Influence of the Gut Microflora and of Biliary Constituents on Morphological Changes in the Small Intestine in Obstructive Jaundice

Increased amounts of intestinal endotoxin are absorbed in obstructive jaundice. The precise mechanism is not known but the increased absorption may arise from alterations in the luminal contents, in the intestinal flora, in the gut wall or in interactions between all three. To examine the effects of the intestinal flora we have compared the morphological changes in the small intestine in obstructive jaundice in germ free and conventional rats while the effects of bile constituents have been examined by addition of bile constituents to the diet of bile duct ligated rats. Changes in the intestine were examined, histologically, by enzyme histochemistry, and by transmission and scanning electron microscopy. The results showed no differences in response between germ free and conventional rats. Feeding of diets containing bile salts exacerbated the lesion. Feeding of diets containing cholesterol, however, reduced the degree of intestinal changes produced by cholestasis and completely antagonised the increase in damage caused by feeding of bile salts.


verseas Publi
hers Association) Amsterdam B.V. Published in The Netherlands by Harwood Academic Publishers GmbH

INTRODUCTION

There is a high incidence of complications following diagnostic and therapeutic intervention in patients with obstructive jaundice ,2.There is considerable evidence to implicate endotoxaemia in the pathogenesis of the renal3,4, hepatic5, immunological and coagulative dis- orders frequently seen in these patients.emia results from an increased absorption, ofluminal endotoxin into the portal circulation and its decreased clearance by the Kupffer cells 1,1 allowing it to spill over into the systemic circulation.However, the nature of the intestinal changes which allow increased absor- Atddressforcorrespondence: Mr. M.E.Bailey, Department ofSurgery, Royal Surrey Hospital, Guildford,Surrey GU2 5XX.U.K. ption of endotoxin during cholestasis are not fully understood.

In theory an increase in endotoxin absorption could arise from alterations in the luminal contents, in the intestinal flora, in the gut wall or interactions between the three.It has been postulated that the absence of bile increases the absorption of end toxin 9,2 but clinical studies have yielded conflicting results following admi- nistration of bile salts13-5.Furthermore in a clinical study no alteration in the small bowel flora was found in jaundiced subjects when compared to controls3.

Earlier work by our group has provided morpho- logical evidence for changes in the intestine of bile duct ligated rats indicative of increased erosion of the microvilli of the enterocytesTM.This would seem para- doxical because bile salts are strong detergents and direct administration is known to damage the wall of the intestine 7 so it would be expected that their removal would, if anything, decrease the absorption of high molecular weight materials such as endotoxins.There is hence a possibility that other factors, for example, an alteration in the bacterial flora in the small intestine might pay a role, although no evidence for such a change has been found in human pa

tive con
ribution of these factors, we have studied the effects of removing intestinal organisms and of the addition of bile constituents on the damage to the wall of the small intestine which occurs in cholestasis.


MATERIALS AND METHODS


Animals

Conventional male Lister Hooded rats weighing approximately 250 g were obtained from the University of Surr y Experimental Biology Unit.Unless other- wise mentioned, the rats were fed standard rat diet (CRM diet, Labsure, Manea, Cambs.) and allowed free access to water.The animal rooms were main- tained at 22 + 2C with a 12h: 12h light: dark cycle.

Male germ free rats (University of Surrey, Experi- mental Biology Unit) were housed in a pressurised polyvinyl chloride isolator in cages with mesh wire bottoms.Incoming air was filtered through glass wool and bacterial filters.The animals were kept on irradi- ated rat diet (Labsure, Manea, Cambs) until 24 hours before experiments.As standard rat diets are not en- dotoxin-free, the rats, from then on, were fed sterile liquid diet (Fortison with Fibre, Cow and Gate, UK) and mixed grains which were washed and baked in oil at 180C.They were allowed free access to autoclaved water which had been supplemented with vitamins.Sterility was checked every three days by faecal cul- ture and, at termination, by culturing the caecal con- tents and faeces of all the rats.Group 2 Bile duct ligation followed by administration of control diet.Group 3 Bile duct ligation followed by administration of a control diet supplemented wi h 1% bile salts.Group 4 Bile duct ligation followed by administration of a control diet supplemented with 1% cholesterol.Group 5 Bile duct ligation followed by administration of a control diet supplemented with 1% bile salts and 1% cholestrol.

The bile salt mixture contained cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid in the same proportions as found in normal rat bileTM.All procedures were carried out under pentobarbital anae- sthesia (Sagatal, May and Baker, Dagenham, UK), a dose of about 6 mg/100g body weight normally being appropriate.Germ free rats were operated on under strict sterile conditions inside the isolators.A 2 cm long pper mid line incision was made and the common bile duct was identified, ligated at two points and divided19.In animals undergoing sham ligation, the common bile duct was mobilised and a suture passed under it but not tied, and then removed.

