Early weaning stress impairs development of mucosal barrier function in the porcine intestine

Abstract

Early life stress is a predisposing factor for the development of chronic intestinal disorders in adult life. Here, we show that stress associated with early weaning in pigs leads to impaired mucosal barrier function. Early weaning (15- to 21-day weaning age) resulted in sustained impairment in intestinal barrier function, as indicated by reductions in jejunal transepithelial electrical resistance and elevations in mucosal-to-serosal flux of paracellular probes [(3)H]mannitol and [(14)C]inulin measured at 5 and 9 wk of age, compared with that shown in late-weaned pigs (23- to 28-day weaning age). Elevated baseline short-circuit current was observed in jejunum from early-weaned pigs and was shown to be mediated via enhanced Cl(-) secretion. Jejunal barrier dysfunction in early-weaned pigs coincided with increased lamina propria immune cell density particularly mucosal mast cells. The mast cell stabilizer drug sodium cromoglycolate ameliorated barrier dysfunction and hypersecretion in early-weaned pigs, demonstrating an important role of mast cells. Furthermore, activation of mast cells ex vivo with c48/80 and corticotrophin-releasing factor (CRF) in pig jejunum mounted in Ussing chambers induced barrier dysfunction and elevations in short-circuit current that were inhibited with mast cell protease inhibitors. Experiments in which selective CRF receptor antagonists were administered to early-weaned pigs revealed that CRF receptor 1 (CRFr1) activation mediates barrier dysfunction and hypersecretion, whereas CRFr2 activation may be responsible for novel protective properties in the porcine intestine in response to early life stress.

Fig. 1.

Effects of weaning age on postweaning barrier function in porcine jejunum. Pigs were subjected to different weaning ages (at 15, 18, 21, 23, and 28 days old), and jejunal tissues were harvested from 35-day-old pigs for measurement of transepithelial electrical resistance (TER) (A), mucosal-to-serosal [³H]mannitol flux (B), and mucosal-to-serosal [¹⁴C]inulin flux (C). Values are means ± SE (n = 6 pigs per weaning age treatment). ^a,b,cSignificantly different (P < 0.05) by ANOVA.

Fig. 2.

Effects of weaning age on postweaning short-circuit current (I_sc) in porcine jejunum. Pigs were subjected to different weaning ages (15, 18, 21, 23, and 28 days old), and jejunal tissues were harvested from 35-day-old pigs for measurement of I_sc. Values are means ± SE (n = 6 pigs per weaning age treatment). ^a,b,cSignificantly different (P < 0.05) by ANOVA.

Fig. 3.

Effects of weaning age on long-term mucosal barrier function in porcine jejunum. Pigs were weaned either at 15 days of age (early weaning) or 28 days of age (late weaning), and jejunal tissues were harvested from 9-wk-old pigs for measurement of TER (A) and mucosal-to-serosal [³H]mannitol flux (B). ^aSignificantly different (P < 0.05) by ANOVA.

Fig. 4.

Mechanisms of altered I_sc as influenced by weaning age. A: jejunum from 35-day-old pigs that were early weaned (at 15 days of age) was mounted on Ussing chambers. After a 30-min equilibration period, normal Ringer solution was replaced with Cl⁻-free Ringer solution, and resultant changes in I_sc were monitored. Data represent averages from 5 pigs and were analyzed using repeated-measures ANOVA. Treatments differed by P < 0.05; pooled SE value = 4.3. B: jejunum from late-weaned pigs (at 28 days of age) was treated with the K⁺ channel blocker BaCl₂ on the mucosal surface, after which changes in I_sc were monitored. Data represent averages from 5 pigs and were analyzed using repeated-measures ANOVA. BaCl₂ treatment induced a positive deflection in I_sc (P < 0.05).

Fig. 5.

Histological analysis of porcine jejunum as influenced by weaning age. Results show representative jejunal sections from 35-day-old pigs previously subjected to different weaning ages. A: weaning at 15 days old. B: weaning at 18 days old. C: weaning at 23 days old. D: lamina propria cell counts. Notice the increased cellularity of the lamina propria in jejunum from early-weaned pig compared with jejunum from pigs weaned at older ages. Values are means ± SE (n = 6 pigs per weaning age treatment). ^a,b,cSignificantly different (P < 0.05) by ANOVA.

Fig. 6.

