Okajimas Folia Anat. Jpn., 73(1): 37-54, May, 1996

The Distribution and Architecture of Lymphatic Vessels in the Rat as Revealed by an Enzyme-Histochemical Method

By

Rui-Cheng JI, Seiji KATO, Masahiro MIURA and Takeshi USUI

Departments of Anatomy and Human Biology, Oita Medical University, 1-1, Idaigaoka, Hasama-machi, Oita-gun, Oita, 879-55, Japan

-Received for Publication, February 16,1996-

Key Words: Lymphatic vessels, Stomach, 5′-nucleotidase, Alkaline phosphatase, Histochemistry

Summary: Enzyme-histochemical methods of staining for 5′-nucleotidase (5′-Nase) and alkaline phosphatase (ALPase) were successfully applied to study the distribution and architecture of lymphatic vessels and their relationships to blood vessels in the rat stomach. Extensively lymphatic capillary networks were found in the gastric wall, but there were significant differences in their extent, pattern, distribution and structure in the four different zones: -stomach

(E-S), forestomach-corpus (F-C), corpus-antrum (C-A) and antrum- (A-D). 5′-Nase-ALPase double staining revealed that the 5′-Nase-positive lymphatic vessels run in close proximity to ALPase-positive arteries and veins. The fine blood capillary network was located superficially to the lymphatic network within the same layer in the gastric wall. The abundant lymphatic network located in the deep and the lamina muscularis mucosa was always closely associated with the base of the lowest , and yet no interglandular lymphatic capillaries were encountered in the corpus or antrum. In contrast, fewer lymphatic capillaries were present in the lamina propria beneath the squamous of the forestomach. The distribution of the well-developed lymphatic networks with valve-like structures in the and subserosa exhibited typical features, i.e., the distribution was annular in the submucosa and fan-shaped in the subserosa in the antrum near the duodenum. Open junctions of lymphatic endothelial cells were seen in the deep lamina propria and submucosa. Collecting lymphatics containing valves were mainly located deep in the submucosa and subserosa. The deep lamina propria and submucosa may play a key role in lymph formation and interstitial fluid homeostasis as well as in pathological processes in certain diseases. The present findings obtained by interstitially injecting ultra-fine carbon particle suspensions or Evans blue showed that a great deal of lymph drained into the lymphatics accompanying the left gastric artery. The existence of a forestomach may explain the complicated organization and constitution of lymphatic networks in the rat stomach.

The rat stomach, whose most distinctive feature is food depot and enables prolonged salivary amylase an extensive forestomach, is fundamentally different activity (Kunstyr et al. , 1976).The corpus and antrum from the human stomach. The forestomach and its of the stomach are sites of further digestion, and the limiting ridge are lined with stratified squamous limiting ridge occupies a physiological borderline epithelium similar to that of the esophagus. The which is subjected to continuous mechanical and squamous epithelium undergoes an abrupt transition chemical irritation (Isomaki, 1973). Thus, it would in the esophagus-stomach (E-S) and forestomach- be of interest and importance to identify the micro- corpus (F-C) zones, and the number of the glandular vascular system, especially the lymphatic network, cells observed in the corpus-antrum (C-A) zone de- in the transitional zones of the rat stomach. Studies creases markedly as one proceeds from the corpus to on the lymphatic network of the stomach have been the antrum even though all of the epithelial cell conducted in some mammalian species, mainly based types ultimately arise from the same population of on such methods as parenchymal or vascular injection undifferentiated cells (Wattel and Geuze, 1977). A (Ohtani and Murakami, 1987), hydrogen peroxide study on aluminum hydroxide uptake in the rat treatment (Noguchi et al., 1990), and fluorescent stomach showed different degrees of permeability of stream investigation (Nagata and Guth, 1984). Many the mucosa in the two zones (More et al. , 1992). of these studies have focused on the lymphatic dis- Functionally, the forestomach serves as an initial tribution in the deen lamina nronria of the stomach

