- Email: [email protected]
Demonstration of the functional anatomy of the canine gastric antrum
Demonstration
of the Functional
of the Canine I. Operative
Gastric
Technics Requiring
Antrum* Gastrotomy
ROGER E. MOE, M.D., P. J. KI,OPPER, M.D., AND LLOYD M. Nytrus,
From the Laboratory for Experimental Surgery, Surgical Clinic, Wilhelmina Gasthuis, University of Amsterdam, and the Department of Surgery, University of Washington, Seattle, Washington. This study was aided in part by funds accruinP from National Institutes of Health Grant $A M-04010, &d Initiative 171, State if Washington, and Research Training Grant GM-325.
I
Anatomy
M.D., Seattle,
Washingtolz
the antrum brown, and the intervening boundary formed a sharp continuous line [Z]. Later, Lowicki and Littlefield reported topical application of Congo red to gastric mucosa of human stomachs, staining the corpus black or dark blue and the antrum red [3]. Their experiment represents the first study devoted to in viva demonstration of the gastric antrum for facilitation of complete antrectomy during clinical surgery. Confirmatory findings were published by Moe, Nyhus, and Harkins [P] and by Smith and Howes [5] using dogs. Smith and Howes employed Congo red as an aid in construction of experimental antral pouches. Other indicators (topical bromphenol blue, intravenous fluorescein, litmus paper, and universal indicator paper) have been used by Capper et al. to detect the gastric “acid-alkaline junction” in patients with peptic ulcers [6]. Furthermore, they discovered that the gastric acid-alkaline junction can be detected through a gastrotomy by directly measuring mucosal pH with an electronic meter. In the present investigation, dyestuffs have been utilized to show anatomic limits of the gastric antrum in dogs at surgery. The basic method of this technic is new. Rather than depending upon different colors of dyestuffs at different pH ranges, the present method depends upon differences in elimination of intravenous dyestuffs via the gastric corpus and antrum. Various workers have studied gastric excretion of many dyestuffs after intravenous injec-
T is common knowledge that anatomic limits
of the gastric antrum in dogs and human subjects cannot be visually discerned consistently and accurately during a surgical operation unless the gastric mucosa is modified in some way. One way to modify gastric mucosa in V&IOinvolves the use of dyestuffs providing contrasting colors in the gastric corpus and antrum which are visualized by exposing gastric mucosa through a gastrotomy. In most publications up to now, the basic method has featured a dyestuff which exhibits one color in a given pH range and another color in a different pH range. Hence, inside the stomach the color of such a dyestuff over an acid-secreting area (corpus) differs from the color over a non-acid-secreting area (antrum). Claude Bernard first published this observation after he demonstrated which part of the stomach in a rabbit produces acid. After intravenous injection of appropriate reagents, Prussian blue appeared in part of the stomach, signifying an acid area, while the remainder of the stomach did not become blue [I]. Using dogs in more detailed studies, Brenckmann and Deloyers stained gastric mucosa topically with Prussian blue reagent. The gastric corpus became blue,
* Presented at the annual meeting of the Pacific Coast Surgical Association, Vancouver, British Columbia, February 22-25, 1965. Vol.110,August 1965
277
Moe,
278
Klopper,
and Nyhus
6.0 P~locdrpme
50-
T
1% 6mqm
loluidine
blue IOcc
I.V. \
SQ t + Copious
secretion of dye t Entire MAAG Good corpus-antrum border Grey-blue I” I min Free dye in corpus, not in ontrum Per~gtols~s-------Peristalsis continuous---------------
1 I1 I I1 I1 I1 1, I I I I,, 8 16 24 32 40 48 56 64 72
I I I,,,‘,,,,,,,,
00
88
96
104
II2
120
128 136
Minutes
Fro 1. Record of corpus and antrum pH during toluidine blue excretion in one dog. tion [7-IO]. Some dyestuffs readily
appeared
in
secretions from corpus pouches but appeared minimally or not at all in secretions from antral pouches in dogs 171.Two of those dyestuffs were employed here-toluidine blue and neutral red. Since these dyestuffs are excreted differently by the corpus and antrum, it should be possible to delineate these gastric areas visually when mucosa is exposed. However, this delineation is unreliable since colors from toluidine blue and neutral red are obscured by the color of gastric mucosa. This problem is corrected when a layer of moist white gauze is applied to gastricmucosa before injecting dyestuffs. As a result, toluidine blue and neutral red are easily visualized in gauze over corpus mucosa and, likewise, the area where these dyes do not appear (antrum) is also easily apparent; a continuous boundary between the corpus and the antrum is accurately delineated. This work also includes further investigations of the use of fluorescein [S] and Congo red for revealing the gastric antrum in dogs [3]. MATERIALS
AND
METHODS
Solutions of dyestuffs were made as follows: toluidine blue or neutral red was dissolved in 5 per cent dextrose and water to make a 1 per cent solution which was filtered to prevent microembolism from undissolved dye particles; Congo red was dissolved in water with moderate warming to make a 6 to 7 per cent solution; aqueous fluorescein was studied in two dilutions, 5 per cent and 0.5 per cent. One half hour before gastrotomy, gastric secretion was stimulated with histamine base, 0.04 mg./kg.
body weight [11] injected subcutaneously or 0.015 mg./min. intravenously. The intravenous route was more reliable. Some dogs were given pyrilamine maleate, 25 mg. intramuscularly or intravenously, prior to histamine injection to minimize systemic effects of histamine. Another way to avoid any systemic effects of histamine is to inject 100 mg. of Histalog*@ one hour before gastrotomy instead of using histamine [12,1?]. Thirty-four dogs were studied. Each had received no food for twenty-four hours but had free access to water. Anesthesia was achieved by slow intravenous injection of a combination of alpha-chloralose (50 mg./kg. body weight) and urethan (500 mg./ kg.) mixed in propylene glycol. This anesthesia does not inhibit gastric secretion [14]. Intravenous thialbarbitone sodium can also be used satisfactorily. Atropine and similarly acting drugs were avoided. Through a midline abdominal incision a longitudinal anterior gastrotomy was made with a scalpel or scissors between crushing clamps. This gastrotomy was about 10 cm. long, extending proximally from a point 2 cm. above the pylorus. After meticulous hemostasis the gastrotomy edges were held apart by retraction sutures extending from the stomach to the abdominal wall. Gastric mucosa was then lavaged with warm saline solution and gently sponged to remove food debris. A plug of gauze was placed near the cardia to block fluid which sometimes emerged from the esophagus. A piece of white gauze was spread apart to obtain a single (very thin) layer. This gauze was moistened and laid upon the gastric mucosa, conforming closely to mucosal contours. The mucosa was covered only where a corpus-an&urn boundary might be anticipated. * Manufactured as betazole by Eli Lilly, Indianapolis, Indiana. American
Journal of Surgery
Functional
FIG. 2. Corpus-antrum I’urplc-stained gauze
Vol.
I If),
ArLgusl
1965
Anatomy
of Gastric
Antrum
boundary after intravenous injection of 10 cc. of 1 per cent is over the corpus, while unstained gauze is over the antrum.
neutral
red.
