Functional and morphological changes of the gut barrier during the restitution process after hemorrhagic shock

Abstract

Aim: To investigate the functional, morphological changes of the gut barrier during the restitution process after hemorrhagic shock, and the regional differences of the large intestine and small intestine in response to ischemia/reperfusion injury.

Methods: Forty-seven Sprague-Dawley rats with body weight of 250-300 g were divided into two groups: control group (sham shock n = 5) and experimental group (n = 42). Experimental group was further divided into six groups (n = 7 each) according to different time points after the hemorrhagic shock, including 0(th) h group, 1st h group, 3rd h group, 6th h group, 12th h group and 24th h group. All the rats were gavaged with 2 mL of suspension of lactulose (L) (100 mg/2 mL) and mannitol (M) (50 mg/each) at the beginning and then an experimental rat model of hemorrhagic shock was set up. The specimens from jejunum, ileum and colon tissues and the blood samples from the portal vein were taken at 0, 1, 3, 6, 12 and 24 h after shock resuscitation, respectively. The morphological changes of the intestinal mucosa, including the histology of intestinal mucosa, the thickness of mucosa, the height of villi, the index of mucosal damage and the numbers of goblet cells, were determined by light microscope and/or electron microscope. The concentrations of the bacterial endotoxin lipopolysaccharides (LPS) from the portal vein blood, which reflected the gut barrier function, were examined by using Limulus test. At the same time point, to evaluate intestinal permeability, all urine was collected and the concentrations of the metabolically inactive markers such as L and M in urine were measured by using GC-9A gas chromatographic instrument.

Results: After the hemorrhagic shock, the mucosal epithelial injury was obvious in small intestine even at the 0(th) h, and it became more serious at the 1st and the 3rd h. The tissue restitution was also found after 3 h, though the injury was still serious. Most of the injured mucosal restitution was established after 6 h and completed in 24 h. Two distinct models of cell death-apoptosis and necrosis-were involved in the destruction of rat intestinal epithelial cells. The number of goblet cells on intestinal mucosa was reduced significantly from 0 to 24 h (the number from 243+/-13 to 157+/-9 for ileum, 310+/-19 to 248+/-18 for colon; r = -0.910 and -0.437 respectively, all P<0.001), which was the same with the large intestine, but the grade of injury was lighter with the values of mucosal damage index in 3 h for jejunum, ileum, and colon being 2.8, 2.6, 1.2, respectively. The mucosal thickness and the height of villi in jejunum and ileum diminished in 1 h (the average height decreased from 309+/-24 to 204+/-23 microm and 271+/-31 to 231+/-28 microm, r = -0.758 and -0.659, all P<0.001; the thickness from 547+/-23 to 418+/-28 microm and 483+/-45 to 364+/-35 microm, r = -0.898 and -0.829, all P<0.001), but there was no statistical difference in the colon (F = 0.296, P = 0.934). Compared with control group, the urine L/M ratio and the blood LPS concentration in the experimental groups raised significantly, reaching the peak in 3-6 h (L/M: control vs 3 h vs 6 h was 0.029+/-0.09 vs 0.063+/-0.012 vs 0.078+/-0.021, r = -0.786, P<0.001; LPS: control vs 3 h vs 6 h was 0.09+/-0.021 vs 0.063+/-0.012 vs 0.25+/-0.023, r = -0.623, P<0.001), and it kept increasing in 24 h.

Conclusion: The gut barrier of the rats was seriously damaged at the early phase of ischemic reperfusion injury after hemorrhagic shock, which included the injury and atrophy in intestinal mucosa and the increasing of intestinal permeability. Simultaneously, the intestinal mucosa also showed its great repairing potentiality, such as the improvement of the intestinal permeability and the recovery of the morphology at different phases after ischemic reperfusion injury. The restitution of gut barrier function was obviously slower than that of the morphology and there was no direct correlation between them. Compared with the small intestine, the large intestine had stronger potentiality against injury. The reduction of the amount of intestinal goblet cells by injury did not influence the ability of intestinal mucosal restitution at a certain extent and it appeared to be intimately involved in the restitution of the epithelium.

Figure 1

Sections of ileum following ischemia-reperfusion injury after resuscitation of hemorrhagic shock. A: Shedding of cell at the tip of the villi is apparent (1 h); B: Notes the sloughed epithelial cells detached from the villi and some regions of the villi are destroyed, goblet cells reside throughout the length of the villi (3 h); C, D: extensive denudation and collapse of the villi and the enterocytes in the upper of the villi had lifted and detached from the villi (3 h); E: the tip of the villi was denuded, but some of the goblet cells still remained on the surface (3 h); F: Restitution is nearly completed and the area of the villi covered by goblet cells and their number are decreased (6 h); G: The restitution of the villi is complete except for the shortening of the villous height and goblet cells cover the villi (12 h). Epithelial cells are lifted off the basement membrane after 1 h. Original magnification: ×40 (A), ×100 (B, C, D, G, H); ×200 (E).

Figure 2

Electron microscopic examination of ileum following ischemia-reperfusion injury after resuscitation of hemorrhagic shock. The apoptosis and necrosis were main manifestation in the intestinal mucosal injury. A: Many enterocytes show compaction and segregation of chromatin against the nuclear envelope, but still preserve mcrovilli and intact mitochondria; B: Showing the appearance of membrane blebs which cause detachment from the basement membrane; C: Some enterocytes show the swelling of mitochondria and endoplasmic reticulum, scarce and lodging microvilli; D: the opening of tight junction among the cells. Original magnification: ×2 500 (A, B), ×4 000 (C,D).

Figure 3

Mucosal damage index in small intestine and colon following resuscitation after hemorrhagic shock. Bars are mucosal damage index, the color represented different sites.

Figure 4

Linear fitness between the standard amount of LPS and absorbance A-value of LPS in portal vein plasma.