Early weaning stress induces chronic functional diarrhea, intestinal barrier defects, and increased mast cell activity in a porcine model of early life adversity

Abstract

Background: Early life adversity (ELA) is a risk factor for development of gastrointestinal disorders later in life. The underlying mechanisms through which ELA and sex interact to influence disease susceptibility remains poorly understood.

Methods: Utilizing a porcine early weaning stress (EWS) model to mimic ELA, we investigated the long-term effects of EWS on functional diarrhea, ileal permeability, mast cell activity and mast cell relationship with enteric ganglia.

Key results: Juvenile and adult EWS pigs exhibited chronic, functional diarrhea (EWS 43.6% vs late wean control(LWC) 4.8%, P<.0001), increased intestinal permeability (2 fold increase EWS vs LWC, P<.0001), and mast cell numbers (at 7 weeks and 20 weeks ~1.6 fold increase EWS vs LWC, P<.05). Compared with EWS male castrates (Male-C), females EWS pigs exhibited more frequent diarrhea (58.8% vs 29.9%, P=.0016), and increased intestinal permeability (1-2 fold higher in EWS females, P<.001). Increased mast cell numbers and their enhanced co-localization with neuronal ganglia were observed in both Male-C and female EWS pigs; however, female pigs exhibited greater release of mast cell tryptase upon activation with c48/80 (~1.5 fold increase, P<.05), compared with Male-C pigs.

Conclusions and inferences: These data demonstrate that pigs exposed to ELA exhibit increased vulnerability to functional diarrhea, intestinal permeability and mast cell activity. Further, these studies also showed that EWS female and Male-C pigs exhibited dimorphic responses to EWS with female piglets exhibited greater susceptibility and severity of diarrhea, intestinal permeability and mast cell tryptase release. Together, these findings mimic some of the key pathophysiologic findings in human functional GI disorders functional gastrointestinal disorders (FGIDs) suggesting that the EWS porcine model could be a valuable preclinical translational model for FGID research associated with ELA.

Keywords: developmental origins of health and disease; early life adversity; intestinal permeability; large animal model; mast cell; mast cell plexitis; translational research.

Figure 1. Influence of early weaning stress in pigs on diarrhea frequency and intestinal histopathology

Stool scores were conducted in pigs between 16–20 weeks of age using the Bristol Stool Scale Scoring System. A) % of days with diarrhea (stool score ≥ 6) in LWC and EWS pigs, B) Comparison of Male-C and female pigs (combined LWC and EWS pig data) C) % of days with diarrhea for Male-C and female pigs; D) % of days with severe diarrhea (stool score ≥ 7) in EWS Male-C and females. E&F) Representative Bristol Stool Form Scores over a 25-d period (dotted line indicating threshold for diarrhea). G–H) Representative H&E stained sections of ileum mucosa. I–J) Representative H&E stained sections of colonic mucosa. Scale bar = 100 μM. Data (A–D) are means ± standard error. Student’s T-test (A,B, and D) *** p<0.001, * p<0.05; Letters “a, b, c” (Panel C) indicate statistical significance between groups by Two way ANOVA with Fisher’s LSD post hoc test. LWC = Late Weaned Control, EWS = Early Weaning Stress, Male-C = male castrated pigs. LWC=Late.

Figure 2. Influence of early weaning stress in pigs on ileal permeability

Ileal mucosa from pigs were mounted on Ussing Chambers for evaluation of permeability. A) FD4 flux in 7 week old Male-C and female pigs, data presented as fold change relative to respective to LWC control. B) FD4 flux in 20 week old Male-C and female pigs, C) ³H-mannitol flux in 20 week old Male-C and female pigs. D) Ileal transepithelial resistance (TER) in 7 week old Male-C and female pigs. E) Ileal TER in 20 week old Male-C and female pigs. Data presented are means ± standard error (n=6/experimental group). Two way ANOVA with Fisher’s LSD post hoc test for panels A–E. ****p<0.0001, **p<0.01,*p<0.05. LWC=Late Weaned Control. EWS=Early Weaning Stress, Male-C = male castrated pigs.

Figure 3. Influence of early weaning stress in pigs on ileal and colonic mast cell numbers

Ileum sections from pigs were stained for mast cell tryptase and tryptase stained cells were quantified to determine mast cell numbers. A) Ileal mast cell (MC) numbers in 7 week old LWC and EWS pigs. Ai) Ileal mast cell numbers in 7 week old Male-C and female pigs. Aii) LWC ileum mucosa and Aiii) EWS ileum mucosa (inset is 100x image demonstrating tryptase-positive mast cell morphology). Arrows indicate red stained tryptase positive mast cells. B) Ileal mast cell numbers in 20 week old pigs. Bi) Ileal mast cell numbers 20 week old Male-C and female pigs. Bii) LWC ileum mucosa and Biii) EWS ileum mucosa. Arrows indicate red staining mast cells. C) Ileal mast cell numbers determined by Toluidine blue staining in 7 week old pigs. Ci) Ileal mast cell numbers determined by Toluidine blue staining in 7 week old Male-C and female pigs. D) Colonic mast cell numbers determined in 7 week old pigs. Di) Colonic mast cell numbers in 7 week old Male-C and female pigs. Data presented are means ± standard error (n=6/experimental group) Two way ANOVA with Fisher’s LSD post hoc test. **p<0.01,*p<0.05. LWC=Late Weaned Control. EWS=Early Weaning Stress, Male-C = male castrated pigs. All images 20x magnification. Scale bar = 50 μm. LWC=Late Weaned Controls. EWS=Early Weaning Stress.

Figure 4. Influence of early weaning stress on ileal mast cell tryptase release

Ileal mucosal explants from 20 week old pigs were treated with secretagogue c48/80 and tryptase was measured in the supernatant via a substrate-based tryptase activity assay. A) EWS pigs tend to release more mast cell tryptase when treated with c48/80. B) EWS female ileum sections released more mast cell tryptase into supernatants compared LWC females and EWS Male-C pigs. Two Way ANOVA with Fisher’s LSD post hoc test (*p<0.01); (**p<0.005). LWC=Late Weaned Controls. EWS=Early Weaning Stress, Male-C = male castrated pigs

Figure 5. Influence of early weaning stress on the numbers of enteric ganglia-associated mast cells in pigs

Ileal mucosa/submucosa segments from 7 week old EWS and LWC pigs (Male-C and female pigs combined within each weaning experimental group) were stained for mast cell tryptase or T-blue and the number of mast cells (MC) co-localized with neuronal ganglia in the submucosal plexus (SMP) and myenteric plexus (MP) were quantified. A) SMP-associated MCs (tryptase-positive) in LWC and EWS pigs Ai) LWC ileum SMP tryptase stained, Aii) EWS ileum SMP tryptase stained. B) SMP-associated MCs (T.blue stained) in LWC and EWS pigs. Bi) LWC ileum SMP, T-blue stained. Bii) EWS ileum SMP, T-blue stained. C) MP-associated MCs (tryptase-positive) in LWC and EWS pigs Ci) LWC ileum MP tryptase stained, Cii) EWS ileum MP tryptase stained. B) SMP-associated MCs (T.blue stained) in LWC and EWS pigs. Images are 40x. Scale bar = 25 μm. Black arrows indicated periphery of ganglia. Black arrowheads identify mast cells. Data (A–C) are means ± standard error. Students T-test (* p<0.05) LWC=Late Weaned Controls. EWS=Early Weaning Stress.