Moe,
Klopper, and Nyhus
RESULTS
Tolw’dine Blue. After intravenous injection of 10 cc. over a period of two minutes, gauze over the corpus area was stained pale blue in approximately five minutes while the antrum area remained unstained. Blue color over the corpus developed more intensely over a fifteen to thirty minute period. Further increments of dye (up to a total of 50 cc.) were used in several instances for greater intensity of color although this was not usually necessary when active gastric secretion existed. With two dogs the protocol was modified slightly to study factors which might relate especially to the excretion of toluidine blue. One dog was not given histamine but still demonstrated a clear antrum-corpus color boundary after 10 cc. of intravenous toluidine blue. Similarly, a second dog was not given histamine but instead received 6 mg. of pilocarpine subcutaneously. After 10 cc. of intravenous toluidine blue, this dog excreted dye copiously in the corpus while the corpus mucosa and antral mucosa maintained a pH between 7.0 and 7.1 by direct measurement with a glass electrode. (Fig. 1.) The protocol was otherwise unchanged. hleutral Red. Intravenous injection of neutral red was performed in the same manner described for toluidine blue. Gauze over the corpus became violet in about three minutes, while antrum gauze remained unstained. (Fig. 2.) Rarely a faint yellow hue appeared on antrum gauze adjacent to the corpus-antrum boundarv. This dye emerges more profusely than toluidine blue. No dog stomachs failed to excrete neutral red. Some dogs excreted this dye in apparently small quantities, but a sharp corpus-antrum boundary was still discernible. One dog failed to reveal a definite corpusantrum boundary. In that stomach, dye was present but faded out gradually near the antrum. That dog had been “wormed” twelve hours before operation. The stomach contained dead worms and feculent material, and the mucosa was dark red, friable, and bled easily. Congo Red. Best results occurred when this dye solution was applied topically as a fine spray from a nebulizer rather than by lavaging or sponging the dye onto the mucosa. When applied directly to the mucosa, Congo red stained the corpus black and the antrum red. When a single thickness of gauze was placed
upon the mucosa before spraying dye, this dye stained the corpus blue and the antrum red. When a more dilute solution of Congo red was used directly upon the mucosa [j], the corpusantrum boundary was not as precisely discernible because the colors were less vivid than when a saturated solution was used. However, when that same diluted dye solution was sprayed onto a thin gauze overlay inside the stomach, the corpus-antrum boundary was more precisely and clearly apparent. Fluorescein. After the intravenous injection of 2 cc. of 5 per cent solution, mucosa of the entire stomach and small intestine fluoresced with a green color under an ultraviolet light source (Philips type no. 57236 E/70 HPW 125 W) in a dark room. Some fluorescein appeared also in free fluid secreted by the corpus and in secretions from the biliary tree. Fluorescence of gastric mucosa was not destroyed when 0.1 N HCl was dropped directly onto the mucosa. In contrast to the results just noted, the injection of 0.5 per cent fluorescein in 2 cc. increments presented a different mucosal appearance. Within one minute, mucosa of the first part of the duodenum fluoresced as far proximally as the boundary between the duodenum and antral mucosa. (Care was taken to prevent contamination from biliary fluid by means of a gauze plug in the second part of the duodenum.) Next, antral mucosa fluoresced while corpus mucosa did not. The corpusantrum boundary was not quite as sharp or abrupt as boundaries from the other three dyestuff s mentioned herein. Continued injection of fluorescein later was followed by fluorescence of the corpus and fundus, obscuring any delineation of the antrum from the corpus. These results were in accordance with those in our initial study of ten rabbits. Acrid&e Orange. This fluorescent dye was also injected intravenously, but a corpusantrum boundary was not revealed in dogs or in rabbits. Controls. Four or five Nylon@ or silk marker sutures were placed through the stomach wall where a corpus-antrum boundary was detected in each specimen. Histology: The marker sutures consistently appeared within 1 cm. on either side of the histologic boundary, a site where the number of corpus parietal cells abruptly falls. The majority of the markers were 3 mm. or less from the Am&can
Jouvnul of Surnery
Functional
Anatomy of Gastric Antrum
2x1
852
30
SK.
I
CORPUS’
PH 2
NTRUM
4
6
FIG. 3. Continuous
tracing of pH values as a glass electrode is moved 1 centimeter back and forth at the location of a corpus-antrum boundary marker suture.
histologicIboundary; one marker was as far as 1 cm. from the histologic boundary. Comparison of boundary sites: All three dyestuffs, toluidine blue, neutral red, and Congo red, yielded the same boundary site when used during the same operation on each of three different dogs. PH ~vttlunlion: Color boundaries obtained by the presence or absence of dye excretion in the corpus and antrum, respectively, corresponded with pH-dependent boundaries detected by Congo red, Universal indicator paper, or direct pH measurement with continuous recording. (Fig. 3.) Stability: Color boundaries visualized through a gauze overlay have been observed in several animals for one hour with no shift in color on the gauze overlays. When dye excretion was unusually heavy in some animals, a thicker layer of gauze minimized flow of secretion along the mucosa. This is not a problem ordinarily, however, because in such cases the corpusantrum border can be marked before movement of secretion ensues. Reproducibility: A corpus-antrum boundary was demonstrated in three dogs. Each boundary was marked with vitallium sutures. One month later, each dog was reoperated upon. Neutral red was again used in two dogs and toluidine blue in the third dog. In all three dogs the second operation revealed a corpus-antrum boundary remarkably close to the original site, differing by only 0 to 4 mm. in biopsies from each animal. COMMENTS
The principle of differential excretion of dyestuffs from the gastric corpus and antrum can be exploited for reliable delineation of the funcVol.
110,
August
1965
tional anatomy of the gastric antrum. Results described herein indicate that toluidine blue or neutral red can be used for this purpose in experimental animals. Evidence that these two dyes are, in fact, excreted by the corpus but not by the antrum rests upon failure to detect dye in gauze over the gastric antrum under the conditions specified. Also, Dawson and Ivy found that neutral red is readily excreted by Pavlov pouches but not by pyloric pouches unless the pyloric pouches were “activated” by continuous application of gastric juice [7]. They did not study toluidine blue in pyloric pouches. Mucosal PH. Do pH differences in gastric mucosa influence color boundaries obtained with differentially excreted dyestuffs in the stomach? This question is part of the basis for the choice of dyestuffs used here. One dyestuff, toluidine blue, is excreted by gastric mucosa but does not change color as a pH indicator. The second dyestuff, neutral red, is excreted by gastric mucosa and can also change color as a pH indicator. The third dyestuff, Congo red, is not excreted by gastric mucosa after intravenous injection but serves only as a pH indicator [7,8]. Since the corpus-antrum boundary is the same from all three dyestuffs, the conclusion is reached that color boundaries obtained from differentially excreted dyestuffs do not depend upon gastric pH differences per se although pH differences do, indeed, exist at the corpus-antrum boundary. Color boundaries from differential dye excretion depend more particularly, of course, upon active secretion of corpus fluid which might or might not be acidic. From studies including microscopy some workers (but not all) report that neutral red is excreted by parietal cells