This work was supported in part by a Grant-in-aid for Scientific Research (No. 05670019) from the Ministry of Education, Science and Culture of Japan. Send off print requests to: Prof. S. Kato, Department of Human Biology, Oita Medical University, Oita 879-55 , Japan

37 38 R.C. Ji et al:

as a means of elucidating special pathological pro- for only 2-5% of the whole E-S zone, whereas in cesses in gastric cancer (Lehnert et al., 1985;Listrom the latter two zones (E-C and E-A), the epithelium and Fenoglio-Preiser, 1987; Sakuma et al. , 1985). of the esophagus undergoes an abrupt transition to Little information is available on lymphatic vessels the columnarepithelium lining the corpusand antrum. in the submucosa, the muscularis externa and the We concentrated our study on the E-C and E-A subserosa. To the best of our knowledge, there have zones not only because they have structurally charac- been no studies on the distribution and architecture teristic features, but because they have extensive of lymphatic vessels in the four zones, especially in transitional zones (60-70% and 25-35% , respec- the transitional zones (E-C, E-A, F-C and A-D) of tively) as well. the stomach. Recent reports show that 5'-nucleotidase The specimens were immersed in 0.1 M cacodylate (5 '-Nase), an important enzymein nucleotide metabo- buffer (7% sucrose) supplemented with 0.1% Triton lism, is being widely employed not only to differ- X-100(Kodak) and the above-mentioned fixative for entiate between blood capillaries and lymphatic 1 hour and 2 hours, respectively, at 4°C. To expose capillaries (Werner et al. , 1987; Kato and Miyauchi, the submucosa, the serosa and muscularis externa 1989), but to differentiate between lymphatic endo- were peeled off in one sheet from ten whole-mount thelia from different sites (De Pierre and Karnovsky, preparations under a dissectingmicroscope. The tissue 1973; Kato and Miyauchi, 1989; Kato, 1990a,b). removed was spread out with the serosal surface In this context, the enzyme-histochemical method facing up, and the specimens were refixed in the was applied to research on lymphatic networks. In same fixative for 30-60 minutes at 4°C. view of uncertainties or lack of information on the After fixation, the tissue blocks from the four extent, pattern and distribution of lymphatic capil- zones of some specimens were excised, embedded in lanes in the stomach, we chose the four zones of the cold glycol methacrylate resin (JB-4, Polysciences, rat stomach because of its specific structural and Inc., Pa, USA) at 4°C for over 10 hours or frozen in functional importance as the subject of this study OCT compound (Miles, USA) at a temperature of and used 5'-Nase and ALPase staining methods to —80°C. Plastic sections 5-7 tim thick were cut with a identifythe lymphaticcapillaries and blood capillaries. steel blade on a microtome for JB-4 blocks or with a cryostat (Bright 5030 microtome, Bright Inst. Co. Ltd. , Huntingdon, England) at a cabinet temperature Materials and Methods of —18°Cfor OCT blocks. To obtain better histo- chemical staining results, 50-um-thick fixed sections Experimental animals and tissue preparations were prepared for transmission electron microscopy Forty adult Wistar rats of both sexes, weighing in a microslicer(Dosaka EM Co. Ltd., Tokyo, Japan). 250 to 300gm, were examined. All experiments were carried out according to the Guidelines for Animal Enzyme-histochemicalstaining Experimentation, Oita Medical University. The rats a) 5' -Nase staining for light microscopy. Both the were anesthetized with diethyl ether and successively spread tissues and sections were incubated in the perfused via the thoracic aorta with 0.1 M cacodylate standard medium (Wachstein and Meisel, 1957) for buffer containing 7% sucrose (pH 7.2) and cold 5'-Nase activity with lead nitrate as the capture formaldehyde CaC12fixative (2% paraformaldehyde, agent and adenosine 5'-monophosphate (AMP, 1% CaC12)in the same buffer. For whole mount sodium salt, Sigma Chemical Co., St. Louis, USA) preparations of intact or peeled tissues, the stomach as the substrate. The standard medium was sup- with the distal end of the esophagus and the proximal plemented with 2 mM L-tetramisole, an inhibitor of portion of the duodenum attached was removed and nonspecific ALPase, in order to obtain the specific opened along the greater curvature, and then care- reaction for 5'-Nase activity. The incubation time at fully stretched out over the steels spines on a plastic 37°C for the spread tissues, the OCT sections and plate with the serosa side facing up, allowing the the JB-4 sections in the reaction medium was 30, 50, four zones of the stomach, i.e., the esophagus-stomach 60-90 minutes, respectively. After rinsing with 0.1 M (E-S), forestomach-corpus (F-C), corpus-antrum cacodylate buffer containing 7% sucrose or with (C-A) and antrum-duodenum (A-D) to be easily distilledwater, they were immersed in 1% ammonium discerned (terminology after Robert, 1971). How- sulfidesolution for 2-3 minutes at room temperature. ever, the esophagus-stomach zone of the rat consists b) ALPase staining for light microscopy. After of three parts, the esophagus-forestomach (E-F), the 5'-Nase histochemical staining, some spread tissues esophagus-corpus (E-C) and the esophagus-antrum and sections were incubated in reaction medium for (E-A), depending on which division of what connects ALPase activity with naphthol AS-MX phosphate with the esophagus (Figs. la ,b). The E-F has the (sodium salt, Sigma) as the substrate and Fast blue same stratified squamous epithelium and accounts BB (Sigma) as the coloring agent for, 30-60 minutes Lymphatic Vessels in the Rat Gastric Wall 39

