Hyperthermia induces injury to the intestinal mucosa in the mouse: evidence for an oxidative stress mechanism

Abstract

Loss of the intestinal barrier is critical to the clinical course of heat illness, but the underlying mechanisms are still poorly understood. We tested the hypothesis that conditions characteristic of mild heatstroke in mice are associated with injury to the epithelial lining of the intestinal tract and comprise a critical component of barrier dysfunction. Anesthetized mice were gavaged with 4 kDa FITC-dextran (FD-4) and exposed to increasing core temperatures, briefly reaching 42.4°C, followed by 30 min recovery. Arterial samples were collected to measure FD-4 concentration in plasma (in vivo gastrointestinal permeability). The small intestines were then removed to measure histological evidence of injury. Hyperthermia resulted in a ≈2.5-fold elevation in plasma FD-4 and was always associated with significant histological evidence of injury to the epithelial lining compared with matched controls, particularly in the duodenum. When isolated intestinal segments from control animals were exposed to ≥41.5°C, marked increases in permeability were observed within 60 min. These changes were associated with release of lactate dehydrogenase, evidence of protein oxidation via carbonyl formation and histological damage. Coincubation with N-acetylcysteine protected in vitro permeability during hyperthermia and reduced histological damage and protein oxidation. Chelation of intracellular Ca(2+) to block tight junction opening during 41.5°C exposure failed to reduce the permeability of in vitro segments. The results demonstrate that hyperthermia exposure in mouse intestine, at temperatures at or below those necessary to induce mild heatstroke, cause rapid and substantial injury to the intestinal lining that may be attributed, in part, to oxidative stress.

Fig. 1.

A: average time profiles of core temperature during the heating protocol (T_max: = 42.4°C). B: plasma levels of 4 kDa FITC dextran (FD-4) following in vivo heat treatment and 30 min recovery. **P < 0.01 compared with time-matched controls. C: intestinal permeability, measured in vitro, 4 kDa rhodamine-dextran (TD-4) following in vivo hyperthermia (P = 0.5) (n = 6 hyperthermia; n = 4 controls).

Fig. 2.

Representative hematoxylin and eosin staining of intestinal epithelium from specified regions of control mice (left) and heatstroke mice (right). There was consistent evidence of disorganized and blunted epithelium in the heatstroke animals, with more predominant evidence of injury in the duodenum.

Fig. 3.

A: comparison of intestinal permeability in in vitro intestinal segments when exposed to increasing bath temperatures (n = 4 mice in each); *P < 0.05 from 37°C. B: time course of changes in permeability in intestinal segments exposed to 41.5°C vs. 37°C (n = 4 mice at each time point). C: lactate dehydrogenase (LDH) release over time in segments exposed to 41.5°C or 37°C (n = 4 mice for each time point). For both B and C: ΦP < 0.01 from initial 15 min time point; *P < 0.05 for 41.5°C vs. 37°C at 90 min; +P < 0.05 from initial 15-min time point in 37°C-treated group.

Fig. 4.

Effects of hyperthermia on protein oxidation (carbonyl formation). A: densitometry averages in segments studied over time **P < 0.01 (n = 6 for each time point) B: left gel representative lanes from the same gel used for grouped densitometry data in A. Right gel is representative of experiments in heated (41.5°C) intestine, with and without coincubation with NAC. These 2 lanes are from a different gel from the series on the left. Dotted vertical lines show where the images were cut to exhibit different lanes from the same gel. The shaded bars in the middle are the approximate location of the bands that were used to generate a summed densitometry signals in each lane. C: grouped effects of NAC on heat-induced carbonyl formation. ***P < 0.001; n = 6 for each treatment.

Fig. 5.

Effects of antioxidant treatment of permeability in in vitro intestinal segments. **P < 0.01; n = 8 for all groups except Tiron and Trolox treatments, where n = 4.

Fig. 6.

A: representative hematoxylin and eosin slides of effects of 90 min incubation of in vitro everted intestinal segments at 37°C compared with 41.5°C (B) and 41.5°C + NAC (C). D: grouped data of overall damage index of histological specimens from intestinal segments in control (n = 10), heated (n = 10), and NAC-heated (n = 8) animals. ***P < 0.001 compared with 37°C control; ΦP < 0.001 compared with 41.5°C control.

Fig. 7.

Calcium chelation by BAPTA-AM (n = 4 in each group). *P < 0.05 and **P < 0.01 compared with 37°C control.