Moe, Klopper, and Nyhus [10,15], the cells which also produce acid. Henning thought neutral red was excreted by pepsin cells [16]. Other research methods indicate that acid secretion and neutral red excretion are not necessarily parallel [17,18]. Evidence from the present work suggests that toluidine blue excretion may not parallel acid secretion either. By direct mucosal pH measurements, the corpus of one dog was not secreting acid but still was vigorously excreting toluidine blue. Gastric cells which excrete toluidine blue have not been identified. If pepsin-secreting cells are involved in dye excretion, this would be of interest since Grossman reported that the presence or absence of pepsin-secreting cells may provide the best histologic criterion for delineating the gastric corpus and antrum [19]. In the case of intravenous neutral red, it was considered that a corpus-antrum color boundary might be obtained partly by differential excretion and partly by color alteration with acidity or alkalinity of the corpus and antrum, respectively. Apparently the corpus-antrum color boundary appears only with differential dye excretion in the vast majority of trials with neutral red. It has been mentioned that intravenous neutral red did not appear in gauze over the antrum. Parenthetically, gastroscopy of human stomachs did not reveal any neutral red excretion from the antrum although the dye could be seen in other parts of the stomach [20]. Is it possible that the dyestuff used in the experiments reported herein had been excreted from the antrum and that the color at alkaline pH’s was too pale to be observed? This is doubtful since the surface fluid over the antrum was often found by direct measurement to be insufficiently alkaline to develop any yellow color in neutral red dye. Also, topical neutral red applied to the antrum usually retains its red or violet (acid) color instead of becoming yellow (alkaline). Furthermore, if excreted neutral red is deliberately allowed to flow from the corpus into the antrum, red dye from the corpus remains red (acidic) in the antrum. (Note that excreted neutral red is still capable of changing color appropriately if an acid or base is mixed into a solution of the excreted dye.) A similar observation was made by earlier workers such as Sax1and Scherf [21] and Piersol, Bockus, and Bank [22] who stated that neutral red injections stained the entire gastric mucosa red, and this red color was particularly intense in the prepyloric region [21,22]. They errone-
ously concluded that the entire gastric mucosa was involved in the excretion of neutral red. The dye was not observed when it first appeared over gastric mucosa; distribution of the dye was noted after the animals were sacrificed, thirty to sixty minutes after neutral red excretion had begun. The neutral red was no longer limited to its site of origin, and over the antrum it showed as a red, not a yellow, color. Thus, if the neutral red had been selectively excreted by the antral mucosa in our experiments, the gauze overlay should have stained red. As indicated previously, this did not occur. As a final check, we applied 0.1 N HCI from an eye dropper directly to the corpus-antrum color boundary so that the acid spread both into the corpus and antrum; a red stain did not appear in the gauze over the antrum, while the red stain over the corpus is intensified. Indeed, this maneuver did not disturb the existing corpusantrum color boundary ! Therefore, under the conditions of our experiments, neutral red was not excreted by the antral mucosa. Comparison of Speed and Accuracy. Certain comparative comments can be made about dyestuffs examined. Congo red delineates the corpus-antrum boundary most rapidly. Neutral red involves a short waiting period for the dye to emerge from the mucosa. Toluidine blue emerges more slowly than neutral red. Dilute fluorescein reveals the antral area as fast or faster than neutral red, but as Capper et al. [6] have indicated, there is a disadvantage in the darkened operating room required during use of an ultraviolet light to detect fluorescence. Toluidine blue, neutral red, and Congo red have a similar accuracy in revealing the corpusantrum border. Variations obtained between the sites of color boundaries and the sites of histologic corpus-antrum boundaries seem to be compatible with the size of a narrow histologic border zone between the corpus and antrum in dogs [15,23] and in human subjects [24,25]. Toluidine blue and neutral red yield boundaries even when acid is not removed from antral fluid by sponging or by other means. Congo red, on the other hand, leads to erroneous boundaries if antral acid (below pH of about 5) exists. Fluorescein did not yield a boundary as sharp or abrupt as that obtained with the other three dyes. Only four dogs were studied with fluorescein, but the results were the same as those in our prior studies in ten rabbits. With respect to reproducibility, neutral red American
Jouvnol of Surgery
Functional
Anatomy
and probably toluidine blue appear to be suitable for experiments involving redocumentation of the corpus-antrum boundary after a time lapse of four to six weeks. Congo red and fluorescein were not studied in this way. Toxicity of Dyestgfs. Toxicity of these dyestuffs cannot be conclusively discussed from this investigation. When these technics were first developed, three dogs died shortly after intravenous injection of 25 cc. of unfiltered neutral red. The cause of death could not be determined. Filtered dye as used in these latter experiments, however, did not lead to apparent adverse effects. Intravenous neutral red is reported to be nontoxic at the dosages studied [7,18]. From toxicity studies of rabbits, Kolmer reported 0.1 g-m. per kilogram body weight as a safe dose of neutral red [26]. Piersol et al. recommended an intravenous dose of 125 mg. for human subjects [ZZ]. Int.ravenous toluidine blue, on the other hand. was described as mildly toxic by Dawson and Ivy [7j. They observed vomiting and inhibition of gastric secretion in five minutes. Dogs used in the current protocol did not vomit after recovery from anesthesia, and their stomachs did not stop secreting toluidine blue during an operation. One conscious dog, 22 kg. in weight, which had not been used in any prior experiments, was given 50 cc. of intravenous 1 per cent toluidine blue in 10 cc. increments five minutes apart and showed no ill effects by crude observation. Our more recent work with dogs (not included in this report) has shown that I per cent toluidine blue can cause cardiac conduction defects, a decrease in heart rate, a fall in blood pressure, increased respiratory rate, but no electroencephalographic changes. More work on toxicity of this dyestuff is required before it can be used in human subjects, but it can be successfully used in laboratory animals when given slowly at lower dosage levels. Perhaps a lower concentration of the dye will prove to be more suitable. Efforts to remove contaminants or impurities in the dye may also be helpful. Congo red and fluorescein did not cause apparent signs of distress in these animals, but studies of toxicity were not made. Brief comments on the pharmacology of dyes mentioned herein are available elsewhere [27]. Distal and Proximal Antral Borders. The distal border of the gastric antrum and duodenum was not demonstrable with neutral red or Congo red. This border can be transitorily Vol.110,August 1965
of Gastric
283
Antrum
localized with fluorescein because the first part of the duodenum fluoresces earlier than the gastric antrum. The distal border can also be observed in dogs actively excreting toluidine blue thirty to fifty minutes after high dosage with this dye. Duodenal mucosa becomes a dull blue, contrasting with a gray color of antral mucosa (no gauze overlay). At present this feature is more of academic interest than of practical value since the antrum-duodenum boundary has been found to be localized in human subjects to sites within 5 mm. on either side of the pyloric sphincter [28]. On the other hand, identification of the corpus-antrum boundary using the principle of differential dye excretion in the stomach comprises an important experimental approach to surgical operations requiring complete antrectomy (29,30], construction of antral pouches, and studies of corpus-antrum relations in pathologic states. A method of using these dyes for obtaining quantitative data comparing the relative areas of the gastric corpus and antrum can be found elsewhere [a]. Pertinent data on neutral red excretion in normal and pathologic human stomachs are also available [17.18]. SUMMARY
Technics are described for visual delineation of the corpus-antrum boundary in canine stomachs. These technics involve various dyestuffs, neutral red, toluidine blue, Congo red, and fluorescein. This study shows that differences in intravenous dye excretion in the gastric corpus as compared with the gastric antrum can be exploited to reveal anatomic limits of the antrum. Color differences obtained in this way were observed to correspond to areas of different pH values, but the color differences result basicall> from the presence or absence of dye excretion which is a major feature of this study. Gastric color boundaries from dyestuffs were marked and compared with histologic corpusantrum boundaries. Color boundaries obtained are accurate and reproducible. Furthermore, color boundaries are easily seen; use of a layer of gauze upon gastric mucosa provides a white background against which dyestuffs are vivid. Some comparative features of these dyestuffs are discussed. Possible applicability of these technics includes operations in which the gastric corpus and antrum are to be separated or in which the
Moe, Klopper, and Nyhus corpus-antrum boundary is to be marked future reference in experiments.
for
REFERENCES 1. BERNARD, C. Lerons
2.
3.
4.
5.
6.
7.
8.
9.
10.
11,
12.
13.
14.
15.
16.