at 4°C (Burstone, 1962; Kato and Miyauchi, 1989). trols: a) the substrate (AMP or naphthol AS-MX The whole mount preparations were examined under phosphate) was omitted, b) 50 mM NiC12 or 5 mM a dissecting photomicroscope using fiber optic illumi- L-tetramisole, specific inhibitors of the 5 '-Nase and nation (Zeiss Stemi SV II) and the sections were ALPase, respectively, were added to the standard observed under a light microscope. medium, c) the enzyme was inactivated by heating at c) 5' -Nase staining for transmission electron mi- 60°C for 60 minutes before incubation. croscopy (TEM). Two 5 '-Nase staining methods were used for TEM in these analyses, the 5'-Nase lead- Interstitial injection based method and the 5'-Nase cerium-based method An ultra-fine carbon particle suspension (Mitsu- (Uusitalo and Karnovsky, 1977; Kato et al., 1991). bishi, CH40, 21 nm in diameter) and 0.5% Evans The 50-Rin sections were separately incubated in blue (in 0.9% NaCl) were employed to determine these reaction media containing L-tetramisole for 30 the lymph drainage by interstitially injecting six rats. minutes at 37°C. Following histochemical staining, The injection sites included 39 points in the stomach, the specimens were refixed with 2% paraformal- 3 in the distal esophagus and 3 in the proximal dehyde and glutaraldehyde in 0.1 M cacodylate buffer portion of the duodenum of each animal (Fig. lc). (pH 7.2) for 2-6 hours and postfixed with 2% osmium tetroxide-0.5% potassium ferrocyanide solution for 2 hours at 4°C. Tissues were uniformly Results dehydrated in increasing concentrations of ethanol and embedded in Epok 812 (Oken, Tokyo, Japan). Light microscopy Sections 1-km thick stained with 0.5% toluidine blue 1. Whole mount preparations and Nabox mixture were analyzed to identify the a) Subserosa: Table 1 shows the main differences general organization of the lymphatic capillaries. in the lymphatics in the subserosa. Strong 5'-Nase Ultrathin sections (850-900 A) cut with a diamond reaction product appeared as a dark-brown precipi- knife on Reichert-Nissei Ultracuts were stained with tate of lead sulfide on the wall of the lymphatics; uranyl acetate or without further staining and were significantly less or no activity was detected in the examined under a JEOL 100 CX or 1200 EX trans- blood vessels (Fig. 2a). When the materials were mission electron microscope. then incubated in azo-dye reaction medium to visual- d) Control experiments for 5'-Nose and ALPase ize ALPase activity, the blood vessels stained blue staining. The followings were done to provide con- (Fig. 2b).The size of the lymphatic and blood capil-

