Chronic stress-associated visceral hyperalgesia correlates with severity of intestinal barrier dysfunction

Abstract

In humans, chronic psychological stress is associated with increased intestinal paracellular permeability and visceral hyperalgesia, which is recapitulated in the chronic intermittent water avoidance stress (WAS) rat model. However, it is unknown whether enhanced visceral pain and permeability are intrinsically linked and correlate. Treatment of rats with lubiprostone during WAS significantly reduced WAS-induced changes in intestinal epithelial paracellular permeability and visceral hyperalgesia in a subpopulation of rats. Lubiprostone also prevented WAS-induced decreases in the epithelial tight junction protein, occludin (Ocln). To address the question of whether the magnitude of visceral pain correlates with the extent of altered intestinal permeability, we measured both end points in the same animal because of well-described individual differences in pain response. Our studies demonstrate that visceral pain and increased colon permeability positively correlate (0.6008, P = 0.0084). Finally, exposure of the distal colon in control animals to Ocln siRNA in vivo revealed that knockdown of Ocln protein inversely correlated with increased paracellular permeability and enhanced visceral pain similar to the levels observed in WAS-responsive rats. These data support that Ocln plays a potentially significant role in the development of stress-induced increased colon permeability. We believe this is the first demonstration that the level of chronic stress-associated visceral hyperalgesia directly correlates with the magnitude of altered colon epithelial paracellular permeability.

Fig. 1. WAS-induced visceral hypersensitivity is decreased with lubiprostone treatment

Rats were subjected to sham stress (controls, Ctrl, n=11) or WAS (n=11) for 1 hr/day for 10 consecutive days. WAS rats received MCT vehicle (WAS) or lubiprostone (WAS+L, n=15) treatment twice daily. 24 hrs after WAS/sham stress, electromyographic response (EMR) to colorectal distention at 20, 40, and 60 mmHg of pressure was tested using surgically implanted electrodes in the abdominal wall muscle. Data were analyzed with two-way ANOVA, * p ≤ 0.02, ** p ≤ 0.0001. (A) Differences in EMR were observed between Ctrl/Ctrl+L and WAS at 40 and 60 mmHg of distention. (B) Lubiprostone treatment significantly decreased VMR only at 60 mmHg distention.

Fig. 2. WAS-induced gut permeability is prevented with lubiprostone treatment

Rats were subjected to sham stress (Ctrl, n=7)) or WAS (n=14) for 1 hr/day for 10 consecutive days while receiving MCT vehicle or lubiprostone (Ctrl+L, n=5) and WAS+L, n=8)) treatment twice daily. On day 11, rats were treated with 4 KDa FITC-Dextran by gavage and after 4 hours blood was collected by heart puncture. Samples were processed, plasma fluorescence levels determined, and the ng/mL calculated using a standard curve. Data was analyzed by one-way ANOVA. A) No significant differences were observed until a sub-group analysis was performed (B). A subset of rats responded to WAS with increased gut permeability (WAS-R, n=7, > 2 SDs from the average Ctrl value, > 499 ng/mL) and the rest were non-responsive to WAS (WAS-NR, n=7, ≤ 2SDs from the average Ctrl value).

Fig. 3. Levels of the tight junction protein Occludin inversely correlated with gut permeability

Western blot analysis of distal colon tissue extracts from control (Ctrl), WAS, and lubiprostone treated WAS (WAS+L) and control (Ctrl+L) rats were performed. A subset of rats responded to WAS with increased gut permeability (WAS-R, > 2 SDs from the average Ctrl value, > 499 ng/mL) and the remaining were non-responsive to WAS (WAS-NR, ≤ 2SDs from the average Ctrl value). A) Representative Western blots of Occludin (Ocln) and β-actin. B) Blots were quantitated, using β-actin as a loading control, with ImageJ. Protein levels are expressed as a percentage of a control sample loaded on all blots (average ± SEM). P-values were calculated using unpaired t-tests, not assuming equal SD due to large, variation between treatment groups, with an adjustment for multiple observations, 2% FDR. C) Occludin (Ocln) protein levels (% control) versus gut permeability to 4 KDa FITC-dextran (ng/mL) was graphed for control (filled circle), control + lubiprostone (open circle), WAS-NR (triangle), WAS-R (square), and WAS+L (diamond) rat groups. The Pearson and Spearman’s rank-order correlations were determined; the Spearman’s correlation was more significant, indicating a non-linear relationship, and is shown.