17.
sur les propriCt&s physiologiques et les alterations pathologiques des liquides de l’organisme. Paris, 1859. vol. 2, p. 375. J. B. Bailliere and Sons. BRENCKMANN, E. and DELOYERS, L. I&de de la topographie des regions hlaboratrices de l’acide chlorhydrique dans l’estomac. Presse mkd., 37: 1086, 1929. LOWICKI, E. M. and LITTLEFIELD, J, B. An experimental method of precisely defining the dimensions of the gastric antrum. S. Forum, 12: 308, 1961. MOE, R. E., NYHUS, L. M., and HARKINS, H. N. The use of dye for differentiating the gastric antrum from the gastric corpus. Bull. Sot. Int. Chir., 22: 424, 1963. SMITH, G. V. and HOWE% E. L. Absence of histamine-reserpine ulcers in pyloric pouches free of acid. Surgery, 55: 262, 1964. CAPPER, W. M., LAIDLAW, C. D’A., BUCKLER, K., and RICHARDS, D. The pH fields of the gastric mucosa. Lance& 2: 1200, 1962. DAWSON, A. B. and IVY, A. C. Contributions to the physiology of gastric secretion. VII. The -limination of dyes by the gastric mucosa. Am. J. Physiol., 73: 304, 1925. MORRISON, S., REEVES, D. L., and GARDNER, R. E. The elimination of various dyes from the Pavlov pouch of dogs. Am. 1. Digest. Dis., 3: 551, 1936. VISSCHER, M. B. The secretion of dyestuffs by the gastric glands and the pancreas. Fed. Proc., 1: 246, 1942: BRADFORD. N. M. and DAVIES. R. E. The site of hydrochloric acid production’ in the stomach as determined by indicators. Biochem. J., 46: 414, 1950. KAY, A. W. Effect of large doses of histamine on gastric secretion of HCI, an augmented histamine test. Brit. M. J.. 2: 77. 1953. ROSIERE, C. E. anh GROSSMAN,M. I. An analog of histamine that stimulates gastric acid secretion without other actions of histamine. Science, 113: 651, 1951. WARD, S., GILLESPIE, I. E., PASSARO, E. P., and GROSSMAN, M. I. Comparison of Histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs. Gastroenterology, 44: 620, 1963. BABKIN, B. P. Secretory Mechanism of the Digestive Glands. New York, 1944. Paul B. Hoeber, Inc. HARVEY, B. C. H. A study of the structure of the gastric Elands of the dog and of the changes which They undergo after gasiroenterostomy and occlusion of the nvlorus. Am. J. Anat.. 6:207, 1906. HENNING, N;. Die Entzundung Des Magens; 1933. Cited by: Lerner, H. H., Asher, L., and Andrews, K. [20]. GILLMAN, T. The excretion of neutral red by the gastric mucosa; a valuable test of gastric function. South African J. M. SC., 8: 50, 1943.
18. GILLMAN, T. A critical evaluation
of the neutral red excretion and acid secretion tests of gastric function in the normal and in subjects with gastric disorders. Gastroenterology, 3 : 188, 1944. 19. GROSSMAN, M. I. and MARKS, I. h’. Secretion of pepsinogen by the pyloric glands of the dog, with some observations on the histology of the gastric mucosa. Gastroenterology, 38: 343, 1960. 20. LERNER, H. H., ASHER, L., and ANDREW% K. The excretion of neutral red by the gastric mucosa as visualized gastroscopically. Am. J.
Digest. Dis., 9: 109, 1942. 21. SAXL, P. and SCHERF, D. fiber die Ausscheidung von Farbstoffen durch den Magensaft. Wien. klin. Wchnschr., 36: 671, 1923. 22. PIERSOL, G. M., BOCKUS, H. L., and BANK, J. The practical value of neutral red as a test for gastric secretory function. Am. J. Med. SC., 170: 405, 1925. 23. EBSTEIN, W. Be&rage zur Lehre vom Bau und den physiologischer Functionen der sog. Magenschleimdriisen. Arch. mikr. Anat., 6: 515, 1870. 24. BERGER, E. H. The distribution of parietal cells in the stomach: A histotopographic study. Am. J.
Anat., 54: 87, 1934. 25. 01, M., OSHIDA, K., and SUGIMURA,S. The location of gastric ulcer. Gastroenterology, 36: 45, 1959. 26. KOLMER, J. A. Personal communication to Piersol, G. M., Bockus, H. L., and Bank, J. [22]. 27. SOLLMAN, T. A Manual of Pharmacology and its Anplications to Therapeutics and Toxicology. p. 831. Philadelphia, 1957. W. B. Saunders Co. 28. 01, M. and SAKURAI, Y. The location of duodenal ulcer. Gastroenterology, 36: 60, 1959. 29. HARKINS, H. N., JESSEPH, J. E., STEVENSON,J. K., and NYHUS, L. M. The “combined” operation for peptic ulcer. Arch. Surg., 80: 743, 1960. 30. EDWARDS, L. W., HERRINGTON, J. L., JR., STEPHENSON, S. E., JR., CARLSON, R. I., PHILLIPS, R. J., JR., CATE, W. R., JR., and SCOTT, H. W., JR. Duodenal ulcer : treatment by vagotomy and removal of the gastric antrum. Ann. Surg., 145: 738, 1957. DISCUSSION
Alto, Calif.): The authors have superbly demonstrated the use of simple technics to delineate the anatomic limits of the gastric antrum and have emphasized the application of such methods to experimental and clinical gastric surgery. Our experience in dogs has been limited to Congo red used locally and neutral red intravenously in experiments in which accurate localization of the antral-fundic junction is required. We have had less success than the authors in consistently obtaining a sharp contrast of the antralfundic boundary. Spreading of secretions when Congo red has been applied and occasional obscuring of the contrast by the color of the gastric mucosa when neutral red is injected intravenously have contributed to our difficulties. However, these problems may in part be related to the type of anesthesia employed since gastric secretory activity to histamine is largely or completely inhibited by HARRY A. OBERHELMAN, JR. (Palo
American
Journal
of Surgery
Functional Anatomy of Gastric Antrum ether in contrast to barbiturates or alpha-chloralose. In addition, the inability to obtain a sharp delineation may be attributed to the failure to utilize the gauze overlay technic used by the authors. We have similarly found an excellent correlation between the histologic antral-fundic junction and that boundary not only obtained with the aforementioned technics but ;also resulting from bleeding dogs. As a result of such studies and the reproducibility obtained in the same animal, experiments utilizing isolated or excluded innervated antra are less subject to error, a criticism that has previously existed when gross estimates onlv were made of this junction. I wish to congratulate the authors on the development. of a new technic which has led to more accurate and reproducible results than others. LLOYD M. NYHUS (closing) : Gastric physiologists have pointed out the importance of the distal portion of the stomach in the over-all stimulation of gastric acid secretion. In fact, the vagal-antral phase (that portion of acid stimulation related to the phenomenon of the vagal release of gastrin) and the local antral phase (that portion of acid stimulation related to local effects, that is, distention and secretogogues) are considered by some physiologists to encompass a major portion of the spectrum of acid stimulation. What are the clinical implications of this study? When the siandard operative procedure for the
Vol. 110, August
1965
285
treatment of gastric or duodenal ulcer was three quarters or two thirds distal gastrectomy, these technics were of little clinical value. However, since the advent of the combined operation, selective vagotomy plus antrectomy, the need for a technic such as the one described becomes apparent. I want to warn you, however, that the technics of demarcation remain crude; the intravenous dye methods must await further study prior to direct clinical application. There is no contraindication to the use of topical application of Congo red or blue litmus as recently suggested hy Bergstrom and Broome. Moe and Klopper have used the following technic without gastrotomy in eight patients. A tube is passed into the stomach. Congo red is sprayed on the gastric mucosa through the nasogastric tube. When a light source behind the stomach transluminates it, the antral corpus border is clearly seen. They have shown this border to be accurate by microscopic study after gastric resection. The technics described by Dr. Moe would allow for surgeons to resect the entire antrum accurately, thus leaving a significant gastric remnant as a reservoir. Finally, it is our belief that the technics described will help to answer certain other questions brought up by Dr. Oberhelman, both in the experimental and clinical areas.
of the Functional
of the Canine I. Operative
Gastric
Technics Requiring
Antrum* Gastrotomy
ROGER E. MOE, M.D., P. J. KI,OPPER, M.D., AND LLOYD M. Nytrus,
From the Laboratory for Experimental Surgery, Surgical Clinic, Wilhelmina Gasthuis, University of Amsterdam, and the Department of Surgery, University of Washington, Seattle, Washington. This study was aided in part by funds accruinP from National Institutes of Health Grant $A M-04010, &d Initiative 171, State if Washington, and Research Training Grant GM-325.