Table 1. Distribution and architecture of lymphatics and their draining direction in the subserosa

S: stomach, E: esophagus, F: forestomach, C: corpus, A: antrum, D: duodenum 40 R.C. Ji et al. lanes differed, the lymphatic capillaries being several lymphatic capillary networks on both sides. Lym- times larger than the blood capillaries. The lymphatic phatic islands could also be seen in this region, but walls were usually bordered by a wavy outline. In they were less characteristic than in the E-C and E-A the E-C and E-A zones, the distal end of the eso- zones. phagus was found to have a loose, irregular lymphatic The lymphatic networks in the C-A zone were network. Pocket-like blind terminals were often seen much fewer and more irregular in distribution. The in this region. Three or four main lymphatics joined slender lymphatic capillaries occupied a larger area, the dense network in the corpus and antrum. A making it appear as though some spaces had no highly developed and irregularly distributed 5'-Nase- lymphatic capillary supply at all. A ring-like or ellip- positive lymphatic network was observed in the corpus tical connection appeared along the lymphatic capil- and antrum. Each initial lymphatic appeared typi- laries but not in their apical parts. cally tubulo-saccular in shape and proceed to branch In the A-D transitional zone (Fig. 4b), 9-13 like antlers (Fig. 4a). So-called "lymphatic islands" lymphatic capillaries were distributed fan-like all with peculiar 5'-Nase-positive tubules or sacs were over the antral region and converged towards the found to be isolated from the main lymphatic net- median line and the distal part of the antrum. Each work. The lymphatic capillaries in the subserosa lymphatic capillary gave off two or three branches were larger than in other regions. The main lymphatic which supplied a distinctive draining area, and the vessels running along the lesser curvature of the tapered terminals ended directly in the interstitium. stomach lay parallel to the longitudinal axis of eso- No lymphatic network could be found in the area. In phagus near the gastroesophageal junction. the proximal portion of the duodenum of this zone, The 5'-Nase-positive lymphatics in the F-C tran- on the other hand, 5'-Nase-positive lymphatic capil- sitional zone (Fig. 2a,b) were generally lobular in laries usually drained into the large lymphatic vessels distribution, and lymphatic capillary networks with which ran along the transitional line. These lymphatic much more knob-like branches were regularly located capillaries seldom foimed a network. in certain regions. They merged into the larger lym- b) Submucosa after peeling off the muscle: Table phatic vessels which run toward the lesser curvature 2 shows differences between the lymphatics in the in radial fashion and had a dilated, beaded profile submucosa. The distal esophagus in the E-C and due to repeated constriction, suggesting the presence E-A zones displayed a well-developed lymphatic of valve-like structures at these sites. A distinctive network with rod- or knob-like ramifications (Figs. lymphatic vessel was sometimes seen along the limit- 6a,b). The main lymphatics ran along the longitudinal ing ridge which seemed to connect evenly with the axis of the esophagus, and valve-like structures were

Table 2. Distribution and architecture of lymphatics and their draining direction in the submucosa