Effect of weaning age on mast cell (MC) numbers and activation. Porcine jejunum from early-weaned (A and C) and late-weaned (B and D) pigs were fixed in Carnoy's fixative, sectioned, and stained with toluidine blue to visualize MCs. Arrows indicate toluidine blue-positive MCs. Increased numbers of degranulated MCs were observed in tissues from early-weaned pigs (C; ×100 magnification) compared with jejunum from late-weaned pigs (D). E: MC counts were performed in sections from 4 pigs and are expressed as the number of toluidine blue-positive cells/mm² tissue. ^a,b,cSignificantly different (P < 0.05) by ANOVA.

Fig. 7.

MC tryptase expression in porcine jejunum. Porcine jejuna mucosa from pigs weaned at different ages was harvested from pigs at 35 days of age, and Western blot analysis was performed to assess MC tryptase expression levels. Jejunal MC tryptase expression decreased as weaning age increased as determined by SDS-PAGE (A) and densitometric analysis relative to β-actin (B). AU, arbitrary units. ^a,b,cSignificantly different (P < 0.05) by ANOVA.

Fig. 8.

Effect of in vivo MC inhibition on intestinal barrier function and I_sc in early-weaned porcine jejunum. Values represent means ± SE; n = 6 animals. Pigs (35 days of age) were injected with cromolyn (20 mg/kg ip) or saline vehicle (control) 24, 16, and 8 h before mounting jejunum on Ussing chambers. Compared with jejunum from late-weaned control (23 day weaning), jejunum from early-weaned (15 day weaning) pigs treated with saline vehicle had lower TER and greater mucosal-to-serosal [³H]mannitol flux and I_sc. Cromolyn treatment restored TER, [³H]mannitol flux, and I_sc to late-weaned control levels. ^aP < 0.05 (ANOVA).

Fig. 9.

Effect of weaning age on hypothalamic-pituitary-adrenal axis activity. Serum samples from 35-day-old pigs that were weaned at 15, 18, and 23 days of age were analyzed for corticotrophin-releasing factor (CRF) and cortisol levels. Early-weaned pigs (at 15 and 18 days) exhibited higher baseline serum CRF (A) and cortisol (B) than late-weaned pigs. Jejunal CRF was measured using ELISA methods (C), which demonstrated that early-weaned pigs (at 15 and 18 days) exhibited higher CRF levels than late-weaned pigs. Values are means (n = 6 animals/treatment) ± SE. ^aP < 0.05 (ANOVA).

Fig. 10.

Effects of CRF on I_sc (A) and barrier function (B) in porcine jejunum mounted on Ussing chambers. Early-weaned porcine jejunum was mounted on Ussing chambers and treated with CRF (1 μM) or the MC activator c48/80 (5 μg/ml) on the serosal surface of tissues. Cromolyn (10⁻⁴ M) and protease inhibitors (Roche Complete Mini tablets) were added 30 min before CRF. Values represent means (n = 6 animals/treatment) ± SE. ^a,bSignificantly different (P < 0.05) by ANOVA.

Fig. 11.

CRF receptor subtype and glucocorticoid receptor (GR) expression in jejunum from early- and late-weaned pigs. A: Western analysis of CRFr1 (50–55 kDa), CRFr2 (50–55 kDa), and GR (95 kDa) in porcine jejuna mucosa. B: densitometric analysis revealed enhanced expression of CRFr2 in jejunum from late-weaned pigs, whereas GR expression was elevated but decreased as weaning age increased. Values, expressed relative to β-actin control (n = 6 animals/treatment), are means ± SE.

Fig. 12.

Effect of in vivo CRF receptor antagonists on intestinal barrier function and I_sc in early-weaned porcine jejunum. Values represent means ± SE; n = 8 animals. Pigs (35 days of age) were injected intraperitoneally with saline vehicle, astressin B (CRFr1/r2 antagonist, 30 μg/kg), or astressin 2B (CRFr2 antagonist, 30 μg/kg) 24, 16, and 8 h before jejunum was mounted on Ussing chambers. Compared with jejunum from late-weaned controls (23 day weaning), jejunum from early-weaned pigs (15 day weaning) treated with saline vehicle had lower TER (A) and greater mucosal-to-serosal [³H]mannitol flux (B) and I_sc (C). Astressin B treatment restored TER, [³H]mannitol flux, and I_sc to late-weaned control levels. Astressin 2B treatment enhanced barrier dysfunction, as indicated by reductions in TER and elevations in I_sc compared with tissues from late-weaned controls. ^a,bSignificantly different (P < 0.05) by ANOVA.