I
Anatomy
M.D., Seattle,
Washingtolz
the antrum brown, and the intervening boundary formed a sharp continuous line [Z]. Later, Lowicki and Littlefield reported topical application of Congo red to gastric mucosa of human stomachs, staining the corpus black or dark blue and the antrum red [3]. Their experiment represents the first study devoted to in viva demonstration of the gastric antrum for facilitation of complete antrectomy during clinical surgery. Confirmatory findings were published by Moe, Nyhus, and Harkins [P] and by Smith and Howes [5] using dogs. Smith and Howes employed Congo red as an aid in construction of experimental antral pouches. Other indicators (topical bromphenol blue, intravenous fluorescein, litmus paper, and universal indicator paper) have been used by Capper et al. to detect the gastric “acid-alkaline junction” in patients with peptic ulcers [6]. Furthermore, they discovered that the gastric acid-alkaline junction can be detected through a gastrotomy by directly measuring mucosal pH with an electronic meter. In the present investigation, dyestuffs have been utilized to show anatomic limits of the gastric antrum in dogs at surgery. The basic method of this technic is new. Rather than depending upon different colors of dyestuffs at different pH ranges, the present method depends upon differences in elimination of intravenous dyestuffs via the gastric corpus and antrum. Various workers have studied gastric excretion of many dyestuffs after intravenous injec-
T is common knowledge that anatomic limits
of the gastric antrum in dogs and human subjects cannot be visually discerned consistently and accurately during a surgical operation unless the gastric mucosa is modified in some way. One way to modify gastric mucosa in V&IOinvolves the use of dyestuffs providing contrasting colors in the gastric corpus and antrum which are visualized by exposing gastric mucosa through a gastrotomy. In most publications up to now, the basic method has featured a dyestuff which exhibits one color in a given pH range and another color in a different pH range. Hence, inside the stomach the color of such a dyestuff over an acid-secreting area (corpus) differs from the color over a non-acid-secreting area (antrum). Claude Bernard first published this observation after he demonstrated which part of the stomach in a rabbit produces acid. After intravenous injection of appropriate reagents, Prussian blue appeared in part of the stomach, signifying an acid area, while the remainder of the stomach did not become blue [I]. Using dogs in more detailed studies, Brenckmann and Deloyers stained gastric mucosa topically with Prussian blue reagent. The gastric corpus became blue,
* Presented at the annual meeting of the Pacific Coast Surgical Association, Vancouver, British Columbia, February 22-25, 1965. Vol.110,August 1965
277
Moe,
278
Klopper,
and Nyhus
6.0 P~locdrpme
50-
T
1% 6mqm
loluidine
blue IOcc
I.V. \
SQ t + Copious
secretion of dye t Entire MAAG Good corpus-antrum border Grey-blue I” I min Free dye in corpus, not in ontrum Per~gtols~s-------Peristalsis continuous---------------
1 I1 I I1 I1 I1 1, I I I I,, 8 16 24 32 40 48 56 64 72
I I I,,,‘,,,,,,,,
00
88
96
104
II2
120
128 136
Minutes
Fro 1. Record of corpus and antrum pH during toluidine blue excretion in one dog. tion [7-IO]. Some dyestuffs readily
appeared
in
secretions from corpus pouches but appeared minimally or not at all in secretions from antral pouches in dogs 171.Two of those dyestuffs were employed here-toluidine blue and neutral red. Since these dyestuffs are excreted differently by the corpus and antrum, it should be possible to delineate these gastric areas visually when mucosa is exposed. However, this delineation is unreliable since colors from toluidine blue and neutral red are obscured by the color of gastric mucosa. This problem is corrected when a layer of moist white gauze is applied to gastricmucosa before injecting dyestuffs. As a result, toluidine blue and neutral red are easily visualized in gauze over corpus mucosa and, likewise, the area where these dyes do not appear (antrum) is also easily apparent; a continuous boundary between the corpus and the antrum is accurately delineated. This work also includes further investigations of the use of fluorescein [S] and Congo red for revealing the gastric antrum in dogs [3]. MATERIALS
AND
METHODS
Solutions of dyestuffs were made as follows: toluidine blue or neutral red was dissolved in 5 per cent dextrose and water to make a 1 per cent solution which was filtered to prevent microembolism from undissolved dye particles; Congo red was dissolved in water with moderate warming to make a 6 to 7 per cent solution; aqueous fluorescein was studied in two dilutions, 5 per cent and 0.5 per cent. One half hour before gastrotomy, gastric secretion was stimulated with histamine base, 0.04 mg./kg.
body weight [11] injected subcutaneously or 0.015 mg./min. intravenously. The intravenous route was more reliable. Some dogs were given pyrilamine maleate, 25 mg. intramuscularly or intravenously, prior to histamine injection to minimize systemic effects of histamine. Another way to avoid any systemic effects of histamine is to inject 100 mg. of Histalog*@ one hour before gastrotomy instead of using histamine [12,1?]. Thirty-four dogs were studied. Each had received no food for twenty-four hours but had free access to water. Anesthesia was achieved by slow intravenous injection of a combination of alpha-chloralose (50 mg./kg. body weight) and urethan (500 mg./ kg.) mixed in propylene glycol. This anesthesia does not inhibit gastric secretion [14]. Intravenous thialbarbitone sodium can also be used satisfactorily. Atropine and similarly acting drugs were avoided. Through a midline abdominal incision a longitudinal anterior gastrotomy was made with a scalpel or scissors between crushing clamps. This gastrotomy was about 10 cm. long, extending proximally from a point 2 cm. above the pylorus. After meticulous hemostasis the gastrotomy edges were held apart by retraction sutures extending from the stomach to the abdominal wall. Gastric mucosa was then lavaged with warm saline solution and gently sponged to remove food debris. A plug of gauze was placed near the cardia to block fluid which sometimes emerged from the esophagus. A piece of white gauze was spread apart to obtain a single (very thin) layer. This gauze was moistened and laid upon the gastric mucosa, conforming closely to mucosal contours. The mucosa was covered only where a corpus-an&urn boundary might be anticipated. * Manufactured as betazole by Eli Lilly, Indianapolis, Indiana. American
Journal of Surgery
Functional
FIG. 2. Corpus-antrum I’urplc-stained gauze
Vol.
I If),
ArLgusl
1965
Anatomy
of Gastric
Antrum
boundary after intravenous injection of 10 cc. of 1 per cent is over the corpus, while unstained gauze is over the antrum.
neutral
red.