S: stomach, E: esophagus, F: forestomach, C: corpus, A: antrum, D: duodenum Lymphatic Vessels in the Rat Gastric Wall 41 seen in the region near the corpus and antrum. The The results of 5'-Nase-ALPase double staining 5 '-Nase-positive lymphatics of the corpus and antrum showed that the main lymphatics were usually ac- exhibited many more connections with each other companied by blood vessels, especially in the F-C than the accompanying 5'-Nase-negative blood capil- and C-A zones. The fine blood capillary network lanes, which had few connections. The typical ring- was more superficiallydistributed than the lymphatic or knob-like blind endings could be easily visualized network in the same layer. Valve-like structures in the apical part of the initial lymphatic capillaries. were never detected in the blood capillary network. Valve-like structures were also demonstrated at greatly constricted points along the lymphatic capil- 2. Tissue sections laries which drained into the lymphatic vessels in the Staining the sections for lymphatic 5'-Nase activity lesser curvature or the esophageal side. A large generally yielded the same results as in the whole collateral connection usually appeared along the mount preparations. The 5'-Nase reaction products course of the lymphatics. were visible as a dark-brown or black lead sulfide In the F-C transitional zone, one or two lymphatics precipitate on the walls of the lymphatics, with much sometimes accompanied by blood vessels, passed less or no activity detected in the blood vessels. through the transitional zone as linking lymphatics 5'-Nase-positive lymphatic capillaries were observed and drained the lymph from the lymphatic capillaries in the deep lamina propria and in the superficial of the forestomach and corpus. The lymphatic capil- submucosa adjacent to the lamina muscularis mucosa laries in the forestomach were more distinct and (Fig. 5a). The collecting lymphatics located in the thicker than in the corpus (Figs. 3a,b). The former deep submucosa near the muscularis interna pene- were usually accompanied by blood capillaries on trated through the muscularis externa perpendicu- both sides or were covered by them. The latter larly or obliquely to merge into the subserosal began as a blind endings and fonned circular anas- lymphatic network. Lymphatics with irregular con- tomoses by themselves. tours were also situated between the inner circular Two distribution patterns of lymphatic capillaries and outer longitudinal muscularis or between the characterized the C-A zone. In one pattern, two muscular bundles. In the subserosa, clusters of lym- lymphatic capillaries followed a blood capillary from phatics with 5'-Nase reaction products were often both sides and usually crossed beneath or over the detected in the E-C and E-A zones (Fig. 5b). With capillary to form a typical coarse network with valve- 5'-Nase-ALPase double staining, the 5'-Nase-positive like structures. A circular connection and a long rod- lymphatic vesselswere seen to run in close proximity like blind terminal on one side of the lymphatic to the ALPase-positive arteries and veins, especially capillary appeared to be typical features of this pat- in the submucosa of the F-C and C-A zones. tern. The lymph drainage seemed to follow the same No 5'-Nase-positive staining of the lymphatics pattern as the blood supply. In the second pattern, was seen in the control experiments either in whole the rich, honeycomb-like lymphatic capillary net- mount preparations or in tissue sections incubated in work was also regionally distributed. A longer lym- substrate (AMP)-free medium, treated at 60°C for phatic capillary was often seen to interconnect two 60 minutes or treated with NiC12before being incu- larger or smaller ring-like structures. Dilated initial bated in intact reaction medium. The blood vessels lymphatic terminals but no lymphatic islands could also failed to stain in the control group by the be found. ALPase method. Annular lymphatic capillaries, 6-7 in number, ran parallel to each other about 0.5-1.1 mm apart around Transmission electron microscopy the adjacent to the duodenum on the antral The lymphatic capillary distribution was demon- side of the A-D zone (Fig. 7), each annular lymphatic strated by both the lead-based and the cerium-based capillary possessed ring- or rod-like structures. 