traumatic, chemical, bacterial, toxic, thermal, circulatory or

AN EXPERIMENTAL STUDY OF THE RELATION OF BILE TOULCERATION OF THE MUCOUS MEMBRANE OF THESTOMACH.*

GEO. MILTON SMITH, M.D.(From the Department of Pathology of Washington University, St. Louis.)

The various views regarding the origin of gastric ulcer inman and also the data relating to the experimental produc-tion of lesions of the mucous membrane of the stomachhave been recently discussed in papers published by Moeller 1and Scagliosi.2 It is therefore not essential for the presentpurpose to describe again in detail the numerous experi-ments that have been performed by different investigatorswith the aim of producing ulcers of the stomach, so manyof which have led to a more complete understanding of thefactors underlying the disease in man.

Most theories as to the cause of gastric ulcer assume thatthere occurs a primary impairment of vitality of the gastricmucosa or of the deeper lying tissues of the wall of thestomach which precedes the digestion of the affected tissuesand the formation of the characteristic area of ulceration.It is generally admitted as improbable that gastric juice willdigest the intact normal tissues of the stomach. The initialimpairment of vitality or the actual necrosis of gastricepithelium has been ascribed to numerous causes, chiefly oftraumatic, chemical, bacterial, toxic, thermal, circulatory ornervous nature. The tendency of ulcer of the stomach toincrease its size and also its failure to heal have been attrib-uted to the continued action of the primary cause of cellularnecrosis and also to the effect of various other factors ofsecondary nature. Among the secondary factors which areinstrumental in effecting delay in the processes of repairmay be mentioned as examples of the more important thechanges in blood vessels occurring in the region of the ulcer,gastric hyperacidity and hypersecretion and the associated

* Received for publication Fep. 10, I914.('47)

SMITH.

anemic conditions of the blood. In addition, the result ofthe recent investigations of Aschoff' and Stromeyer4 havepointed out that mechanical conditions influencing the formand mobility of the stomach may affect the healing of ulcersin the stomach; while the experiments of Katzenstein 5 havesuggested that a disturbed relation between the pepsin ofgastric juice and the antipepsin normally present in the wallof the stomach and in the blood may be a factor in thepersistence of the ulcer.The purpose of the present paper is to record a number

of experimental observations on the relation of bile in thepresence of an excess of hydrochloric acid of .5 per centstrength to necrosis and ulceration of the mucous membraneof the stomach; to describe the character of the lesionsproduced by the interaction of bile with hydrochloric acidupon the epithelial surface of the stomach, and to definesome of the conditions under which such lesions were mostreadily produced.The fact that duodenal contents with bile and pancreatic

juice may regurgitate through the pylorus under variousconditions has long been recognized. Beaumont I in hisstudies on the physiology of digestion based on observationsand experiments on Alexis St. Martin, makes a number ofallusions to the presence of bile in St. Martin's stomach.On page 2 1 of the Introduction to "Experiments andObservations on the Gastric Juice and the Physiology ofDigestion" the following appears:"On laying him (St. Martin) horizontally on his back,

pressing the hand upon the hepatic region, agitating a little,and at the same time turning him to the left side, brightyellow bile appears to flow freely through the pylorus andpasses out through the tube (introduced into the gastric fis-tula). Sometimes it is found mixed with the gastric juicewithout this operation. This is, however, seldom the case,unless it has been excited by some other cause."Again (p. 95) Beaumont said: " Bile is not essential to

chymification. It is seldom found in the stomach exceptunder peculiar circumstances. I have observed that when

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AN EXPERIMENTAL STUDY OF BILE.

the use of fat or oily food has been persevered in for sometime there is generally the presence of bile in the gastricfluid. Whether this be a pathological phenomenon, inducedby the peculiarly indigestible nature of oily food, or whetherit be a provision of nature to assist the chymification of theparticular kind of diet, I have not as yet satisfied myself. Oilis affected by the gastric juice with considerable difficulty.The alkaline properties of the bile may render it more sus-ceptible of solution in this fluid by altering its chemicalcharacter. Irritation of the pyloric extremity of the stomachwith the end of the elastic tube, or the bulb of the thermome-ter, generally occasions a flow of bile into this organ. Exter-nal agitation, by kneading with the hand on the right sideover the regions of the liver and pylorus, produces the sameeffect. It may be laid down as a general rule, however, sub-ject to the exceptions above mentioned, that bile is not neces-sary to the chymificatien of food in the stomach. Magendiesays, I believe, that in certain morbid conditions the bile isnot introduced into the organs (the stomach), inferring thatin a healthy state it is always to be found there. There canhardly be a greater mistake. With the exceptions that Ihave mentioned it is never found in the gastric cavity, in astate of health, and it is only in ' certain morbid conditions '

that it is found there. When bile is found with the gastricjuice the acid taste is diminished, and the flavor of the bileprevails in proportion to the quantity in the mixture."

Finally (p. IO9), Beaumont wrote: " After excessiveeating or drinking chymification is retarded, and, althoughthe appetite be not always impaired at first, the fluids becomeacrid and sharp, excoriating the edges of the aperture, andalmost invariably produce aphthous patches and the otherindications of a diseased state of the internal membranementioned above. Vitiated bile is also found in the stomachunder these circumstances, and flocculi of mucus are muchmore abundant than in health."

Pavlov 7 a number of years ago called attention to the factthat if olive oil is introduced into the stomach of a dog witha gastric fistula and the gastric contents is drawn off, an

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SMITH.

emulsified fluid with bile and pancreatic juice may be obtainedfrom the stomach in the course of one to two hours.

It remained, however, for Boldyreff8 to define numerousexperimental conditions under which bile, pancreatic andintestinal juices may pass from the duodenum into the stom-ach. In a series of experiments upon dogs with gastricfistula he found, following meals very rich in fats, or after theintroduction of large amounts of acids, or during starvation,that bile and pancreatic juice mixed with intestinal juicesmight be found in the stomach.From his experiments in which dogs were fed various

kinds of fat, Boldyreff was led to conclude that the regurgi-tation of bile, pancreatic and intestinal fluids into the stom-ach did not occur through an antiperistaltic movement, butrather in the form of a strong uninterrupted stream beginningshortly after the introduction of the fats into the stomach.He believes that a gastric digestion of fats takes place in largemeasure through the agency of the pancreatic fermentswhich have found their way into the stomach. Boldyrefffound that the introduction of large amounts of acids suchas one hundred to two hundred cubic centimeters of hydro-chloric acid, one per cent of lactic acid or acetic into eitherthe empty or digesting stomach was followed also by theregurgitation of duodenal contents and bile into the stomach.He noted also the interesting fact that the injection of weakacids (.OI-.15 per cent hydrochloric acid) into the lumenof an isolated loop of duodenum was followed by a regurgi-tation into the stomach of mixture of bile, pancreatic andintestinal juices in as large amounts as one hundred, onehundred and fifty or two hundred cubic centimeters. Thechemical analysis of products of digestion under these cir-cumstances showed that digestion had advanced to such adegree as to resemble in results the processes of digestionusually found in the small intestine rather than that of thestomach. Boldyreff suggests that the explanation for thebackward flow of duodenal contents into the stomach, whenthe latter has received injections of acids, lies in the fact thatan attempt is being made on the part of the body to neutralize

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the unusual acidity in the stomach with the regurgitationof an alkaline secretion normally found in the duodenum.He points to the fact that the delicate intestinal wall doesnot readily bear the presence of even weak acids. Acidfoods are retained in the stomach a long while and then dis-charged into the intestine in small amounts. In an experi-ment in which he introduced two hundred cubic centimetersof .5 per cent of hydrochloric acid into the stomach of a dog,Boldyreff made the important observation that only afteracidity had fallen to a strength of .2 per cent to .I5 per centdid gastric content begin to leave the stomach. Boldyreffdescribed also the regurgitation of pancreatic juice and bileinto the stomach as occurring in fasting dogs, and found thatas much as one hundred cubic centimeters of a mixture ofbile, pancreatic and intestinal juices were obtained from thegastric fistula of a fasting dog in the course of twelve tofifteen hours. The presence of pancreatic juice and bile inthe stomach was associated with a marked production ofmucus lasting from two to three days.The toxicity of bile for various tissues of the body has

long been recognized, but has been especially evident sincethe investigation of Rywosch I and the more recent studiesby Meltzer and Salant.10The destructive action of bile on epithelium and parenchy-

matous tissue has been clearly demonstrated in acute diseaseof the pancreas. Opie 1 was the first to explain the condi-tion of acute hemorrhagic necrosis of the pancreas in man,by showing that if bile is directed into the pancreatic ductby a biliary calculus lodged in the diverticulum of Vater, awidespread hemorrhagic necrosis may result. A lesion ofthe pancreas, similar to that seen in acute hemorrhagicnecrosis in man, was experimentally produced by Opie inthe dog by injecting bile into the pancreatic duct. Withintwenty-four hours after the injection of five cubic centimetersof bile into the pancreatic duct of a dog the animal died,exhibiting a marked disintegration of the pancreas. Flex-ner 12 demonstrated that the toxic action of bile upon pan-creatic tissue was due to the presence of bile salts.