Moe,
Klopper, and Nyhus
RESULTS
Tolw’dine Blue. After intravenous injection of 10 cc. over a period of two minutes, gauze over the corpus area was stained pale blue in approximately five minutes while the antrum area remained unstained. Blue color over the corpus developed more intensely over a fifteen to thirty minute period. Further increments of dye (up to a total of 50 cc.) were used in several instances for greater intensity of color although this was not usually necessary when active gastric secretion existed. With two dogs the protocol was modified slightly to study factors which might relate especially to the excretion of toluidine blue. One dog was not given histamine but still demonstrated a clear antrum-corpus color boundary after 10 cc. of intravenous toluidine blue. Similarly, a second dog was not given histamine but instead received 6 mg. of pilocarpine subcutaneously. After 10 cc. of intravenous toluidine blue, this dog excreted dye copiously in the corpus while the corpus mucosa and antral mucosa maintained a pH between 7.0 and 7.1 by direct measurement with a glass electrode. (Fig. 1.) The protocol was otherwise unchanged. hleutral Red. Intravenous injection of neutral red was performed in the same manner described for toluidine blue. Gauze over the corpus became violet in about three minutes, while antrum gauze remained unstained. (Fig. 2.) Rarely a faint yellow hue appeared on antrum gauze adjacent to the corpus-antrum boundarv. This dye emerges more profusely than toluidine blue. No dog stomachs failed to excrete neutral red. Some dogs excreted this dye in apparently small quantities, but a sharp corpus-antrum boundary was still discernible. One dog failed to reveal a definite corpusantrum boundary. In that stomach, dye was present but faded out gradually near the antrum. That dog had been “wormed” twelve hours before operation. The stomach contained dead worms and feculent material, and the mucosa was dark red, friable, and bled easily. Congo Red. Best results occurred when this dye solution was applied topically as a fine spray from a nebulizer rather than by lavaging or sponging the dye onto the mucosa. When applied directly to the mucosa, Congo red stained the corpus black and the antrum red. When a single thickness of gauze was placed
upon the mucosa before spraying dye, this dye stained the corpus blue and the antrum red. When a more dilute solution of Congo red was used directly upon the mucosa [j], the corpusantrum boundary was not as precisely discernible because the colors were less vivid than when a saturated solution was used. However, when that same diluted dye solution was sprayed onto a thin gauze overlay inside the stomach, the corpus-antrum boundary was more precisely and clearly apparent. Fluorescein. After the intravenous injection of 2 cc. of 5 per cent solution, mucosa of the entire stomach and small intestine fluoresced with a green color under an ultraviolet light source (Philips type no. 57236 E/70 HPW 125 W) in a dark room. Some fluorescein appeared also in free fluid secreted by the corpus and in secretions from the biliary tree. Fluorescence of gastric mucosa was not destroyed when 0.1 N HCl was dropped directly onto the mucosa. In contrast to the results just noted, the injection of 0.5 per cent fluorescein in 2 cc. increments presented a different mucosal appearance. Within one minute, mucosa of the first part of the duodenum fluoresced as far proximally as the boundary between the duodenum and antral mucosa. (Care was taken to prevent contamination from biliary fluid by means of a gauze plug in the second part of the duodenum.) Next, antral mucosa fluoresced while corpus mucosa did not. The corpusantrum boundary was not quite as sharp or abrupt as boundaries from the other three dyestuff s mentioned herein. Continued injection of fluorescein later was followed by fluorescence of the corpus and fundus, obscuring any delineation of the antrum from the corpus. These results were in accordance with those in our initial study of ten rabbits. Acrid&e Orange. This fluorescent dye was also injected intravenously, but a corpusantrum boundary was not revealed in dogs or in rabbits. Controls. Four or five Nylon@ or silk marker sutures were placed through the stomach wall where a corpus-antrum boundary was detected in each specimen. Histology: The marker sutures consistently appeared within 1 cm. on either side of the histologic boundary, a site where the number of corpus parietal cells abruptly falls. The majority of the markers were 3 mm. or less from the Am&can
Jouvnul of Surnery
Functional
Anatomy of Gastric Antrum
2x1
852
30
SK.
I
CORPUS’
PH 2
NTRUM
4
6
FIG. 3. Continuous
tracing of pH values as a glass electrode is moved 1 centimeter back and forth at the location of a corpus-antrum boundary marker suture.
histologicIboundary; one marker was as far as 1 cm. from the histologic boundary. Comparison of boundary sites: All three dyestuffs, toluidine blue, neutral red, and Congo red, yielded the same boundary site when used during the same operation on each of three different dogs. PH ~vttlunlion: Color boundaries obtained by the presence or absence of dye excretion in the corpus and antrum, respectively, corresponded with pH-dependent boundaries detected by Congo red, Universal indicator paper, or direct pH measurement with continuous recording. (Fig. 3.) Stability: Color boundaries visualized through a gauze overlay have been observed in several animals for one hour with no shift in color on the gauze overlays. When dye excretion was unusually heavy in some animals, a thicker layer of gauze minimized flow of secretion along the mucosa. This is not a problem ordinarily, however, because in such cases the corpusantrum border can be marked before movement of secretion ensues. Reproducibility: A corpus-antrum boundary was demonstrated in three dogs. Each boundary was marked with vitallium sutures. One month later, each dog was reoperated upon. Neutral red was again used in two dogs and toluidine blue in the third dog. In all three dogs the second operation revealed a corpus-antrum boundary remarkably close to the original site, differing by only 0 to 4 mm. in biopsies from each animal. COMMENTS
The principle of differential excretion of dyestuffs from the gastric corpus and antrum can be exploited for reliable delineation of the funcVol.
110,
August
1965
tional anatomy of the gastric antrum. Results described herein indicate that toluidine blue or neutral red can be used for this purpose in experimental animals. Evidence that these two dyes are, in fact, excreted by the corpus but not by the antrum rests upon failure to detect dye in gauze over the gastric antrum under the conditions specified. Also, Dawson and Ivy found that neutral red is readily excreted by Pavlov pouches but not by pyloric pouches unless the pyloric pouches were “activated” by continuous application of gastric juice [7]. They did not study toluidine blue in pyloric pouches. Mucosal PH. Do pH differences in gastric mucosa influence color boundaries obtained with differentially excreted dyestuffs in the stomach? This question is part of the basis for the choice of dyestuffs used here. One dyestuff, toluidine blue, is excreted by gastric mucosa but does not change color as a pH indicator. The second dyestuff, neutral red, is excreted by gastric mucosa and can also change color as a pH indicator. The third dyestuff, Congo red, is not excreted by gastric mucosa after intravenous injection but serves only as a pH indicator [7,8]. Since the corpus-antrum boundary is the same from all three dyestuffs, the conclusion is reached that color boundaries obtained from differentially excreted dyestuffs do not depend upon gastric pH differences per se although pH differences do, indeed, exist at the corpus-antrum boundary. Color boundaries from differential dye excretion depend more particularly, of course, upon active secretion of corpus fluid which might or might not be acidic. From studies including microscopy some workers (but not all) report that neutral red is excreted by parietal cells