Fig. 4. WAS rats with increased intestinal permeability, demonstrated a decrease in Occludin protein levels in colon epithelial cells, which was prevented with lubiprostone treatment

Immunostaining for Ocln protein is shown for sham stress rats (Ctrl), sham stress rats treated with lubiprostone (Ctrl+L), and WAS rats with and without lubiprostone treatment (WAS+L). Tissue from rats that demonstrated increased gut permeability due to WAS, WAS-R, or did not demonstrate increased gut permeability (WAS-NR), are shown separately. Both sagittal (first column) and transverse (2^nd and 3^rd column) views are shown. Yellow arrows indicate examples of the normal localization of Ocln protein, small white arrows indicate examples of abnormal localization or decreased stained areas, and large white arrows indicate examples of increased Ocln staining or localization at the basal side of the colon.

Fig. 5. Level of visceral pain correlates with the magnitude of change in colon permeability

Rats were subjected to sham stress (Ctrl, n=8) or WAS (n=10) for 1 hr/day for 10 consecutive days. Visceral motor response (VMR) was measured using the non-invasive manometric method on the day before stress/sham stress and 24 hrs after the last WAS/sham stress session. Each rat served as its own control, with data expressed as a percentage of the average 60 mmHg pre-stress measurement (C and D). 24 hrs post WAS/sham stress, the distal colon was removed, the serosal musculature stripped away, and the tissue put into an Ussing Chamber to determine the mucosal to serosal transport of 4 KDa FITC-dextran, apparent permeability (Papp, A and B), and transepithelial resistance (TER, E and F) for each rat. A subgroup analysis based on permeability was performed to separate rats into WAS-responders (WAS-R, n=3), those with Papp values greater than > 2 SDs from the average Ctrl value, and WAS non-responsive (WAS-NR, n=7), those with Papp values ≤ 2 SDs from the average Ctrl (B,D, and F). All data is expressed as mean ± SD. A student t-test (2 groups) or one-way ANOVA analysis (3 groups) was performed to determine significance. The Pearson and Spearman’s rank-order correlations were determined for Papp vs. VMR (G) and Papp vs. TER (H); the Pearson correlation was more significant, indicating a linear relationship, and is shown (n=17).

Fig. 6. Knockdown of Ocln protein with si-RNA in the colon of control rats leads to increased visceral pain

Control rats were treated intrarectally with Ocln-specific siRNA (Ocln siRNA, n=3) or non-specific, scrambled siRNA (Ctrl siRNA, n=3). A) VMR to CRD was measured one day before treatment and 48 hrs after treatment. Each rat served as its own control, with VMR expressed as the percent of the pre-treatment 60 mmHg distention value. After VMR to CRD measurements, colon epithelium was collected from the distal (D) and proximal (P) colon for each rat. Epithelial extracts were analyzed in duplicate by Western blot at two different exposures within the linear range. C and D) Western blots of Occludin (Ocln) and β-actin are shown. Blots were quantitated, using β-actin as a loading control, with ImageJ. Protein levels are expressed as a percentage of proximal colon epithelial levels. B) In a second group of rats, 48 hrs after Ocln siRNA (n=5) or control siRNA (n=5) treatment, the distal colon was removed, the serosal musculature stripped away, and the tissue put into an Ussing Chamber to determine the mucosal to serosal transport of 4 KDa FITC-dextran, apparent permeability (Papp). Some bars are too small to see. * p < 0.001, t-test with a 1% FDR.