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SMITH.

In an earlier publication 'I dealing with the transplantationof epithelial tissues into the gall bladder I noted that necro-sis of the epithelium of grafted tissues took place whenepithelium lay in contact with bile, whereas epithelium lyingdeep between folds of mucous membrane and protected frombile apparently survived for considerable periods of time.

In studying experimentally the effect of bile upon thegastro-intestinal tract Rywosch found that necrosis occurredin the crop of chickens and also in the stomach of rabbitswhen bile was fed in large amounts to these animals. In hisexperiments with chickens, ox-bile was fed in the form ofpills. The chickens were found dead on the fourth day afterthe administration of bile, and the mucous membrane of thecrop presented a deep red appearance and was necrosed inplaces. The stomach and the intestine were stained withbile. In experiments with rabbits, ox-bile was introduceddaily into the stomach in amounts of fifty cubic centimeters,death occurring on the fourth day. The stomach of allanimals treated in this way showed a marked inflammationof the mucous membrane, and in one instance a gangrene ofthe wall of the stomach. No histological study of the tissuesis recorded. Rywosch drew the conclusion that largeamounts of bile introduced into the stomach might becomethe source of injury to the stomach and cause death; whereassmall amounts of bile produced no injurious effects.

There is much evidence to support the view that the pres-ence of bile in the stomach of the higher animals and in manmay not be associated with any injurious effects upon thegastric mucous membrane when this is in a normal condition.Oddi 14 fed dogs large amounts of ox-bile for many days;and also diverted bile into the stomach by anastomosingthe gall bladder with the stomach, without noting injuriesto the gastric mucosa. Dastre1 after ligating the commonbile duct in a dog anastomosed the gall-bladder with thestomach, and in this way allowed bile to be in constantcontact with the gastric mucous membrane. The animalthus operated on showed no gastric disturbance, and chemi-cal analysis of the products of digestion showed no changes

I 5 2


from the normal. According to Kaiser,16 bile may be regu-larly observed in the stomach of patients upon whom agastro-enterostomy has been performed. Carle and Fantino 7found bile present in the gastric contents of all patients withgastro-enterostomy examined by them. Kausch18 found itpresent for many years in a patient with gastro-enterostomy.Boldyreff is the only observer who speaks of a transientcatarrhal condition of the gastric mucosa following the pres-ence of pancreatic juice and bile in the stomach in the dog.The present investigation was undertaken to determine

whether or not the action of bile on the stomach mucousmembrane, although at times clearly harmless, could not beintensified under certain abnormal conditions, so that itmight cause ulceration of the gastric mucous membrane.

Methods: The animals used for experiments were thecat and the dog, chiefly the former. It was found early inthe series of experiments that the gastric mucous membraneof the dog showed a greater resistance to injury produced bybile and hydrochloric acid than did the stomach of the cat.This was due in part to the presence of abundant mucusfrequently found in the dog's stomach and to active secretionof mucus which often quickly followed the introduction ofbile and acid into the dog's stomach. As will be noted inthe protocols that follow, the application of bile and hydro-chloric acid to the stomach was performed in several differ-ent ways: (a) by direct application to the gastric mucousmembrane, after the anterior wall of the stomach had beenincised during an operation; (b) by stomach tube; (c)after opening the abdomen by injecting into the stomach bileand acid through an aspirating needle passed through thewall of the stomach; (d) by injecting bile and acid back-ward into the stomach through the pylorus by means of anaspirating needle, passed through the wall of the duodenum;(e) by anastomosing the gall bladder with the stomach, afterligating the common bile duct, and subsequently introducingacid into the stomach of the animal by means of a stomachtube. Of these methods that in which bile and acid were

I5 3

SMITH.

introduced into the stomach by way of the duodenum andthe pylorus was the one most frequently employed. Themethod allowed the use of the animal's own bile, whichcould be obtained by aspiration from the gall bladder. Nohandling of the stomach was necessary in this experiment.The injecting needle was pushed through the wall of theduodenum at the level of the entrance of the common bileduct and pointed in the direction of the pylorus. The bilewas allowed to flow back slowly through the pylorus into thecavity of the stomach, traversing in this way a region of thegastro-intestinal tract, which it would naturally take in caseof regurgitation into the stomach. When the amount of bileavailable in the animal's gall bladder was insufficient for thepurpose of the experiment, fresh bile from another cat orfrom a dog was used. Unless otherwise stated, hydrochloricacid was used in a strength of .5 per cent, which is withinthe range of normal acidity of gastric juice for both the dogand the cat. In those experiments in which artificial gastricjuicewasemployed,thiswas prepared from acid infusionsofthegastric mucous membrane of the cat or the dog, hydrochloricacid being used in solutions varying in strength from .25 percent to .5 per cent. Fresh ox-bile was used in the experimentsperformed for the purpose of studying the effect on gastricmucous membrane of large amounts of bile when confinedin the stomach after ligating the esophagus and pylorus.In most of the experiments recorded in the present paper,animals were killed within the first twenty-four hours afterbeginning the experiment. Numerous control experimentswere made under conditions exactly those of the test experi-ment.

EXPERIMENTAL OBSERVATIONS. - Application of bile and.5 per cent hydrochloric acid to the mucous membrane ofthe stomach.

If the mucous membrane of the stomach of the cat or dogbe exposed by operation and a drop of bile, previously re-moved by aspiration from the gall bladder, be placed upon itssurface, the bile causes no marked changes in the appearance

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of the mucosa. The drop of bile preserves its outlinefor a few minutes, and then slowly diffuses into the mucuswhich lies upon the surface of the epitheliuml, giving it ayellowish tint. If the surface of the mucous membrane isheld in a slightly slanting position the bile readily flows alongthe surface of the epithelium, usually following the directionof some groove formed by two adjacent folds of mucousmembrane. If a drop of .5 per cent hydrochloric acid beplaced by means of a pipette upon the mucous surface of theliving stomach no striking changes in the appearance of theepithelial surface occur. The drop may retain its form fora short while, usually slowvly spreads over the mucous surfaceor flows along between adjacent folds of the mucous mem-brane. At times the surface of the mucous membrane,especially if several drops of acid are superadded, may assumea grayish tint, probably due to the precipitation of proteinscontained in the mucus lying in contact with the epithelium.No gross or histological injury of the tissue is producedunder these circumstances by either bile or hydrochloric acid,providing the tissues are handled with care and receive notrauma.When, however, a drop of bile is placed on the mucous

surface of the stomach and immediately afterward a drop of.5 per cent hydrochloric acid is added to it, there soonappears at the surface of the epithelium evidence of well-defined reaction consisting in the formation of a yellowish orgreenish-yellow precipitate which corresponds in outline tothe area occupied bv the combined drops of bile and acid.The color of this yellow area becomes intensified with theaddition of more bile and hydrochloric acid, so that it maytake the form of a greenish-yellow film or pellicle closelyadherent to the epithelial surface. Hammersten 19 has notedthat in the course of normal digestion when acid chymeenters the duodenum and mingles with bile, a precipitate isformed of proteins of the food and bile acids. According toHammersten this precipitation of proteins occurring underphysiological conditions when bile meets the acid contents ofthe stomach in the duodenum is "re-dissolved in an excess

I55

SMITH.

of bile and also in the NaCl formed in the neutralization ofthe hydrochloric acid of the gastric juice." In a somewhatsimilar way under experimental conditions, when bile in thepresence of an excess of hydrochloric acid comes in contactwith the gastric mucous membrane, a precipitation ofproteins contained in bile and in the gastric mucus occurs.The precipitate forms a thin cohesive membrane in immediatecontact with the epithelium, dipping into or bridging overthe openings of glands and the minute grooves of the mucosa.

If the material forming the greenish film of precipitate iscollected by gently scraping the mucous membrane it isfound to be insoluble in hydrochloric acid (.5 per cent). Itis readily dissolved in alkaline solutions such as .5 per centsodium carbonate; but dissolves slowly in water and aneutral sodium chloride solution (.85 per cent). It is slowlysoluble in an excess of bile. It is digested by gastric juice.The close contact of the pellicle formed by the precipitation

of bile and hydrochloric acid to the epithelial surface is mostmarked in the presence of a scanty amount of mucus. Whenmucus is abundant, which is often the case, especially in thedog, the yellowish green pellicle is less closely adherent tothe epithelium. It tends to dissolve with an accumulation ofalkaline mucus so that at first its edges become lifted fromthe epithelial surface and finally the whole pellicle maybecome entirely detached. Films of precipitate cast off fromthe surface of the mucous membrane may contain numerousepithelial cells. The relative quantity of mucus appears tobe an important factor in determining how closely in contactwith the epithelium the interaction of bile and acid is to occur.

Although the interaction of bile and hydrochloric acidupon the epithelial surface of the stomach may occur withoutinjury to the epithelium, under certain conditions which willbe defined, areas of necrosis of gastric epithelium are formed.The chemical injury produced by the combined action ofbile and hydrochloric acid is of such a nature that gastricepithelium succumbs either directly from the effect of thesesubstances or subsequently to the action of gastric juice.