Moe, Klopper, and Nyhus [10,15], the cells which also produce acid. Henning thought neutral red was excreted by pepsin cells [16]. Other research methods indicate that acid secretion and neutral red excretion are not necessarily parallel [17,18]. Evidence from the present work suggests that toluidine blue excretion may not parallel acid secretion either. By direct mucosal pH measurements, the corpus of one dog was not secreting acid but still was vigorously excreting toluidine blue. Gastric cells which excrete toluidine blue have not been identified. If pepsin-secreting cells are involved in dye excretion, this would be of interest since Grossman reported that the presence or absence of pepsin-secreting cells may provide the best histologic criterion for delineating the gastric corpus and antrum [19]. In the case of intravenous neutral red, it was considered that a corpus-antrum color boundary might be obtained partly by differential excretion and partly by color alteration with acidity or alkalinity of the corpus and antrum, respectively. Apparently the corpus-antrum color boundary appears only with differential dye excretion in the vast majority of trials with neutral red. It has been mentioned that intravenous neutral red did not appear in gauze over the antrum. Parenthetically, gastroscopy of human stomachs did not reveal any neutral red excretion from the antrum although the dye could be seen in other parts of the stomach [20]. Is it possible that the dyestuff used in the experiments reported herein had been excreted from the antrum and that the color at alkaline pH’s was too pale to be observed? This is doubtful since the surface fluid over the antrum was often found by direct measurement to be insufficiently alkaline to develop any yellow color in neutral red dye. Also, topical neutral red applied to the antrum usually retains its red or violet (acid) color instead of becoming yellow (alkaline). Furthermore, if excreted neutral red is deliberately allowed to flow from the corpus into the antrum, red dye from the corpus remains red (acidic) in the antrum. (Note that excreted neutral red is still capable of changing color appropriately if an acid or base is mixed into a solution of the excreted dye.) A similar observation was made by earlier workers such as Sax1and Scherf [21] and Piersol, Bockus, and Bank [22] who stated that neutral red injections stained the entire gastric mucosa red, and this red color was particularly intense in the prepyloric region [21,22]. They errone-
ously concluded that the entire gastric mucosa was involved in the excretion of neutral red. The dye was not observed when it first appeared over gastric mucosa; distribution of the dye was noted after the animals were sacrificed, thirty to sixty minutes after neutral red excretion had begun. The neutral red was no longer limited to its site of origin, and over the antrum it showed as a red, not a yellow, color. Thus, if the neutral red had been selectively excreted by the antral mucosa in our experiments, the gauze overlay should have stained red. As indicated previously, this did not occur. As a final check, we applied 0.1 N HCI from an eye dropper directly to the corpus-antrum color boundary so that the acid spread both into the corpus and antrum; a red stain did not appear in the gauze over the antrum, while the red stain over the corpus is intensified. Indeed, this maneuver did not disturb the existing corpusantrum color boundary ! Therefore, under the conditions of our experiments, neutral red was not excreted by the antral mucosa. Comparison of Speed and Accuracy. Certain comparative comments can be made about dyestuffs examined. Congo red delineates the corpus-antrum boundary most rapidly. Neutral red involves a short waiting period for the dye to emerge from the mucosa. Toluidine blue emerges more slowly than neutral red. Dilute fluorescein reveals the antral area as fast or faster than neutral red, but as Capper et al. [6] have indicated, there is a disadvantage in the darkened operating room required during use of an ultraviolet light to detect fluorescence. Toluidine blue, neutral red, and Congo red have a similar accuracy in revealing the corpusantrum border. Variations obtained between the sites of color boundaries and the sites of histologic corpus-antrum boundaries seem to be compatible with the size of a narrow histologic border zone between the corpus and antrum in dogs [15,23] and in human subjects [24,25]. Toluidine blue and neutral red yield boundaries even when acid is not removed from antral fluid by sponging or by other means. Congo red, on the other hand, leads to erroneous boundaries if antral acid (below pH of about 5) exists. Fluorescein did not yield a boundary as sharp or abrupt as that obtained with the other three dyes. Only four dogs were studied with fluorescein, but the results were the same as those in our prior studies in ten rabbits. With respect to reproducibility, neutral red American
Jouvnol of Surgery
Functional
Anatomy
and probably toluidine blue appear to be suitable for experiments involving redocumentation of the corpus-antrum boundary after a time lapse of four to six weeks. Congo red and fluorescein were not studied in this way. Toxicity of Dyestgfs. Toxicity of these dyestuffs cannot be conclusively discussed from this investigation. When these technics were first developed, three dogs died shortly after intravenous injection of 25 cc. of unfiltered neutral red. The cause of death could not be determined. Filtered dye as used in these latter experiments, however, did not lead to apparent adverse effects. Intravenous neutral red is reported to be nontoxic at the dosages studied [7,18]. From toxicity studies of rabbits, Kolmer reported 0.1 g-m. per kilogram body weight as a safe dose of neutral red [26]. Piersol et al. recommended an intravenous dose of 125 mg. for human subjects [ZZ]. Int.ravenous toluidine blue, on the other hand. was described as mildly toxic by Dawson and Ivy [7j. They observed vomiting and inhibition of gastric secretion in five minutes. Dogs used in the current protocol did not vomit after recovery from anesthesia, and their stomachs did not stop secreting toluidine blue during an operation. One conscious dog, 22 kg. in weight, which had not been used in any prior experiments, was given 50 cc. of intravenous 1 per cent toluidine blue in 10 cc. increments five minutes apart and showed no ill effects by crude observation. Our more recent work with dogs (not included in this report) has shown that I per cent toluidine blue can cause cardiac conduction defects, a decrease in heart rate, a fall in blood pressure, increased respiratory rate, but no electroencephalographic changes. More work on toxicity of this dyestuff is required before it can be used in human subjects, but it can be successfully used in laboratory animals when given slowly at lower dosage levels. Perhaps a lower concentration of the dye will prove to be more suitable. Efforts to remove contaminants or impurities in the dye may also be helpful. Congo red and fluorescein did not cause apparent signs of distress in these animals, but studies of toxicity were not made. Brief comments on the pharmacology of dyes mentioned herein are available elsewhere [27]. Distal and Proximal Antral Borders. The distal border of the gastric antrum and duodenum was not demonstrable with neutral red or Congo red. This border can be transitorily Vol.110,August 1965
of Gastric
283
Antrum
localized with fluorescein because the first part of the duodenum fluoresces earlier than the gastric antrum. The distal border can also be observed in dogs actively excreting toluidine blue thirty to fifty minutes after high dosage with this dye. Duodenal mucosa becomes a dull blue, contrasting with a gray color of antral mucosa (no gauze overlay). At present this feature is more of academic interest than of practical value since the antrum-duodenum boundary has been found to be localized in human subjects to sites within 5 mm. on either side of the pyloric sphincter [28]. On the other hand, identification of the corpus-antrum boundary using the principle of differential dye excretion in the stomach comprises an important experimental approach to surgical operations requiring complete antrectomy (29,30], construction of antral pouches, and studies of corpus-antrum relations in pathologic states. A method of using these dyes for obtaining quantitative data comparing the relative areas of the gastric corpus and antrum can be found elsewhere [a]. Pertinent data on neutral red excretion in normal and pathologic human stomachs are also available [17.18]. SUMMARY
Technics are described for visual delineation of the corpus-antrum boundary in canine stomachs. These technics involve various dyestuffs, neutral red, toluidine blue, Congo red, and fluorescein. This study shows that differences in intravenous dye excretion in the gastric corpus as compared with the gastric antrum can be exploited to reveal anatomic limits of the antrum. Color differences obtained in this way were observed to correspond to areas of different pH values, but the color differences result basicall> from the presence or absence of dye excretion which is a major feature of this study. Gastric color boundaries from dyestuffs were marked and compared with histologic corpusantrum boundaries. Color boundaries obtained are accurate and reproducible. Furthermore, color boundaries are easily seen; use of a layer of gauze upon gastric mucosa provides a white background against which dyestuffs are vivid. Some comparative features of these dyestuffs are discussed. Possible applicability of these technics includes operations in which the gastric corpus and antrum are to be separated or in which the
Moe, Klopper, and Nyhus corpus-antrum boundary is to be marked future reference in experiments.
for
REFERENCES 1. BERNARD, C. Lerons
2.
3.
4.
5.
6.
7.
8.
9.
10.
11,
12.
13.
14.
15.
16.
