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. 1998 Mar;42(3):366–373. doi: 10.1136/gut.42.3.366

Focal reduction of villous blood flow in early indomethacin enteropathy: a dynamic vascular study in the rat

D Kelly 1, C Piasecki 1, A Anthony 1, A Dhillon 1, R Pounder 1, A Wakefield 1
PMCID: PMC1727016  PMID: 9577343

Abstract

Background—Oral indomethacin causes villous shortening, microvascular damage, and distortion, which might induce mucosal ischaemia and necrosis. 
Aims—In order to determine the early events in indomethacin induced jejunal injury we examined the temporal relations between morphological damage and changes in villous blood flow following indomethacin. 
Methods—In anaesthetised rats, mid jejunal villi were exteriorised in a chamber and observed by fluorescence microscopy. Blood flow in surface capillaries was calculated from velocities and diameters. Indomethacin was applied by both luminal and intravenous routes for 90 minutes, after which the animal was perfusion fixed and the villi were processed for histological examination. Control animals received intravenous or luminal bicarbonate (1.25%). 
Results—Blood flow slowed in individual villi at 20 minutes, and progressed to complete stasis (in another group) by 45 minutes. Histological examination at 20 minutes revealed microvascular distortion, but no villous shortening: crypt depth:villous height ratios were 0.356 (0.02) in test and 0.386 (0.01) in surrounding villi (p>0.5). At stasis, the villi under study showed epithelial clumping and were shortened: crypt depth:villous height ratios were 0.92 (0.2) in test and 0.42 (0.06) in surrounding villi (p<0.02). Vehicle alone had no effect on either blood flow or histology. 
Conclusions—Focal slowing of villous blood flow and microvascular distortion precede villus shortening and epithelial disruption, and indicate that damage to surface microvasculature is an early event in indomethacin induced mucosal injury in this model. 



Keywords: indomethacin; jejunum; villi; microcirculation; endothelium; microthrombi

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Figure 1 .

Figure 1

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Apparatus used for in vivo microscopy. The water jacket is attached to the side of the animal and a loop of jejunum exteriorised and placed on an observation peg. A small water bath is then placed using micromanipulators onto the mucosal surface of the jejunum and test solutions placed into this bath. 


Figure 2 .

Figure 2

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Effect of either luminal or intravenous indomethacin (100 µg/ml, 2.8 × 10−4M) and bicarbonate control (0.034%) on jejunal villous blood flow. Each point expressed as mean (SEM) of five rats (p=0.9, NS). 


Figure 3 .

Figure 3

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Normal jejunal villous tip from a rat which received luminal indomethacin (100 µg/ml, 2.8 × 10−4M) and was perfusion fixed 90 minutes after dosing. Subepithelial capillaries are cleared of blood (arrow) indicating no vascular occlusion/blockage, with good preservation of villous architecture. Haematoxylin and eosin, original magnification ×250. 


Figure 4 .

Figure 4

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Effect of a combined intravenous and luminal dose of indomethacin (100 µg/ml, 2.8 × 10−4M) and bicarbonate control on jejunal villous blood flow. Each point expressed as mean (SEM) of five rats. *p<0.02, †p<0.005. 


Figure 5 .

Figure 5

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(A) Video image obtained from a normal in vivo villus prior to application of indomethacin; the vasculature is highlighted with FITC-dextran in the plasma. Bottom left: line diagram showing the vascular anatomy of the villus in A. Notice the central artery ascending the centre of the villus and dividing at the tip as a T junction to form the arcade artery. This runs along to the left along the tip to supply the left half of the villus, and along the right tip for the right half of the villus. (B) The same villus after application of indomethacin: the darkened area in the upper left half is an area of blood stasis; this is shown more clearly in the line drawing. 


Figure 6 .

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(A) An isolated villus from rat jejunum after combined indomethacin, taken from the left half of the villus which showed stasis and vessel hyperfluorescence (arrow), at which point the villus was perfuse fixed; the area of fluoresence to the left of this vessel is epithelium; original magnification ×600. This is more clearly seen in the line drawing. (B) Image from other right side of the villus which had normal flow and no fluorescence except for surface mucus; original magnification ×600. This is more clearly shown in the line drawing. (C) Transverse confocal image of an isolated villus from rat jejunum after combined luminal (100 µg/ml, 2.8 × 10−4M) and intravenous (15 mg/kg) indomethacin, and perfusion fixed as blood flow slowed at 30 minutes. Image is taken from the right side of the villus which exhibited slowing of flow and hyperfluorescence of the arcade artery (arrow), which is surrounded by epithelium showing slight fluoresence due to surface mucus; original magnification ×600. This is more clearly shown in the line drawing. (D) The left side of this villus which maintained normal flow. The only fluorescence again comes from surface mucus on the epithelium; original magnification ×600. This is more clearly shown in the line drawing. 


Figure 7 .

Figure 7

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Villous tip from rat jejunum showing blood stasis after combined luminal (100 µg/ml, 2.8 × 10−4M) and intravenous (15 mg/kg) indomethacin. The surface epithelium is beginning to lift and fall away from the lamina propria. There is contraction of surface smooth muscle cells manifest by prominent contractile elements (arrow 1) causing occlusion of surface vessels (arrow 2); there is also degeneration of the lamina propria; original magnification ×250. 


Figure 8 .

Figure 8

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Effect of a combined intravenous and luminal dose of indomethacin (100 µg/ml) and bicarbonate control on jejunal villous blood flow. Each point expressed as mean (SEM) of five rats. *p<0.03. 


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