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Bile diverted into the stomach by anastomosing the gallbladder with the stomach was found to be non-injurious togastric mucous membrane. Single injections of bile intothe stomach by way of the duodenum were without harmfuleffect.

Twelve experiments in the nature of controls were donetesting the effect of bile alone upon the gastric mucousmembrane. Bile was diverted into the stomach, as in theexperiments of Oddi and Dastre, by anastomosing the gallbladder with the stomach and at the same time ligating anddividing the common bile duct. The animals used were thedog and the cat. They were allowed to live for periods oftime varying from two days to six weeks. No condition ofulceration of the stomach was found in these experiments.In several of the animals the mucus contained in thestomach appeared to be in excess of the normal, and alsounusually viscid.

In experiments in which from three to five cubic centime-ters of bile were injected into the stomach by way of theduodenum during the later stages of digestion no injury tothe mucous membrane resulted.

Hydrochloric acid in weak dilutions of .5 per cent wasfound to be non-injurious to the mucous membrane of thestomach.

The acidity of gastric juice for the dog is given by Pavlovas varying from .5 per cent to .6 per cent, and for the cat as.52 per cent. According to Moeller the work of numerousinvestigators has shown that an experimental increase in theamount of hydrochloric acid alone is never sufficient to pro-duce ulceration of the mucous membrane of the stomach.Thus Matthes O found that the daily introduction into thestomach of dogs of three hundred and fifty grams of .56per cent hydrochloric acid by stomach tube was able todelay the healing of wound previously made in gastricmucosa, but failed of itself to produce any lesion in the

15 7

SMITH.

intact mucous membrane. Numerous experimnents wereperformed testing the effect of .5 per cent hydrochloric acidupon the gastric mucous membrane of the cat and the dogwhen the acid was introduced into the stomach duirinigvarious stages of digestion. In none of these experimentsdid ulceration of the gastric mucosa result. As a rule, acidin amounts from five cubic centimeters to ten cubic centime-ters was used. In one experiment as much as seventy cubiccentimeters of hydrochloric acid were given by stomach tubeto a cat four hours after a meal of milk without causing injuryto the mucosa. In another experiment carried out in boththe cat and the dog a solution of .5 per cent hydrochloricacid was applied drop by drop to a certain point on the sur-face of the mucous membrane of the stomach for one-halfhour, and no injury of the mucosa was found on examinationof the stomach twenty-four hours after the operation. Evenwhen .5 per cent hydrochloric acid was introduced into afasting stomach together with an amount of artificial gastricjuice, and both these fluids were confined in the stomach fortwenty-four hours by ligating the esophagus and theduodenum just beyond the pylorus, no lesion of the mucousmembrane developed. Such was the case in the followingexperiment:

Experiment I.- Cat; male; weight, 2,800 grams.No food for twenty-four hours. Dec. 20, 1913. 2 P.M., the abdomen

was opened. The duodenum just below the pyloric valve was incised.The stomach was irrigated by means of a small soft catheter introducedthrough the pylorus with a warm solution of .25 per cent of hydrochloricacid until the return flow was clear. Five cubic centimeters of .5 per centhydrochloric acid were then introduced into the stomanh, followed byeight cubic centimeters of artificially prepared gastric juice. The gastricjuice was prepared from an infusion of mucous membrane of the cat'sstomach with .5 per cent of hydrochloric acid for twenty hours. Theesophagus was tied off with a silk suture at a point just below thediaphragm; the duodenum was ligated immediately below the pylorus.The duodenal and the abdominal wounds were then closed.The animal was killed twenty-four hours later. No lesion of the

stomach was found.

With the introduction of bile and .5 per cent hydrochloric

I5 8


acid into the stomach lesions occurred. The following pro-tocols are illustrative of lesions in the stomach produced bybile in the presence of an excess of hydrochloric acid.

Necrosis of gastric epithelium following direct application of bile andhydrochloric acid to the mucous membrane:

Experiment 2 - Cat; male; weight, 2,800 grams.April 21, I913. At noon received a meal consisting of meat. At

4 P.M. a median laparotomy was performed and about 2.5 cubic centi-meters of bile aspirated from the gall bladder. The stomach was thenopened by a transverse incision, 4 centimeters in length, in the middle ofthe anterior wall. The stomnach contained a considerable amount of par-tially digested food, some of which was carefully removed in order toexpose the mucous membrane of the posterior wall. Several drops ofbile were allowed to fall from the aspirating needle upon the gastricmucous membrane, followed immediately afterward by several drops of.5 per cent hydrochloric acid applied by means of a pipette. An irregular-shaped greenish yellow area appeared at the epithelial surface, at the pointwhere bile and acid became mingled. This area was intensified subse-quently by the repeated addition of drops of bile alternating with aciduntil a pellicle formed in close contact with the epithelial surface of themucous membrane. At the end of ten minutes the gastrotomy woundwas closed with a double layer of fine silk sutures, followed by the closureof the abdominal wound.

April 22, 1913. 7 30 A.M. the animal was killed. The stomach con-tained a slight residue of food. The gastrotomy wound showed no hyper-emia or excoriation. The mucous membrane of the posterior wall ofthe stomach at the point of application of bile and hydrochloric acidshowed an irregular area measuring 3 x 2 5 centimeters, which had a redexcoriated appearance, especially well marked along the grooves betweensmall folds of the mucosa. In one place, measuring 3 x 4 millimeters.,there was a round area of superficial ulceration about whose edges healthytissue had become somewhat elevated, giving the small ulcer a depressedappearance. A microscopic examination was made of the tissue takenfrom this region. The mucous membrane presents a sharply circum-scribed area of necrosis which reaches two-thirds of the distance to thesubmucosa. The most superficial part of the necrotic area consists of astructureless mass, at the margins of which large numbers of polymorpho-nuclear leucocytes and extravasated red blood cells with fibrin have col-lected. In the middle third of the mucosa the structure of the tissues canstill be made out, but most of the cells take the stain poorly, show unusu-ally large vacuoles, and are variously distorted. Parietal cells appear bestpreserved, yet frequently show irregular and distorted cell bodies andnuclei. It is only near the muscularis mucosa that the epithelium appearsuninjured, yet even here the tubules of glands appear irregular in shape, at

I 5 9

SMITH.

times dilated or obliquely placed, containing in their lumen a granularsubstance and occasionally desquamated cells. Leucocytes are found inconsiderable numbers in the deeper parts of the mucosa between thetubules. The capillaries are much congested and red blood cells havefound their way in small collections at different points into the interglandu-lar tissues. Healthy mucous membrane lies on each side in contact withthis large area of necrosis, showing, however, much engorgement ofcapillaries and a moderate number of polynuclear leucocytes. Severalother areas of necrosis appear in the section, much less wide in extent yetequally deep. These are narrow wedge-shaped regions affecting in eachinstance the epithelium lying in a minute depression formed by lateralfolds of the mucous membrane. The histological characters of thesesmall areas are much the same as that already described. The submu-cosa throughout the section is edematous, its vessels distended with blood.The muscularis is normal. A few small collections of leucocytes appearjust below the serosa.

Application of bile in the presence of an excess ofhydrochloric acid of .5 per cent strength to the mucousmembrane of the stomach of the cat resulted in the produc-tion of an injury to the epithelium, characterized on grossappearance by small areas of ulceration and microscopicallyby a well defined circumscribed area of hemorrhagic necro-sis involving the mucous membrane to two-thirds of itsdepth. The superficial parts of the necrotic area appearedhomogeneous and structureless as if in part digested awayby the gastric juice, and polynuclear leucocytes were foundsurrounding the area of hemorrhagic necrosis.

In all, forty experiments were performed employing theabove method of applying bile and hydrochloric aciddirectly to the gastric mucous membrane after incising theanterior wall of the stomach. In twenty-one experimentslesions were produced which varied considerably in severity.At times the lesions took the form of areas of hyperemia orof small hemorrhages into the mucosa, which histologicallyshowed a superficial necrosis; in other experiments lesionswere more severe in character and small ulcers were producedwith sharply defined margins and a dark hemorrhagic base.Microscopically the necrosis of tissues was found alwayslimited to the mucous membrane.

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Necrosis of epithelium in the pyloric portion of the stomach, small super-ficial ulcers in the body and the fundus of the stomach:

Experiment 3.- Cat; male; weight, 3,500 grams.Aug. I, 1913. II A.M., a meal of milk was given. At 2 P.M. the

abdomen was opened, 3 cubic centimeters of bile from the animal's gallbladder were injected into the stomach from the duodenum. Five cubiccentimeters of .5 per cent hydrochloric acid were injected into the stomachimmediately afterward.Aug. 2, 1913. 9 A.M. the animal was killed. At the pylorus are a

number of interlacing reddish lines with four small points of ulceration(Fig. i, Plate i). Two lines of erosion extend along the lesser curvature*toward the body of the stomach. These measure at the widest point3 millimeters in diameter. The more marked lesions are on the anteriorsurface of the body of the stomach. Here a number of irregular-shapedsmall ulcers are found. The largest of these measures 7 x 2 millimeters.Two smaller ulcers near by measure 4 x 2 millimeters. Each ulcer hassmall dark red lines extending radially outward from its margins, whichinterlace with other fine lines. Where minute hemorrhages have occurredinto the mucosa on the posterior surface of the fundus there is anothersmall ulcer, measuring 5 x 4 millimeters in diameter.