The animals were sacrificed by anaesthetic overdose three weeks after operation.Blood was collected by cardiac puncture.Where appropriate lml was placed in endotoxin-free tubes containing 30U of sodium heparin; the remaining blood was aliquoted into lithium- heparin tubes and used for biochemical assays.In ex- periment I, 1 ml of blood was drawn from the portal vein into endotoxin-free tubes containing 30U sodium heparin.Segments of small intestine were taken from the duodenum (immediately distal to the point of entry of the bile duct), jejunum (measured as 28 cm from the entry of the bile duct) and from d

tal ileum (5 cm from
ileo-caecal junction) and prepared for light and electron microscopy.The caecum was removed for esti- mation of endotoxin content and, in the case of germ free animals for culture to confirm their microbiological status.


Surgical Procedures

This study consists of two separate experiments.The first experiment was designed to compare the response of germ free rats and conventional rats.Each group consisted of 12 animals, 6 of which were subjected to bile duct ligation and 6 to sham operation.The second study investigated the ro

of bile salts
nd cholesterol in modulating intestinal damage.The study involved 5 experimental groups each of three animals treated according to the following protocol:-Group 1 Sham operation followed by administration of control diet.

Blood Analysis
Blood collected on the day of the autopsies was ana- lysed by standard techniques for total bilirubin, alanine aminotransferase and alkaline phosphatase on a Fal- con autoanalyser (Instrumentation Laboratories, Spokane, WA).


Endotoxin Assay

Endotoxins in portal and systemic blood were mea- sured using a chromogenic limulus amoebycyte lysate test [Coatest Endotoxin, Kabivitrum, Uxbridge, UK] using microtitre plates.Endotoxin levels in faeces were measured by making a suspension ofcaecal contents in endotoxin free water (Sigma, Pool

Dorset) in
a con- centration of 10 mg/ml.This was then centrifuged and supernatants collected.Serial dilutions were made to bring the endotoxin levels to within the meas rable range of the assay.


Microscopy

Specimens for light microscopy were immediately fixed in neutral buffered 10% formalin.They were th n pro- cessed, embedded in paraffin and sectioned at 5 tm.

Sections were stained routinely with haematoxylin and eosin, by the periodic acid-Schiffs technique (PAS) and with 3% alcian blue stains2.

Sections for electron microscopy were prepared by an adaptation of the method of Mann et a121.Small segments of intestine, approximately 1.5 cm long were immediately perfused with a solution of4% glutaralde- hyde buffered with sodium cacodylate (pH 7.4) at room temperature and left overnight in the fixative.They were then washed in 0.1M cacodylate buffer for 24 hours.Small rings, approximately 3 mm long, were cut from the intestine and counterfixed in 2% buffered osmium tetroxide for 2h.They were then dehyd ated in a graded series of ethanol and propylene oxide and embedded in Epon 812.Sections were cut, collected on copper grids, counter stained with uranyl acetate 22 and lead citratC and examined.

Specimens for scanning electron microscopy were prepared in the same way up to the completion of dehydration with ethanol.The se ments were then opened and immersed in acetone, critically point dried, mounted on aluminium stubs, sputter coated with gold and examined in a Cambridge Instruments scanning electron microscope.

Specimens for enzyme histochemistry were frozen in hexane at -80C and then stored at -80C until stained24.Staining was carried out within 28 days of autopsies.10 tm sections were cut in a cryostat.In most cases the sections were stained according to the proto- cols given by Chayen and Bitensky 24 except that when staining for alkaline phosphatase the sections were incubated in th

3 minutes, and when staining for 5'-Nucleotidase, bromote
ramisole was omitted from the incubation medium and replaced by 100 mM L-phenylalanine and staining was done for 5 minutes.


RESULTS


1) Comparison of the Intestinal Effects of Cholestasis in

Germ Free and Conventional Rats Following bile duct ligation there was a fall in body weight in both germ free and conventional rats.Routine histological examination revealed no alterations in the intestines following ligation of the common bile duct.Examination following staining by the periodic acid-Schiffs technique or with alcian blue showed an increase in size ofthe goblet cells in the bile duct ligated animals.Consistent with these results, scanning elec- tron microscopy showed an increase in the mucus ad- herent to the surface of the cells and also showed an increase in desquamating cells and a shortening and irregularity in the microvilli ofbile duct ligated animals (Figures a, 2a, b).This was confirmed by transmission electron microscopy (Figures b, 2c, 2d) which also showed a thickening of the terminal web and hypertro- phy of the golgi apparatus in bile duct ligated animals.In addition there was some increase in the incidence of damaged mitochondria in bile duct ligated animals and, in some cells, the mitochondria were markedly condensed.Histochemical staining showed a patchy loss of the brush border

ent with
the results of elec- tron microscopy.No difference was observed between germ-free and conventional animals in any of these studies.


