Intestinal mucus layer preservation in female rats attenuates gut injury after trauma-hemorrhagic shock

Abstract

Background: We tested the hypothesis that females are more resistant to trauma-hemorrhagic shock (T/HS)-induced gut injury than males, and this is related to better preservation of their intestinal mucus layer, which is influenced in turn by the estrus cycle stage at the time of injury.

Methods: Male, proestrus and diestrus female rats underwent a laparotomy (trauma) and 90 minutes of shock ( approximately 35 mm Hg). At 3 hours after reperfusion, terminal ileum was harvested and stained with Carnoy's Alcian Blue for mucus assessment, hematoxylin and eosin, and periodic acid schiff for villous and goblet cell morphology and injury. Ileal permeability was measured in separate intestinal segments using the ex vivo everted gut sac technique.

Results: When compared with males, proestrus female rats were significantly more resistant to T/HS-induced morphologic gut injury, as reflected in both a lower incidence of villous injury (14% vs. 22%; p < 0.05) and a lesser grade of injury (1.0 vs. 2.8; p < 0.05) as well as preservation of gut barrier function (17.9 vs. 32.2; p < 0.05). This resistance to gut injury was associated with significant preservation of the mucus layer (87% vs. 62%; p < 0.05) and was influenced by the estrus cycle stage of the female rats. There was a significant inverse correlation between mucus layer coverage and the incidence (r = 0.9; p < 0.0001) and magnitude (r = 0.89; p < 0.0001) of villous injury and gut permeability (r = 0.74; p < 0.001).

Conclusions: The resistance of female rats to T/HS-induced intestinal injury and dysfunction was associated with better preservation of the intestinal mucus barrier and was to some extent estrus cycle-dependent. Preservation of the mucus barrier may protect against shock-induced gut injury and subsequent distant organ injury by limiting the ability of luminal contents such as bacteria and digestive enzymes from coming into direct contact with the epithelium.

Figure 1

(A) In male rats, T/HS increased the number of intestinal villi injured plus the magnitude of villous injury as well as intestinal permeability. **p < 0.01 vs. male T/SS group. *p < 0.05 vs. male T/SS group. Data are expressed as mean ± SD (n = 4–6 rats/group). (B) Representative photomicrographs of terminal ileum from T/SS or T/HS rats obtained 3 hours after reperfusion (200×) showing high-grade villous injury after T/HS manifest as sloughing of villous tips and a severely disrupted mucus layer (arrow).

Figure 2

(A) Males rats subjected to T/HS had a significant reduction in their intestinal mucus layer coverage but no change in goblet cell numbers. **p < 0.01 versus male T/SS groups. Data are expressed as mean ± SD (n = 4–6 rats/group). (B) Representative photomicrographs of terminal ileum 3 hours after reperfusion (100×) showing loss of the mucus layer in the T/HS group.

Figure 3

(A) The incidence of T/HS-induced villous injury was increased to a greater degree in the diestrus than the proestrus rats subjected to T/HS as was gut permeability. *p < 0.05 versus all other groups. Data are expressed as mean ± SD (n = 4–6 rats/group). (B) Representative photomicrographs of terminal ileum 3 hours after reperfusion (200×) showing low-grade villous injury after T/HS in proestrus females manifest as subepithelial spaces at the tips (arrows). The diestrus group shows higher degree of gut injury with subepithelial spaces extending all the way to the crypt (arrow).

Figure 4

(A) After T/HS, the magnitude of loss of the mucus layer was greater in the diestrus than the proestrus females when compared with the T/SS group. *p < 0.05 versus all other groups #p < 0.05 versus T/SS group. Data are expressed as mean ± SD (n = 4–6 rats/group). (B) Representative photomicrographs of terminal ileum 3 hours after reperfusion (100×) showing better preservation of the mucus layer and goblet cells in proestrus compared with diestrus rats subjected to T/HS.

Figure 5

Both proestrus and diestrus female rats subjected to T/HS were more resistant to gut injury (A), loss of gut barrier function (A) or loss of the mucus layer than male rats (B). *p < 0.05 versus all other groups. **p < 0.01 versus all other groups. Data are expressed as mean ± SD (n = 5–6 rats/group).

Figure 6

There is significant inverse correlation between that the degree of mucus layer coverage with the incidence (r² = 0.9; p < 0.0001; A) and grade (r² = 0.89; p < 0.0001; B) of villous injury and gut permeability (r² = 0.74; p < 0.001; C). All T/HS and T/SS animals were used.