Multiple ulcers of the anterior and posterior surface of the stomach follow-ing the injection of bile and hydrochloric acid into the stomach fromthe duodenum:

Experiment 4.-Cat; female; weight, 2,500 grams.October 3, between 10.30 and II A.M., the animal was given a meal of

milk. At 3.30 P.M. the operation was performed. One cubic centimeterof bile, dark green in color, was removed from the gall bladder of theanimal. To this was added 4 cubic centimeters of bile, golden yellow incolor, taken from a dog's gall bladder. The mixture of bile was theninjected into the stomach of the cat from the duodenum. Five cubiccentimeters of .5 per cent hydrochloric acid were injected in the samemanner.

October 4. 8.45 A.M. the animal was killed. Beginning at the pylorusthere are three irregular lines of red excoriation leading from the pylorusalong the lesser curvature to the fundus. Seven small irregular ulcers arefound scattered over the anterior and posterior surface of the stomachlying somewhat nearer the lesser than the greater curvature. The largestulcer measures .5 x .3 centimeter. On' histological examination thenecrosis is limited entirely to the mucosa. The interglandular tissue andepithelium have been destroyed and digested away, but only to a depth ofabout one-half the mucous membrane (Fig. 2). In that part of themucosa that still remains the interglandular tissue takes a pink color withthe eosin stain and frequently presents the appearance as if several massesof tissue have become fused together. Many of the chief and parietalcells lying in the meshes of this tissue are in part disintegrated or exhibit

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SMITH.

various distortions in their form. Polynuclear leucocytes have accumu-lated in considerable numbers about the area of necrosis.

Diffuse hyperemia and necrosis of mucous membrane of posterior surfacenear esophagus; small areas of necrosis in the pylorus and along thegreater curvature of the fundus:

Experiment 5. - Cat; male; weight, 3,600 grams.July 25, 19I3, at II A.M., the animal was given a meal consisting of

chopped meat. At 4 P.M. the abdomen was opened and 3 cubic centime-ters of bile were injected into the stomach via the duodenum This wasfollowed by an injection of 6 cubic centimeters of .5 per cent hydrochloricacid. The bile was taken in part from the gall bladder of the same animal,in part from another cat that had been killed a short time previous.

July 26. 10 30 A.M. the animal was killed. The most marked lesion isnear the esophagus on the lesser curvature. It consists of a dark redexcoriated area measuring 4 x 2.5 centimeters. In this deeply hyperemicregion several small ulcers are seen measuring about 2 millimeters in diam-eter. Along the lesser curvature are several lines of excoriation whichwiden at various points into small ulcers. Along the pylorus as far as thepyloric valve are a number of pink-colored lines which here and thereappear to reach a breadth of I.5 millimeters and at such points give theappearance of superficial ulcers. Microscopic sections taken from the areaof ulceration near the esophagus show numerous sharply circumscribedpatches of hemorrhagic necrosis, in part digested. There is a markedextravasation of red blood cells in such places and at times numerousleucocytes are found as well. The epithelium lying between the variousareas of necrosis is well preserved and looks normal. The submucosa andmuscular coats of the stomach are normal.

Necrosis of epithelium in the pyloric part of the stomach:

Experiment 6. - Cat; female; weight, 2,6o0 grams.July 23, 1913. 2.15 P.M., three hours after a meal of milk, the abdomen

was opened, and 2 cubic centimeters of bile, taken from the animal's owngall bladder, were injected into the stomach by way of the duodenum.This was followed by an injection of 5 cubic centimeters of .5 per centhydrochloric acid in the same manner The abdomen was then closed.

July 24, 1913, 9 A.M. There are a series of irregular red streaks oferosion beginning with the pyloric valve and extending between the foldsof the mucous membrane toward the fundus for a distance of 4 centime-ters. Several of the small red streaks intersect and formn minute areas ofulceration with a diameter of from 2 to 3 millimeters. The mucous mem-brane of the fundus is normal in appearance, the duodenum immediatelybeyond the pyloric ring shows no gross changes. Tissues removed fromthe pyloric region of the stomach show numerous areas of superficialnecrosis (Fig. 3). The regions of necrosis are sharply defined. Epithe-lium at the surface of the mucous membrane is lost and the interglandular

I162


tissue near the surface is homogeneous and structureless, exhibiting hereand there fragmented nuclear material. Polymorphonuclear leucocytesare found in moderate numbers in the area of necrosis. Small hemor-rhages are present in adjacent interglandular tissue which otherwiseappears normal.

A total of sixty-eight experiments were performed on catsusing the method of injecting bile into the stomach from theduodenum. In twenty-eight experiments injury to thegastric mucosa was noted, varying considerably in intensity,yet exhibiting, in general, the characters described in thethree preceding experiments.

Small ulcer on the anterior surface of the stomach of a dog following theinjection of bile and hydrochloric acid into the stomach via theduodenum:

Experiment 7.- Dog; male; weight, 7 kilos.July 25, at II A.M., the animal had received a meal of meat. Operation

at 4 P.M. On opening the abdomen the stomach was found slightly dis-tended with food. Three cubic centimeters of bile removed from thegall bladder were injected into the stomach by passing a needle throughthe wall of the duodenum. Six cubic centimeters of .5 per cent of hydro-chloric acid were injected into the stomach in the same way.

July 26. IO A.M. the animal was killed. There was a small superficialulcer measuring .6 x .3 centimeter situated on the anterior wall of thestomach, I centimeter from the lesser curvature and 6 centimeters fromthe pylorus. The edges of the ulcer and adjacent mucous membrane fora distance of a centimeter from the ulcer were very hyperemic. Thebase of the ulcer was rough and covered with a grayish film. Severalsmall irregular red lines of excoriation were situated about 2 centimetersfrom the ulcer on the anterior surface of the stomach.

Out of a total of twelve dogs receiving injection of bileand hydrochloric acid as in the experiment just described,two only showed lesions of the mucous membrane of thestomach. It is not unlikely that the abundant supply ofmucus often found in the dog's stomach offers strong protec-tion against the chemical injury produced by bile andhydrochloric acid.

In order to study early lesions of the gastric mucousmembrane a number of animals were examined three and sixhours after the introduction of bile and .5 per cent hydro-chloric acid into the stomach.

I63

SMITH.

Early lesions of gastric mucous membrane three hours after injecting bile.and hydrochloric acid into the stomach from the duodenum:

Experiment 8. - Cat; male; weight, 3,800 grams.Dec. 4, 1912. Ir A.M. received meal of milk. At 2.30 P.M., operation.

The abdomen was opened and 3 cubic centimeters of fresh bile previouslytaken from the gall bladder of a dog were injected into the stomach fromthe duodenum. This injection was followed by the injection of 5 cubic-centimeters of .5 per cent hydrochloric acid.At 5.30 P.M. (three hours later) the animal was killed. Numerous.

hyperemic streaks were found on the anterior surface of the stomach, andsome less conspicuous were present in the pylorus.

Microscopic examination of tissues removed from the hyperemic areas.of the fundus shows that lesions are sharply circumscribed and affect onlythe most superficial part of the mucous membrane. The epitheliumcovering the surface of the mucous membrane in the affected areas is lost.Here and there epithelial cells are found in adjacent collections of mucus.The inter-glandular tissue lying directly below the surface epithelium takesthe stain less well than in normal regions. In places the inter-glandulartissue projects slightly outward like short villi, whose tips appear necrosed.The small capillaries in necrosed inter-glandular tissue are dilated andhere and there occupied by hyaline thrombi. The capillaries deeper downin the affected mucosa are greatly distended with blood and numerous redblood cells have found their way into the tissues below the epithelium.Except for numerous mucus cells at the surface which have becomedetached from underlying connective tissue, epithelial cells including thechief parietal cells of the peptic glands are well preserved. The lesions ofthe pyloric part of the stomach are not as well marked. A dense hyalinefilm of mucus is stretched along the surface of the mucous membrane, bridg-ing over in many places the foveole. In regions where shallow grooves.form in the mucous membrane, mucus lies in thicker masses and contains alarge number of red blood cells with occasional epithelial cells. In places.where the epithelium at the surface is detached small hyaline thrombi arefound in the minute capillaries of the reticular tissue. Numerous smalldense spherical mucus bodies of brownish or greenish color are foundscattered along the surface of the mucous membrane usually in contactwith the hyaline layer of mucus already referred to as present at the-surface.