17.
sur les propriCt&s physiologiques et les alterations pathologiques des liquides de l’organisme. Paris, 1859. vol. 2, p. 375. J. B. Bailliere and Sons. BRENCKMANN, E. and DELOYERS, L. I&de de la topographie des regions hlaboratrices de l’acide chlorhydrique dans l’estomac. Presse mkd., 37: 1086, 1929. LOWICKI, E. M. and LITTLEFIELD, J, B. An experimental method of precisely defining the dimensions of the gastric antrum. S. Forum, 12: 308, 1961. MOE, R. E., NYHUS, L. M., and HARKINS, H. N. The use of dye for differentiating the gastric antrum from the gastric corpus. Bull. Sot. Int. Chir., 22: 424, 1963. SMITH, G. V. and HOWE% E. L. Absence of histamine-reserpine ulcers in pyloric pouches free of acid. Surgery, 55: 262, 1964. CAPPER, W. M., LAIDLAW, C. D’A., BUCKLER, K., and RICHARDS, D. The pH fields of the gastric mucosa. Lance& 2: 1200, 1962. DAWSON, A. B. and IVY, A. C. Contributions to the physiology of gastric secretion. VII. The -limination of dyes by the gastric mucosa. Am. J. Physiol., 73: 304, 1925. MORRISON, S., REEVES, D. L., and GARDNER, R. E. The elimination of various dyes from the Pavlov pouch of dogs. Am. 1. Digest. Dis., 3: 551, 1936. VISSCHER, M. B. The secretion of dyestuffs by the gastric glands and the pancreas. Fed. Proc., 1: 246, 1942: BRADFORD. N. M. and DAVIES. R. E. The site of hydrochloric acid production’ in the stomach as determined by indicators. Biochem. J., 46: 414, 1950. KAY, A. W. Effect of large doses of histamine on gastric secretion of HCI, an augmented histamine test. Brit. M. J.. 2: 77. 1953. ROSIERE, C. E. anh GROSSMAN,M. I. An analog of histamine that stimulates gastric acid secretion without other actions of histamine. Science, 113: 651, 1951. WARD, S., GILLESPIE, I. E., PASSARO, E. P., and GROSSMAN, M. I. Comparison of Histalog and histamine as stimulants for maximal gastric secretion in human subjects and in dogs. Gastroenterology, 44: 620, 1963. BABKIN, B. P. Secretory Mechanism of the Digestive Glands. New York, 1944. Paul B. Hoeber, Inc. HARVEY, B. C. H. A study of the structure of the gastric Elands of the dog and of the changes which They undergo after gasiroenterostomy and occlusion of the nvlorus. Am. J. Anat.. 6:207, 1906. HENNING, N;. Die Entzundung Des Magens; 1933. Cited by: Lerner, H. H., Asher, L., and Andrews, K. [20]. GILLMAN, T. The excretion of neutral red by the gastric mucosa; a valuable test of gastric function. South African J. M. SC., 8: 50, 1943.
18. GILLMAN, T. A critical evaluation
of the neutral red excretion and acid secretion tests of gastric function in the normal and in subjects with gastric disorders. Gastroenterology, 3 : 188, 1944. 19. GROSSMAN, M. I. and MARKS, I. h’. Secretion of pepsinogen by the pyloric glands of the dog, with some observations on the histology of the gastric mucosa. Gastroenterology, 38: 343, 1960. 20. LERNER, H. H., ASHER, L., and ANDREW% K. The excretion of neutral red by the gastric mucosa as visualized gastroscopically. Am. J.
Digest. Dis., 9: 109, 1942. 21. SAXL, P. and SCHERF, D. fiber die Ausscheidung von Farbstoffen durch den Magensaft. Wien. klin. Wchnschr., 36: 671, 1923. 22. PIERSOL, G. M., BOCKUS, H. L., and BANK, J. The practical value of neutral red as a test for gastric secretory function. Am. J. Med. SC., 170: 405, 1925. 23. EBSTEIN, W. Be&rage zur Lehre vom Bau und den physiologischer Functionen der sog. Magenschleimdriisen. Arch. mikr. Anat., 6: 515, 1870. 24. BERGER, E. H. The distribution of parietal cells in the stomach: A histotopographic study. Am. J.
Anat., 54: 87, 1934. 25. 01, M., OSHIDA, K., and SUGIMURA,S. The location of gastric ulcer. Gastroenterology, 36: 45, 1959. 26. KOLMER, J. A. Personal communication to Piersol, G. M., Bockus, H. L., and Bank, J. [22]. 27. SOLLMAN, T. A Manual of Pharmacology and its Anplications to Therapeutics and Toxicology. p. 831. Philadelphia, 1957. W. B. Saunders Co. 28. 01, M. and SAKURAI, Y. The location of duodenal ulcer. Gastroenterology, 36: 60, 1959. 29. HARKINS, H. N., JESSEPH, J. E., STEVENSON,J. K., and NYHUS, L. M. The “combined” operation for peptic ulcer. Arch. Surg., 80: 743, 1960. 30. EDWARDS, L. W., HERRINGTON, J. L., JR., STEPHENSON, S. E., JR., CARLSON, R. I., PHILLIPS, R. J., JR., CATE, W. R., JR., and SCOTT, H. W., JR. Duodenal ulcer : treatment by vagotomy and removal of the gastric antrum. Ann. Surg., 145: 738, 1957. DISCUSSION
Alto, Calif.): The authors have superbly demonstrated the use of simple technics to delineate the anatomic limits of the gastric antrum and have emphasized the application of such methods to experimental and clinical gastric surgery. Our experience in dogs has been limited to Congo red used locally and neutral red intravenously in experiments in which accurate localization of the antral-fundic junction is required. We have had less success than the authors in consistently obtaining a sharp contrast of the antralfundic boundary. Spreading of secretions when Congo red has been applied and occasional obscuring of the contrast by the color of the gastric mucosa when neutral red is injected intravenously have contributed to our difficulties. However, these problems may in part be related to the type of anesthesia employed since gastric secretory activity to histamine is largely or completely inhibited by HARRY A. OBERHELMAN, JR. (Palo
American
Journal
of Surgery
Functional Anatomy of Gastric Antrum ether in contrast to barbiturates or alpha-chloralose. In addition, the inability to obtain a sharp delineation may be attributed to the failure to utilize the gauze overlay technic used by the authors. We have similarly found an excellent correlation between the histologic antral-fundic junction and that boundary not only obtained with the aforementioned technics but ;also resulting from bleeding dogs. As a result of such studies and the reproducibility obtained in the same animal, experiments utilizing isolated or excluded innervated antra are less subject to error, a criticism that has previously existed when gross estimates onlv were made of this junction. I wish to congratulate the authors on the development. of a new technic which has led to more accurate and reproducible results than others. LLOYD M. NYHUS (closing) : Gastric physiologists have pointed out the importance of the distal portion of the stomach in the over-all stimulation of gastric acid secretion. In fact, the vagal-antral phase (that portion of acid stimulation related to the phenomenon of the vagal release of gastrin) and the local antral phase (that portion of acid stimulation related to local effects, that is, distention and secretogogues) are considered by some physiologists to encompass a major portion of the spectrum of acid stimulation. What are the clinical implications of this study? When the siandard operative procedure for the
Vol. 110, August
1965
285
treatment of gastric or duodenal ulcer was three quarters or two thirds distal gastrectomy, these technics were of little clinical value. However, since the advent of the combined operation, selective vagotomy plus antrectomy, the need for a technic such as the one described becomes apparent. I want to warn you, however, that the technics of demarcation remain crude; the intravenous dye methods must await further study prior to direct clinical application. There is no contraindication to the use of topical application of Congo red or blue litmus as recently suggested hy Bergstrom and Broome. Moe and Klopper have used the following technic without gastrotomy in eight patients. A tube is passed into the stomach. Congo red is sprayed on the gastric mucosa through the nasogastric tube. When a light source behind the stomach transluminates it, the antral corpus border is clearly seen. They have shown this border to be accurate by microscopic study after gastric resection. The technics described by Dr. Moe would allow for surgeons to resect the entire antrum accurately, thus leaving a significant gastric remnant as a reservoir. Finally, it is our belief that the technics described will help to answer certain other questions brought up by Dr. Oberhelman, both in the experimental and clinical areas.