5'-Nase staining methods. Reaction product was There were straight or oblique short-circuit lymphatic principally localized on the luminal and abluminal capillaries connecting two or three adjacent lym- surfaces of the lymphatic endothelial cells but was phatic circles near the duodenum but many fewer far either absent or extremely faint in the blood capillaries from the duodenum, where the annular lymphatic (Fig. 9a). The cerium-based method, however, en- capillaries were much thinner and formed clear ring- abled better demonstration of the lymphaticcapillaries like connections along their course. The presence of than the lead-based method in terms of 5'-Nase the lymphatic valve-like structures indicated that the reaction products. The 5'-cerium reaction products lymph drained in one direction toward the duodenum. were constantly, uniformly and continuouslyrestricted On the duodenal side, fine lymphatic networks with to the cell membrane of attenuated endothelial cells ring- or rod-like branches were seen to be distributed (Fig. 9a). In the mucosa, the 5 '-Nase-positive lym- inter-regionally in the submucosa. phatic capillaries were always separated from the 42 R.C. Ji et al. glandular cells by about 4-301AM of connective Discussion tissues, and no direct contact could be found between them. However, a rich lymphatic network was found The present study, in which the 5 '-Nase and in the mucosa above the lamina . ALPase histochemical staining methods were used, Lymphatic capillaries also coursed within the lamina successfully demonstrated the fine distribution and muscularis mucosae. architecture of the lymphatics in the rat stomach and A property found to be common to all lymphatic their relationship to the blood vessels. Whole mount vessels was three types of intercellular junctions: preparation techniques combined with tissue sections end-to-end, overlapping and interdigitating, adorned and interstitial injection allowed extensive obser- by granules which reacted strongly for 5'-Nase. The vation of lymphatic capillaries. We were able to latter two contacts, however, accounted for the vast follow the ramification pattern and observe initial majority (94-97%) (Figs. 9b—d). Open (gap) inter- blind sacs and valve-like structures along the lym- cellular junctions with a diameter 400-700 nm in the phatic drainage routes. 5'-Nase-ALPase double lymphatic vessels were labelled with 5'-Nase reaction staining enabled clear demonstration of the distri- precipitates. It was noteworthy that the open junc- bution of the lymphatics and blood vessels in the tions were chiefly present in the mucosal and sub- same preparation (Figs. 2b, 3b, 6b). Ultra-thin sec- mucosal lymphatic networks of the C-A and A-D tions stained for 5'-Nase shed light on certain ques- zones. tions such as whether intercellular open junctions The lymphatic valves consisted of two layers of were present and on the relationship between glandu- endothelial cells separated by a fine lar cells and lymphatic capillaries. Application of core to which the reaction granules were adherent. this method has proved very reliable for studying not Interestingly, the lymphatic valves were mainly lo- only flat membranous structures but hollow or thin- cated in the submucosa and subserosa (Figs. 10a,b). walled visceral organs, such as the intestine, The basement lamellar materials were usually lacking (Kato et al. , 1993). The whole mount preparation or wispy and irregular, whereas the quantity of with enzyme-histochemical staining was also free of anchoring filaments and collagen fibers surrounding some artifacts or misinterpretations which arose when the lymphatic capillaries in the fine connective tissue the interstitial injection technique was used. varied from region to region even on different sides There is no consensus concerning the existence of of the same capillary. Occasionally, reaction products lymphatic microvessels in the between were scattered in adjacent connective tissue. By the glands. Some earlier papers have reported that addition of L-tetramisole to the histochemical lymphatic capillaries extend into the gastric mucosa medium, 5'-Nase staining method, especially the (Eker and Efskind, 1952), while other descriptions cerium-based method, rarely yields nonspecific of the lymphatic distribution in the human stomach reaction deposits. have commented on the almost complete lack of lymphatics in the mucosa and the absence of any Interstitial injection relationship with the glands (Lehnert et al., 1985). Dynamic analysis by interstitially injecting ultra- Still others have rejected the idea that lymphatics fine carbon particle suspension and Evans blue in could be present within the gastric mucosa (Koelliker, living animals showed that the lymphatics of about 1854). Possible reasons for these conflicting reports 90% of the gastric wall drained into the lymphatic are that most of the descriptions are based on routine vessels accompanying the left gastric artery (Figs. lc, light microscope studies or