Early lesions of the gastric mucous membrane six hours after the injectionof bile and hydrochloric acid into the stomach:

Experiment 9.-Cat; male; weight, 3,000 grams.Dec. 6. The animal has been kept on a milk diet for the past two.

days. At 7 A.M. given five ounces of milk. At 9.30 A.M. the abdomenwas opened and 3 cubic centimeters of cat's bile followed by 5 cubiccentimeters of .5 per cent of hydrochloric acid were injected into the.

i64


stomach through the duodenum and the pylorus. At 3.30 P.M. the ani-mal was killed. On the anterior and the posterior wall of the stomachare numerous irregular bright red spots and short streaks of hemorrhageinto the mucous membrane, many of them projecting slightly above thelevel of adjacent mucous membrane (Fig. 4). Sections taken from theregions of hemorrhage into the mucous membrane show small localizedareas devoid of surface epithelium where interglandular tissue has under-gone necrosis (Fig. 5). Many of the capillaries near the surface of suchareas contain hyaline thrombi. Below the necrosed parts of the mucosathere is present a very striking hemorrhage into the interglandular tissue.Polynuclear leucocytes are present in small numbers.

As early as three hours after injection of bile and hydro-chloric acid into the stomach of the cat during a late period ofdigestion, injury to the mucous membrane may be observed.In sharply circumscribed areas the epithelium at the surfaceof the mucous membrane may be lost or seen as cellsdetached from underlying connective tissue. The bloodcapillaries near the surface contain frequently hyalinethrombi and lie directly in or near necrosed interglandulartissue. The capillaries situated deeper in the mucous mem-brane are intensely engorged, and red blood cells are foundin the interglandular tissue. Six hours after injection ofbile and hydrochloric acid the necrosis of the epithelium andinterglandular tissue is more marked, hemorrhages into theaffected area is of greater extent, and inflammatory cellsare found in the region of necrosis.

Bile and .5 per cent hydrochloric acid introduced into thestomach of the cat by a stomach tube, from two and one-half to three hours after meals, resulted in lesions of gastricmucosa in two experiments out of a total of six. In theseexperiments the bile from a cat or a dog was used inamounts of three to five cubic centimeters. From eight toten cubic centimeters of .5 per cent hydrochloric acid wereintroduced into the stomach immediately after the bile.The lesions were slight in character, as seen in the followingexperiment:

Experiment Io. - Cat; male; weight, 2,600 grams.Dec. 19, 1913. 3 P.M., three hours after a meal of milk, the animal

I65

SMITH.

received by stomach tube 5 cubic centimeters of cat's bile and 6 cubiccentimeters of .5 per cent hydrochloric acid.

Dec. 20, 1913. 9 A.M., the animal was killed. A slight superficialerosion is found on the posterior surface of the stomach near the esopha-geal opening. This measures about 5 x 2 millimeters.

Microscopic examination: A localized area of necrosis of epitheliumand interglandular tissue is present, which involves only the outer one-fourth of the mucous membrane.

Bile and .5 per cent hydrochloric acid were introducedinto the stomach of the cat through an injecting needlepassed through the anterior wall of the fundus of thestomach. Out of three experiments two showed injury tothe mucosa. In the following experiment well markedlesions were found:

Experiment iI. Cat; male; weight, 4,000 grams.Nov. 21, 19I3. It A.M., received a meal of milk. 2.30 P.M., the abdo-

men was opened and 2 cubic centimeters of bile were removed from thegall bladder. The bile was injected into the stomach directly through theanterior wall of the fundus 4 centimeters from the esophagus. This wasfollowed by an injection of 4 cubic centimeters of .5 per cent hydrochloricacid at the same point.

Nov. 22, 1913, 9 A.M., the animal was killed. A triangular shapedulcer 7.5 x 6.2 cubic centimeters in diameter was found on the posteriorsurface of the stomach 4 cubic centimeters from the pylorus. Its mar-gins are irregular and very hyperemic. The base of the ulcer is pinkishgray in color. Three smaller ulcers lie below and to the right of thelarger, together with a number of hyperemic lines which on grossinspection show no loss of substance of the mucous membrane.

In eleven experiments an attempt was made to impairthe movements of the stomach by suturing the serosa of theanterior surface of the pyloric part of the stomach to theanterior parietal peritoneum. In this way it was hoped tocause a retention in the stomach of bile and hydrochloricacid over a longer period than in the preceding experiment,where no attempt at interference with peristalsis had beenmade. Bile and hydrochloric acid were introduced into thestomach by way of the duodenum immediately after thesuture of the stomach to the anterior parietal peritoneumhad been performed. In six animals operated on in thismanner lesions of the stomach were produced. In the

166


experiment that follows unusually severe injury to the gas-tric mucous membrane occurred.

Severe ulceration of the anterior and posterior walls of the stomach ofthe cat following the injection of bile and hydrochloric acid throughthe duodenum into the stomach. The lesions in this experimentrepresent the most severe obtained in the series:

Experiment I2. -Cat; male; weight, 3,200 grams.Oct. 10, 1913. Milk given from II.I5 to 11.30 A.M. Operation at

3.30 P.M. The serosa of the anterior surface of pyloric part of stomachwas sutured to the anterior parietal peritoneum with a single silk suture.Two cubic centimeters of bile were removed from the gall bladder andinjected from the duodenum into the stomach. Five cubic centimeters of.5 per cent of hydrochloric acid were immediately injected after this in thesame manner.

October ii; 8.45 A.M., the animal was killed. Both the anterior andthe posterior wall of the fundus are the site of very marked ulceration.Scattered over both surfaces of the stomach are numerous superficialulcers which vary considerably in size and appearance (Fig. 7). Thesmallest measures from I to 2 millimeters in diameter, the largest meas-ures I.5 centimeters by .5 centimeter. The edges of the larger ulcersare sharply defined and elevated. The bases of the ulcers are dark redand clean in appearance. In a number of ulcers the base is covered by athin grayish slough. The lesser and the greater curvatures are the siteof a number of the smaller ulcers.

Microscopical examination: Sections taken from various ulcers on theposterior surface show but slight variation in the character of the lesion.The ulcers are sharply circumscribed and the area of necrosis extends forvarying depths into the mucous membrane. In some places the epitheliumhas been almost completely destroyed as far as the submucosa so that onlyhere and there appear single cells of the chief or the parietal type.Occasionally several cells are grouped together. The necrotic tissue hasin large part been digested away. The submucosa and the muscularis arenormal.

In the course of experiments in which the stomach wasopened and bile and acid applied to the mucous membrane,it became apparent that lesions were most apt to occur whendigestion was in progress and gastric juice was present inthe stomach. With an empty stomach, and especially aftera period of fasting, injury to the mucous surface did notresult. Even after prolonged application of bile and oneper cent hydrochloric acid (nearly twice the acidity of nor-mal gastric jtuice) necrosis of gastric epithelium was not

I67

SMITH.

produced when the stomach was in a state of fasting. Suchwas the case in the following experiment:

Experiment 13. -Cat; female; black; weight, 3,000 grams.The animal had neither food nor drink for forty hours.April 13, I913, at 9.15 A.M., the abdomen was opened and bile aspi-

rated from the gall bladder. The stomach after being incised along themiddle of the anterior surface was found to be free of gastric contents,the surface covered with a moderate amount of mucus. Bile and hydro-chloric acid of I per cent strength were applied to the mucous membraneof the posterior wall at frequent intervals for fifteen minutes, a silk suturebeing placed as a guide 2 centimeters from the point where application ofthe bile and the hydrochloric acid was made. The gastric and theabdominal wounds were then closed.

April I4, 19I3. 7.30 A.M., the animal was killed. The mucous mem-brane showed a slight hyperemia in the region where the bile and acidwere applied. Microscopic examination showed an intact mucous mem-brane.

Lesions were not produced by the direct application ofbile and hydrochloric acid to gastric mucous membrane aftergastrotomy when the stomach contained a large amount offood and digestion was in its earliest stages. An experimentof this nature was the following:

Experiment 14. - Cat; female; weight, 3,000 grams.April i8, 1913. Food was placed in the animal's cage for several hours

before operation. At the time of operation the stomach was greatly dis-tended and contained meat, bread, and curds of milk. The food appearedto be recently ingested, and some of it was removed in order to bring intoview the mucosa of the posterior wall of the stomach. Drops of one percent hydrochloric acid alternating with bile were applied to the gastricmucosa for five minutes. The gastric and the abdominal wounds werethen closed.

April I9, 19I3. The animal was killed. No lesion was found on grossor microscopic examination of the tissues taken from the site of applicationof bile and hydrochloric acid.

The following table (Table I.) shows approximately theperiods after ingestion of food at which lesion of the gastricmucous membrane most frequently occurred. Milk wasgiven the animals in quantities from one hundred to two hun-dred cubic centimeters. In six experiments the animals

I68


received a meal of meat. Bile and hydrochloric acid wereintroduced into the stomach by injection ftom the duodenum.

TABLE I.

Number of Hoursafter Meal.

I ..................

2..................

3 ..................

4 ..................

5---*9X-X...........6 ..................

8 ..................

24 ..................

Lesion occurredhour after feeding.

Total Number of Number of Experi-Experiments. ments with Lesions.

3

3

II

'9

14

6

4

8

0

6

13

6

2

0

Per Cent.

0

33X3%

54.5%68.4%

42.8%

33.3%0

12.5°/0

chiefly between the third and the fifthAt this time after a meal of milk gas-

tric digestion in the cat is well advanced and food has in largepart been passed into the duodenum. Mucus is probablyless abundant in the stomach at this period than in earlierstages of digestion, and gastric juice is present in sufficientquantity to attack epithelium injured by the combined actionof bile and hydrochloric acid.