2.) Endotoxin Levels in Germ Free and Conventional


Animals

To confirm the endotoxin poor status of germ free animals we examined the amount of endotoxin in faces and in portal and systemic blood.Very low levels (37.5 _+ 2.0 ng/g) of endotoxin was detected in the cae- cal contents of germ free rats but levels were about 0.05% of the levels (77.7 + 9.4 gg/g) found in coven- tional animals.Endotoxin levels in both portal and systemic circulation were below the limits of detection in both SHAM and BDL germ free animals.As micro- biological tests confirmed the germ free status of the animals it was assumed that the caecal endotoxin was derived from the diet on which the animals were r

red.Convent
onal animals showed the expected increase in circulating endotoxin following bile duct ligation, changes being greater in the portal vein than in the systemic circulation (Figure 3). 3) Effects of Bile Salts and Cholesterol on Changes in the Intestinal Morphology Following Bile Duct Ligation Administration of bile salts resulted in a marked exac- erbation of damage to the enterocytes as assessed both by scanning (Figure 4a) and transmission (Figure 4b) electron microscopy and by histochemica staining.Both damage to the brush border and the golgi hypertrophy were increased.Administration of cholesterol, on the other hand appeared to ameliorate the damage.In bile duct ligated animals fed cholesterol-enriched diets the changes were less marked than in animals not receiving the supplement (Figure 5) although there remained significant difference from control animals.Co-administration of cholesterol prevented the in- crease in intestinal damage caused by bile salts, the architecture being indistinguishable from that of bile- duct ligated animals receiving no dietary supplements.b) Figure 2 Electron micrographs of the jejunum in bile duct ligated rats.a) Low magnification scanning electron micrograph showing increased mucus and debris, b) Scanning electron micrograph showing disorganisation of microvilli d) High magnification transmission electron micrograph demonstrating marked Golgi hypertrophy, d) High magnification transmission electron micrograph demonstrating marked Golgi hypertrophy.


DISCUSSION

Absorption of endotoxin increases in obstructive jaundice 8, 9,12 and endotoxaemia has been implicated in the pathogenesis of a number of end organ effects in the presence of cholestasis3-7.Endotoxins are also known to potentiate the effects of ischaemic injury2.In earlier studies we have reported that the increase in endotoxin absorption is accompanied by morphological changes in the intestine 6 and now present the results of studies designed to determine the pathogenesis ofthese changes.

Our results showed that morphological changes in the small intestine during cholestasis were identical in the endotoxin-poor germ free rats and conventional animals falsifying the hypothesis that a significant change in the intestinal flora could explain the alter- ations to intestinal morphology.This is consistent with the findings of a clinical study where no significant d)

Figure 2 (Continued) difference of the gut flora was found between controls and patients with obstructive jaundice 3.

As the intestinal changes in cholestasis do not ap- pear to be associated with changes in the bacterial flora it seemed possible that the structural integrity of the small intestine depends, in some way, on factors present in bile Earlier we suggested that the endotoxaemia of cholestasismight be due to increased absorption secondary to changes in the gut wall.Our results strengthen this hypothesis.The high turnover of intestinal epithelial cells results in many transient breaks in the lining through which high molecular materials may pass.The increase in desquamating cells found in bile-duct ligated animals shows that cell turn- over has, indeed, increased, creating gaps through which endotoxin and other materials may pass to the subepithelium.It is therefore necessary to determine the pathogenesis of this damage.Animal experiments have demonstrated that the flow ofbile into the intestine is important in controlling endotoxaemia 26 and in decreasing the mortality from infection27.Both clinical and experimental studies have shown that bile salt administration protects against the systemic effects of cholestasis 9,13-14.Our results, how- ever, show clearly that, as expected from studies on non-jaundiced animals17,28, bile salts exacerbate the intestinal damage resulting from cholestasis.This drew attention to other bile components.Most of the choles- terol required by the body either derives from the diet or is synthesised in the liver.It therefore appeared possible that biliary cholesterol is required for the maintainance of the enterocytes.This hypothesis was examined and it was found that cholestrol supplementation of diet did indeed ameliorate the damage.The environment in which intestinal enterocytes exist is, inevitably, extremely hostile.Two factors assist in maintaining the epithelial barrier.