed as means SEM. P 0.05 was considered statistically significant. Results Chronic WA stress resulted in increased visceral nociception The serum corticosterone level was significantly higher in chronic WA rats compared with the controls ( 0.05). In control rats, the serum corticosterone level was 97.8 23.2 ng/ml, while it was 310.8 30.9 ng/ml in stressed rats (Fig. 1A). The VMR to grades intensities of CRD was recorded on day 0 before the start of WA stress and sham stress as the baseline level and recorded again on day 11 after chronic stress. In response to CRD, the VMR mean change, expressed as AUC percentage, was significantly higher in the WA stress rats at day 11 compared with the baseline level for the pressures of 20 and 40 mmHg (Fig 1B; 0.05). The AUC in the WA stress rats increased 109.7 37.2% compared with the controls for the pressure 60 mmHg ( 0.01). The VMR in rats following sham stress did not change significantly at day 11 compared with the baseline level. All 8 rats after repeated WA stress showed increased VMR to CRD, which is consistent with visceral hyperalgesia observed in the rats following repeated WA stress.12 Moreover, fecal pellet outputs were significantly increased in rats on day 1, day 5 and day 10 of WA stress compared with the controls (Fig 1C, 0.05), indicating the altered colonic motor function following repeated WA stress. Open in a separate window Number 1 Effect of chronic water avoidance (WA) stress on serum corticosterone level, visceromotor response (VMR) to colorectal distension (CRD), and colonic engine function( 0.05; **, 0.01. Chronic WA stress increased the level of the endocannabinoid anandamide in DRGs Representative chromatographs depicting the content of anandamide in DRG cells components are demonstrated in Fig. 2A. The value from control L6-S2 DRGs was 111.3 ng/g cells weight for anandamide, whereas it was 160.3 ng/g cells weight in chronic WA stress rats, related to 44% higher anandamide content than that of the controls (Fig. 2B). This percentage increase in anandamide content material was similar to the significant increase in anandamide observed in lumbar DRG inside a model of neuropathic pain.21 We also measured the content of 2-arachidonylglycerol (2-AG). However, the levels of 2-AG in DRG components from both control and WA stressed rats were too low to be detected, possibly due to the natural significant lower content material of 2-AG as compared with anandamide in DRGs.21 Open in a separate window Number 2 The level of the endocannabinoid anandamide was increased in L6-S2 DRGs from WA ratsRepresentative chromatograph from DRG extracts from control and WA pressure rats depicting the relative abundance of endocannabinoid anandamide. The pub graph illustrated the increase in anandamide content material in L6-S2 DRGs in rats following chronic WA.The number of CB1 IR-positive neurons was also significantly decreased in stress rats (57.5 5.8 %) compared with the control (84.3 4.2 %) (Fig. from stressed rats. Immunofluorescence and Western blot analysis showed a significant decrease in CB1 and reciprocal increase in TRPV1 manifestation and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats with WIN or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and clogged the up-regulation of TRPV1. Conclusions These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important part in stress-induced visceral hyperalgesia. test was used to examine the data for the manifestation of TRPV1 and CB1 from Western blot and immunohistochemistry, for corticosterone and fecal pellet output measurements. Results were indicated as means SEM. P 0.05 was considered statistically significant. Results Chronic WA stress resulted in improved visceral nociception The serum corticosterone level was significantly higher in chronic WA rats compared with the settings ( 0.05). In control rats, the serum corticosterone level was 97.8 23.2 ng/ml, while it was 310.8 30.9 ng/ml in stressed rats (Fig. 1A). The VMR to marks intensities of CRD was recorded on day time 0 before the start of WA stress and sham stress as the baseline level and recorded again on day time 11 after chronic stress. In response to CRD, the VMR imply change, indicated as AUC percentage, was significantly higher in the WA stress rats at day time 11 compared with the baseline level for the pressures of 20 and 40 mmHg (Fig 1B; 0.05). The AUC in the WA stress rats improved 109.7 37.2% compared with the settings for the pressure 60 mmHg ( 0.01). The VMR in rats following sham stress did not switch significantly at day time 11 compared with the baseline level. All 8 rats after repeated WA stress showed improved VMR to CRD, which is definitely consistent with visceral hyperalgesia observed in the rats following repeated WA stress.12 Moreover, fecal pellet outputs were significantly increased in rats on day time 1, day time 5 and day time 10 of WA stress compared with the settings (Fig 1C, 0.05), indicating the altered colonic motor function following repeated WA stress. Open in a separate window Number 1 Effect of chronic water avoidance (WA) stress on serum corticosterone level, visceromotor response (VMR) to colorectal distension (CRD), and colonic engine function( 0.05; **, 0.01. Chronic WA stress increased the level of the endocannabinoid anandamide in DRGs Representative chromatographs depicting the content of anandamide in DRG cells components are demonstrated in Fig. 2A. The value from control L6-S2 DRGs was 111.3 ng/g cells weight for anandamide, whereas it was 160.3 ng/g cells weight in chronic WA stress rats, related to 44% higher anandamide content than that of the controls (Fig. 2B). This percentage increase in anandamide content material was similar to the significant increase in anandamide observed in lumbar DRG inside a model of neuropathic pain.21 We also measured the content of 2-arachidonylglycerol (2-AG). However, the levels of 2-AG in DRG components from both control and WA stressed rats were too low to be detected, possibly due to the natural significant lower content material of 2-AG as compared with anandamide in DRGs.21 Open in a separate window Number 2 The level of the endocannabinoid anandamide was increased in L6-S2 DRGs from WA ratsRepresentative chromatograph from DRG extracts from control and WA pressure rats depicting the relative.