In order to emphasize the important relation of gastricjuice to the production of lesions of the gastric mucousmembrane with bile and hydrochloric acid, a number ofexperiments were performed in which artificial gastric juicewas introduced into the fasting stonmach in conjunction withbile and hydrochloric acid. Lesions of the gastric mucosacould be readily produced in this way, particularly ifmeasures were taken to confine the fluids to the stomach byligating the esophagus and also the duodenum just belowthe pyloric valve. The following is an illustrative experi-ment:

I69

Experiment I5. - Cat; male; weight, 4,000 grams.The animal received no food for forty-eight hours preceding operation.

Dec. I2, 1913, 2 P.M., the abdomen was opened and the esophagus wasligated below the diaphragm with a silk suture. The duodenum wasopened by a short incision at about the level of the entrance of the com-mon bile duct and a small soft rubber catheter was introduced into theduodenum and through the pylorus. The stomach was washed with a .25per cent hydrochloric acid solution until the returning fluid appeared clear.Two cubic centimeters of bile removed from the animal's gall bladdermixed with X cubic centimeter of bile from a dog were allowed to flow intothe stomach through the rubber tube. Five cubic centimeters of .5 percent hydrochloric acid were then injected. Two minutes later, 8 cubiccentimeters of gastric juice prepared from infusion of cat's stomach werealso injected into the stomach. All these fluids were warmed to a tem-perature of 370 C. before injecting them into the stomach. A silk ligaturewas tied around the duodenum 2 centimeters below the pyloric opening.The duodenal and abdominal wounds were then closed.The animal was killed at 9 A.M. December 13. Five small superficial

ulcers were found scattered over the posterior surface of the stomach,nearer the lesser than the greater curvature. The largest measured 5 x 2millimeters. A number of interlacing pinkish streaks were found alongthe great curvature.

In eight other similar experiments in which small amountsof bile and hydrochloric acid were confined in the fastingstomach in the presence of moderate amounts of acid infu-sion of gastric mucous membrane, lesions closely resemblingthose just described occurred.

In experiments controlling the above, in which animalshad been starved for twenty-four hours, and had receivedhydrochloric acid and the gastric juice but no bile, lesionsdid not result. The washing of the stomach with a weakhydrochloric acid solution preceding the introduction of bilewas done, because at times a little bile is found in thestomach of the cat or dog as a result of regurgitation fromthe duodenum following the struggle during early stages ofanesthesia. Bile can occasionally be found in the stomachin considerable quantity as a result of regurgitation whenthe thorax has been compressed for purposes of artificialrespiration. It was with the purpose of excluding any bilethat had accidentally found its way into the stomach that apreliminary irrigation of the stomach with either weak

170 iSMITH .

AN EXPERIMENTAL STUbY OF BILE.

hydrochloric acid or normal solution sodium chloride wasperfortned. The precaution was taken to measure theamount of fluid used before and after irrigation.

The significance of mucus as a protection of the gastricmucous membrane against injury by bile and .5 percent hydrochloric acid.

A frequent reason for failure to produce lesions of necrosisin the gastric mucous membrane after introduction of bileand hydrochloric acid into the stomach is perhaps the pres-ence of mucus. Whenever the coating of mucus is abun-dant or unusually viscid it affords gastric epithelium ampleprotection against the action of bile, hydrochloric acid orgastric juice. Thus, during the early stages of digestion,when mucus from swallowed salivary secretions is added tomucus already existing in the stomach, injury to gastricepithelium was not apt to occur. When the stomach of theexperimental animals was found to contain parasites or for-eign material, such as a bolus of hair, mucus was usuallypresent in liberal amounts in the stomach and conditionswere not favorable for the experiment. The mucus whichhad been swallowed as a result of the active salivation pro-duced during the early stages of the administration of ether,was believed at times to contribute to the failure of theexperiment. For this reason chloroform was administeredto animals as a routine during the early stages of the anes-thesia and, after unconsciousness developed, the anestheticwas changed to ether. When an unusually thick or viscidbile was used in the experiment, lesions were less apt tooccur than when more fluid bile was employed. The irrita-tion to the gastric mucous membrane resulting from thewearing of a metal gastric canula appeared to increase themucous secretions in an animal's stomach sufficiently to pre-vent injury when bile and hydrochloric acid were introducedinto the stomach.The importance of mucus as a means of protecting the

stomach against injury by chemical, mechanical, and thermalagents has long been recognized and has been the subject of

17I

SMITH.

comment by earlier writers such as Claude Bernard2l andBeaumont. In this connection the experiments which Schiff22performed on dogs with gastric fistulw may be briefly cited.Schiff employed a specially constructed canula supplied withnumerous small openings through which gastric juice mightreadily pass. The canula was so arranged that small amountsof food could be placed in its interior. In the experimentthe canula without food was first introduced into the emptystomach of a dog. At the end of two hours the canula wasremoved and found covered with a membrane of adherentmucus. After placing food into the canula it was once moreintroduced into the dog's stomach, the animal receiving ameal by mouth at the same time. Even at the end of eighthours the particles of food in the canula were found intact,although hydrochloric acid could be detected on the insideof the canula. It was Schiff's opinion that the mucus sur-rounding the canula permitted a diffusion of hydrochloricacid to occur, but prevented pepsin from gaining access tothe food contained in the canula. Recent experiments byKirchheim 3 aim to prove that mucus protects the intestineagainst proteolytic enzymes of the pancreas. Kirchheimplaced flakes of fibrin colored with fuchsin in the bottom ofsmall beakers. He covered the fibrin with mucus which hadbeen carefully removed from the surface of the intestine andthen centrifugalized it. Over the mucus was poured a solu-tion of trypsin, possessing the power of digesting fibrin in afew minutes. The fibrin in the experiment which was sepa-rated from trypsin by a layer of mucus withstood digestionfor sixteen and twenty hours.

Besides affording the gastric epithelium protection throughits physical properties, mucus is thought to protect thestomach against self digestion because it contains, accordingto Danielewsky,% a certain amount of antipepsin, similar toantipepsin first described by Weinland 25 as existing in someintestinal parasites. Ever since Pavlov has established thefact that the secretion of hydrochloric acid in the dog is ofa constant acidity and that the usual variations in the acidityof hydrochloric acid in gastric juice are dependent upon the

I 72


extent of neutralization by alkaline secretion of mucus, ithas become more and more recognized that in man, also,the secretion of gastric mucus bears an important relationto the degree of acidity of gastric juice. Schmidt2 hasfound in the analysis of the gastric contents of the humanstomach that the degree of acidity of hydrochloric acidvaries usually in an indirect ratio with the amount of mucuspresent in the stomach. Of considerable interest in thisconnection are the observations of Kaufman27 based likewiseon the study of the gastric analysis of human subjects, inwhich he has noted that the amount of mucus in thestomach may be greatly diminished in conditions of hyper-acidity and also in cases of gastric ulcer. In the pres-ent experiments the amount of mucus found in the stomachhas its significance chiefly in the fact that an abundant sup-ply prevented the interaction of bile and hydrochloric acidfrom taking place in close contact with epithelium; and,further, because alkaline mucus tended to dissolve thecohesive film of precipitate formed on gastric epithelium bythe mingling of bile with hydrochloric acid.

A series of experiments was performed to study the effectof bile alone on the mucous membrane of the fasting stom-ach, when confined in the stomach by ligating the esophagusand the duodenum. In the following table (II.) the effectof varying amounts of bile in the fasting stomach of the cathave been recorded. The animals received no food forforty-eight hours. Ox-bile was introduced into the stomachby injection through the duodenum. The esophagus andthe duodenum were ligated. The animals were killed twentyhours after the introduction of bile into the stomach.

I 73

174 SMITH.

TABLE II.

Weight of Cat. Amount of Bile. Lesions.

2,800 grams. 40 cc. Present.

2,600 " 30 " 4

3,200 " 30 "

2,400 " 20

2,600 " I5 " Slight.

2,800 " I0

4,100 l1O " Absent.

3,600 lO10

2,200 " 10 " ..

2,100 " 7.5 " Slight.

2,800 " 7.5 " Absent.

2,80o " 5 . Is

3,600 5

2,000 " 5

2,600 5 91

The experiments, as tabulated above, show that bileintroduced in large amounts into the fasting stomach of thecat, and confined there by ligating the esophagus and theduodenum, will cause injury to gastric mucous membrane.Bile in amounts of ten cubic centimeters to 7.5 cubic centi-meters may produce very slight lesions, in smaller amounts,however, it causes no injury to gastric mucosa.The lesions consisted of hemorrhages into the mucous

membrane and superficial necrosis of gastric epithelium,resembling in character the lesions described in earlier experi-ments when bile and hydrochloric acid were introduced intothe stomach in small amounts without the ligation of duo-denum or esophagus.


Another series of experiments was performed testing theeffect of different amounts of bile in the presence of hydro-chloric acid on the mucous membrane of the fasting stomach(Table III.). The animals received no food for forty-eighthours. Bile with .* per cent hydrochloric acid was theninjected into the stomach by way of the duodenum. Theesophagus and the duodenum were ligated.

TABLE III.

Weight of Cat.

2,000 grams.

3,000 "

3,200 "

3,400

3,600 "

3,000 "

3,100 "

3,600 "

3,600

2,100 "

4,000 "

2,600 C

Bile.

10 CC.

lO "

10 "'

75"5 is

5 "

3 "3 cc

0.5% HCI.

I5 cc.

15 "

I5s

I5 "

IO"

10 "

8"

8"75 id8 s

S d

Lesions.

Present.

di

6.96

id

SC

is

..

Absent.

Present.

From the above table it will be seen that bile confinedin the stomach in the presence of hydrochloric acid mayproduce lesions of the gastric* mucosa even when usedin small amounts. It will appear, further, that the combi-nation of bile and hydrochloric acid is more injurious to.gastric epithelium than bile alone. As described in theearlier part of this paper, if to small amounts of bile andhydrochloric acid (such as three cubic centimeters bile andfive cubic centimeters of .5 per cent hydrochloric acid) anacid infusion of gastric mucous membrane is added, lesionsof the mucosa occur with regularity.

175

SMITH.

In a third series, bile in the presence of solution of .5 percent sodium carbonate was studied (Table IV.). The animalsin these experiments, after fasting forty-eight hours, receivedbile and varying amounts of .5 per cent sodium carbonate,injected into the stomach by way of the duodenum. Theesophagus and the duodenum were ligated.

TABLE 1V.

Weight of Cat.

2,I00 grams.

2,0 s"

3,000 "

3,200 "

2,200 "

3,000 "

2,0oo "

2, I00 "

2,000 "

2,400 "

Bile.

20 CC.

I5 St

Io St

I0"Io 6

10 s

7.55 d'

5 "

3 66

Sodium Carbonate.

20 CC.

20 "

15 "

Is5 9

Is5 4

15

10"

Io it

8s

5"

Lesions.

Absent.

Very slight.

Absent.

Bile in the presence of an alkaline solution such as .5 percent sodium carbonate appears less toxic for gastric epithe-lium than either bile alone or bile in the presence of hydro-chloric acid. In only one experiment were lesions producedconsisting histologically of slight hemorrhages into themucosa and of minute areas of necrosis of epithelium at thesurfaces of the mucous membrane.

Summary. -It would appear from the foregoing experi-ments that, by the combined action of bile and of hydrochlo-ric acid used in a strength which is normal for the gastricjuice of the cat and the dog, there may be produced in thegastric mucous membrane areas of hemorrhagic necrosiswhich, upon subsequent digestion, form ulcers. When bile

176


and .5 per cent hydrochloric acid are placed together ongastric mucous membrane a visible change in the appearanceof the epithelial surface occurs, caused by the precipitationof proteins existing in both the bile and the gastric mucus.The precipitate forms a fine cohesive film in close contactwith the underlying epithelium. It is insoluble in weak acids,dissolves readily in alkaline solutions, and can be digested bygastric juice. The experiments show that the presence ofmucus in abundance is frequently sufficient to protect thegastric epithelium against the combined effect of bile andhydrochloric acid.

Lesions of the mucosa of the stomach were most readilyproduced from three to five hours after meals when gastricdigestion was nearing its completion, as at this time a suffi-cient amount of gastric juice was still present in the stomachto insure immediate action upon the epithelium injured bybile and hydrochloric acid. The experiments were unsuc-cessful when attempted soon after the giving of a meal, for atthis time the presence of an increased amount of mucus,derived both from the stomach and the saliva, afford theepithelium a strong protection against injury. As a rule, inthe fasting stomach lesions of the mucous membrane did notoccur after the introduction of bile and acid, because theabsence of gastric juice removes an important factor in theproduction of the lesion. Yet, if artificial gastric juice wasintroduced into the fasting stomach together with bile andhydrochloric acid, lesions were formed.The gross appearance of the lesions produced by bile in

the presence of an excess of .5 per cent of hydrochloricacid acting upon gastric mucous membrane is characterizedby short, irregular red lines of superficial necrosis and bysmall ulcers often following along fine grooves formed bythe mucous membrane. Larger ulcers may be formed at thepoint of confluence of several linear streaks of erosion.Ulcers may be formed apparently in no relation to and dis-tant from the small red streaks of ulceration. The ulcersthough superficial in character have hyperemic edges whichare sharply defined and usually a clean base of grayish red or

177

SMITH.

of dark red color. The areas of ulceration produced by theagency of bile and hydrochloric acid may exist in any regionof either the pylorus or the fundus, but chiefly the latter.Lesions near the esophagus were found to be infrequent,Injury to the duodenum just beyond the pyloric valve didnot occur in any of the experiments. When bile and hydro-chloric acid were applied to the duodenal mucosa of the dogand the cat a precipitate was formed much like that seen in thestomach; it was, however, readily dissolved by the alkalinesecretions of the duodenum in these animals and no injuryto the epithelium resulted.

Histological examination of the early lesions of the gas,tric mucous membrane occurring from three to six hoursafter the introduction of bile and hydrochloric acid into thestomach furnished evidence that superficial epithelial cellsmay already be detached from underlying supporting tissue.Not alone epithelium but the interglandular stroma lyingdirectly beneath the surface epithelium exhibited evidence ofnecrosis. Not infrequently hyaline thrombi were found inthe small capillaries lying in the necrotic connective tissuejust below the epithelium. Eight hours after the introductionof bile and acid into the stomach, extensive capillary hem-orrhages were found and accumulation of inflammatory cellsabout the areas of necrosis had occurred. The destructionof both the chief and the parietal cells were well marked atthis time. Within a period of twenty-four hours the maxi-mum injury to gastric mucous membrane following a singleinjection of bile and hydrochloric acid into the stomach hasusually taken place. At this time necrotic tissue was to agreater or less extent digested away and areas of superficialulceration, never extending deeper than the submucosa, weredefinitely formed.

In the fasting stomach after ligating the esophagus andthe duodenum bile when used in large amounts was found tocause hemorrhages into and necrosis of the mucous mem-brane. Bile in the presence of .5 per cent hydrochloric acidproduced injuries more readily than did bile used alone.Bile in the presence of an alkaline solution, as .5 per cent

I178


sodium carbonate, was found to be non-injurious to gastricmucous membrane even when introduced in considerablequantities into the fasting stomach that had been ligated at theesophagus and at a point just distal to the pylorus.

Significance of experiments for ulcer of the stomach inman.- In the absence of objective evidence it must beregarded as a matter of conjecture whether or not in thestomach of man bile may become a source of injury to themucous membrane and thus produce an ulcer. A numberof facts would suggest that such a cause for gastric ulcermay possibly exist. The usual distribution of ulcer in theupper part of the duodenum above the entrance of the com-mon bile duct and in the pyloric region of the stomachmarks the region which bile must traverse when it flowsbackward into the stomach. The acid gastric juice existshere plentifully during certain phases of normal digestion.The presence of an abnormal amount of hydrochloric acid,which occurs frequently with pathological hyperacidity andhypersecretion in patients the subject of gastric ulcer, mightafford the conditions necessary for regurgitating bile to actinjuriously upon gastric or duodenal mucosa. It is nowknown that bile is found at times in the gastric content ofpatients suffering with ulcer of the stomach. Recentstatistics in the gastric analysis of patients suffering fromulcer of the stomach published by Smithies,28 show that bilewas found in nine and two-tenths per cent of the casesexamined.

Moreover, observations on gastric contents made byKaufman have pointed out that in certain conditions ofhyperacidity as well as in conditions of gastric ulcer, mucusis less plentiful in the stomach than under normal circum-stances. These observations are in harmony with the viewof Schmidt, published a number of years ago, who statesthat gastric acidity in man usually bears an inverse ratio tothe amount of mucus present in the stomach. Schmidt hasdemonstrated by experiments that gastric juice in thepresence of an excess of hydrochloric acid may even digest

I79

SMITH.

mucus obtained from the stomach. If it is true, therefore,that the two conditions, hyperacidity of gastric juice and arelative diminution of mucus, may be present at the sametime in the stomach of man, unusually favorable circum-stances might exist for the production of an injury to themucous membrane through the agency of bile. An excessof hydrochloric acid and a scanty amount of mucus affordedunder experimental conditions the best opportunity for theproduction of a lesion of the gastric mucous membranewhen bile was introduced into the stomach. Finally it ispossible that a hypersecretion of hydrochloric acid mayactually form the stimulus for the flow of bile back into thestomach, a circumstance which finds its parallel in the experi-ments of Boldyreff, already cited, in which bile and pan-creatic juice were found to regurgitate into the stomach whenlarge amounts of weak acids had been introduced into thisorgan.The ulceration produced in the gastric mucous membrane

by the experimental introduction of bile and weak hydro-chloric acid into the stomach are superficial in character and.doubtless readily heal. These lesions, therefore, cannot beregarded as closely resembling the usual form of chroniculcer in man. The experimental ulcers formed with bile andchydrochloric acid bear a greater likeness to the small multi-ple ulcers or erosions which are fotund post-mortemn in thehuman stomach. It is believed that these erosions areformed a short time before death. Ulcers and erosions ofthis character in man show a tendency to occupy the bodyand the fundus of the stomach rather than region of thepylorus and at times are found arranged in rows along thelongitudinal folds of gastric mucous membrane.

CONCLUSIONS.

i. When introduced into the stomach of the cat or the-dog, bile in the presence of an excess of .5 per cent hydro-chloric acid may cause injury to gastric mucous membrane,whereas bile or .5 per cent hydrochloric acid introducedalone into the stomach is without harmful effect.

I80

AN EXPERIMENTAL STUDY OF BILE. i8i

2. Lesions of the gastric mucous membrane produced bybile in the presence of an excess of .5 per cent hydrochloricacid consist of necrosis of epithelium and interglandulartissue with hemorrhages into the mucous membrane, as aresult of which small superficial ulcers may form.

3. Ulceration of gastric mucous membrane, following theintroduction of bile and hydrochloric acid into the stomachinjected by way of the duodenum, is produced most readilybetween the third and the fifth hour after meals; leastreadily in the fasting stomach or shortly after.the ingestionof food.

4. If confined in the fasting stomach by ligating theesophagus and the duodenum, bile in the presence of anexcess of .5 per cent hydrochloric acid is more toxic forgastric epithelium than either bile alone or bile in the pres-ence of an alkaline solution, such as .5 per cent sodiumcarbonate.

5. The presence of mucus in the stomach protects gastricepithelium against injury by bile and hydrochloric acid.

REFERENCES.

i. Moller, S. Die Pathogenese des Ulcus ventriculi mit besondererBeriicksichtigung der neueren experimentellen Ergebnisse. Ergeb. d.Inneren Med. und Kinderheilkunde, 1911, vii, 520.

2. Scagliosi, G. Beitrag zur Atiologie des Duodenal geschwiirs.Virchows Archiv., 1913, CCXiV, 220.

3. Aschoff, L. Ueber die Mechanische Momente in der Pathogenesedes runden Magengeschwiirs und iUber seine Beziehung zum Krebs.Deutsch. Med. Woch., 1912, xxxviii, 494.

4. Stromeyer, F. Die Pathogenese des Ulcus Ventriculi, zugleichein Beitrag zur Frage nach den Beziehungen zwischen Ulcus und Car-cinom. Beitrage zur Pathologischen Anat. und Allegemein. Path., I912,liv, I.

,5. Katzenstein, M. Der Schutz des Magens gegen die selbst verdau-ung nebst einem Vorschlag zur Behandlung des Ulcus Ventriculi. Berl.Klin. Woch., I908, xxxix, 1749.

6. Beaumont, William. Experiments and observations in the gas-tric juice and the physiology of digestion. Plattsburgh, x833, 21.

7. Pavlov, I. P. Die Arbeit der Verdauungsdrnsen. Wiesbaden,1898, 159.

8. Boldyreff, W. Der tbertritt des NatUirlichen Gemisches aus Pan-kreassaft, Darmsaft und Galle in den Magen. Archiv. f. Gesammte Phys-iol. d. Menschen u. d. Thiere, I908, cxxi, 13.

182 SMITH.

9. Rywosch, David. Ueber die giftige Wirkung des Gallensaurennebst einem Anhange Uber die Giftigkeit der Gallenfarbstoffe. Inaugural--Dissertation, Dorpat, I891, 413.

io. Meltzer, S. J., and Salant, W. Studies on the toxicity of bile.Journal of Experimental Medicine, 1906, viii, 127.

ii. Opie, E. L. The etiology of acute hemorrhagic pancreatitis. Bull.of the Johns Hopkins Hospital, igot, xii, i82.

12. Flexner, S. The constituent of the bile causing pancreatitis andthe effect of colloids upon its action. Journal of Experimental Medicine,I9o6, viii, I67.

13. Smith, G. M. Morphological changes in tissue with changes inenvironment. Replacement of surface epithelium of grafted tissue byadjacent epithelium. Journ, of Med. Research, 1913, xxviii, 423.

14. Oddi, R. Azione della bile sulla digestione gastrica, studiata calmezzo dello fistola colecisto-gastrica. Reviewed in Centralbl. f. Phys.i887, Xiv, 312.

15. Dastre, A. Recherches sur la Bile. Archives de Physiologienormale et Pathologique, 1890, V, 315.

i6. Kaiser, D. Ueber die Erfolge der Gastro-Enterostomosis. Deut-*sche Zeitschrift f Chir. 190I, lxi, 294.

17. Carle, A., and Fantino, G. Beitrag zur Pathologie und Therapiedes Magens. Arch. f. Klin. chir. I898, lvi, i.

i8. Kausch, W. Ueber functionelle Ergebnisse nach Operationen amMagen bei gutartigen Erkrankungen. Mittheil a. d. Grenz. d. Med. u.chir., I899, iv, 347.

I9. Hammersten, 0. A text-book of physiological chemistry, I9II,441.

20. Matthes, M. Untersuchungen uber die Pathogenese des Ulcusrotundum ventriculi und iuber den Einfluss von Verdauungsenzym auflebendes und todtes Gewebe. Beitriage z. Path. Anat. u. z. AllgemeinenPathologie, I893, xiii, 309.

2I. Bernard, C. Physiologie Expdrimentale, ii, i856, 408.22. Schiff, M. Autodigestion de l'estomac aprEs la mort et durant la

vie. Gesammalte Beitriige zur Physiologie, I898, iv, 405.23. Kirchheim, L. Uber den Schutz der Darmwand gegen das Tryp-

sin des Pankreassaftes. Archiv. f Experimentelle Pathologie und Pharma-kologie, 1913, lxxi, I.

24. Danilewsky, A. Reviewed in Jahres-Berichte uber die Fortschritted. Thier-chemie, 1904, xxxiii, 556.

25. Weinland, E. Ueber Antifermente. Zeitschr. f. Biologie, 1903,xliv, 1.

26. Schmidt, Adolf. Ueber die Schleimabonderung im Magen.Deutsch Archiv. f. Klin. Med., I896, lxxv, 65.

27. Kaufman, T. Mangel an Magenschleim (Amyxorrhea gastrica),seine pathologische Bedeutung und seine Beziehungen zur Hyperaciditatund zum Magengeschwiir. Archiv. f. Verdauungs-Krankheiten, I907, xiii,6i6.

28. Smithies, F. Gastric ulcer without food retention. A clinicalanalysis of one hundred and forty operatively demonstrated cases. Amer.Journ. of the Med. Sciences, 1913, cxlv, 340.

AN EXPERlMENTAL STUDY OF BILE.

DESCRIPTION OF PLATES.

PLATE XIII., FIG. I. -Stomach of cat nineteen hours after theintroduction of three cubic centimeters of bile and five cubic centimeters.5 per cent hydrochloric acid, by way of duodenum. A network of linesis seen near the pylorus, marking the site of superficial necrosis of epi-thelium and hemorrhages inito the mucous membrane. Larger ulcers areseen in the body of the stomach on both the anterior and the posteriorsurface.FIG. 2.- Microscopic section taken from a superficial ulcer of the

stomach of a cat seventeen hours after the introduction of three cubiccentimeters of bile and five cubic centimeters of .5 per cent hydrochloricacid. Necrotic epithelium and interglandular tissue have become in partdigested. x 300.

PLATE XIV., FIG. 3.-Necrosis of epithelium and interglandular tis-sue in the region of the pylorus of the cat, nineteen hours after theintroduction into the stomach of two cubic centimeters of bile and fivecubic centimeters of .5 per cent hydrochloric acid by way of the duode-num. Polynuclear leucocytes have accumulated in moderate number inthe area of necrosis. x 300.FIG. 4.- Early hemorrhages into the mucous membrane of the

stomach of the cat with necrosis of epithelium, six hours after the intro-duction into the stomach of three cubic centimeters of bile and five cubiccentimeters .5 per cent hydrochloric acid by way of the duodenum.

PLATE XV., FIG. 5. - Early necrosis of epithelium and interglandulartissue in the mucous membrane of the stomach of the cat, six hours afterthe introduction into the stomach of three cubic centimeters of bile and.5 per cent hydrochloric acid. x 300.

FIG. 6. -Ulcers formed on the posterior wall of the stomach of a cateighteen and one-half hours after the injection into the stomach of twocubic centimeters of bile and four cubic centimeters .5 per cent hydro-chloric acid made directly through the anterior wall of the fundus.

PLATE XVI., FIG. 7.- Severe ulceration in the stomach of a cat, seven-teen hours after introducing into the stomach two cubic centimeters ofbile and five cubic centimeters .5 per cent hydrochloric acid by way ofthe duodenum. In this experiment the pyloric part of the stomach wassutured to the anterior parietal peritoneum in order to impair the move-ments of the stomach.

FIG. 8. - Superficial ulcer formed on the posterior wall of the stomach,taken from the same experiment as Fig. 7, Plate XVI. x 20.

FIG. 9.- Edge of an ulcer taken from the same experiment as Fig. 7,Plate XVI. The destruction of epithelium and interglandular tissue islimited to the mucosa. x 300.

183

JOURNAL OF MEDICAL RESEARCH

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