retrograde injection even 8). In the gastric region near the greater curvature though the microvascular structure usually shows the lymphatic capillaries drained into the lymphatics interspecies differences. accompanying the right gastroepiploic artery, then The present findings show a rich lymphatic net- into the lymphatic vessels running along the gastro- work located in the deep lamina propria and the duodenal artery. In the E-S zone, the lymphatic lamina muscularis mucosae. Some lymphatic capil- vessels of the distal esophagus joined the lymphatics lanes were found to penetrate the lamina muscularis of the corpus and antrum, or directly drained into mucosae into the submucosa, but no interglandular the lymphatics accompanying the left gastric artery. lymphatic capillaries were encountered. These find- In the A-D zone, the lymphatic capillaries of the ings coincide with observations made by fluorescent proximal portion of the duodenum joined the lym- stream analysis in vivo after injecting isothiocyanate phatics lying on the A-D transitional zone or into the by manual pressure, but the fluorescent stream inves- lymphatic vessels accompanying the gastroduodenal tigation failed to demonstrate the precise location of artery. One or two lymphatics between the fore- the lymphatic capillaries and their relationship to stomach and the spleen could be found after injecting glandular cells (Nagata and Guth, 1984). Our positive the forestomach near the greater curvature. identification of lymphatic capillaries with 5'-Nase Lymphatic Vessels in the Rat Gastric Wall 43 activity clearly showed that the lymphatic capillaries capillaries, on the other hand, may play an important were associated with the base of the lowest glandular role in lymph drainage in this region. Some studies cells. In addition, the existence of open junctions at have indicated that this type of lymphatic vessel the C-A and A-D zones seems to suggest that the contraction occurs as a result of intrinsic motor mucosa has certain permeability to macromolecules activity itself, not as a result of indirect factors which allows lymph node metastasis by early gastric (Nagata and Guth, 1984). Spontaneous contraction cancer in spite of a much lower risk of metastasis in of lymphatic vessels has also been detected in bovine this period (Lehnert et al., 1985). Furthermore, many mesentery (Ohhashi et al., 1980) and the human lymphatic capillaries were located beneath the thoracic duct (Kinmoth and Taylor, 1956). Recent squamous epithelium in the E-C, E-A and F-C tran- study has shown that higher concentration of lym- sitional zones, but the farther the site was from the phatic vessel-associated neurons in the and transitional point, the fewer lymphatic capillaries of the guinea pig might participate in many were present. Thus, the mucosa of the corpus and complex activities of the (Wang antrum of the rat stomach may play a more important et al. , 1994). Moreover, our present findingsdemon- role in interstitial tissue fluid homeostasis. strate that many open intercellular junctions adorned The microlymphaticorganization of the rat stomach with 5'-Nase reaction precipitates were detected in showed some regional differences, the same as in the the submucosal lymphatic network of the C-A and and colon (Kamei, 1969;Lowden and A-D zones. Thus, it is reasonable to assume that the Heath, 1993). Whole mount preparations after peel- lymphatic capillaries in the submucosa play a crucial ing off the muscle showed the typical ramification role in reabsorbing excess interstitial fluid and trans- pattern and distribution characteristic of lymphatic porting other solutes or local tissue hormones. Un- capillaries in the superficial layer of the submucosa, doubtedly, once gastric cancer invades the submucosa, as summarized in Table 2. Generally speaking, lym- malignant cells have access to numerous lymphatics, phatic capillariesare located deep to blood capillaries thereby facilitating metastasis (Sakuma et al., 1985; in the same layer of the stomach. It was very interest- Listrom and Fenoglio-Preiser, 1987). ing to find that many valve-like structures were Our current findings based on the BEI technique located along the course of the lymphatic capillary (Kato and Gotoh, 1990) and TEM histochemical network in the submucosa.These valve-likestructures method (Kato, 1990a,b) also revealed a 5'-Nase- probably act as tiny pumps to facilitate centripetal positive lymphatic capillary network between the lymph flow (Casley-Smith, 1977) or as a supporting inner circular and outer longitudinal muscular layers frame to prevent the collapse of lymphatic capillaries and along muscular fibers, but failed to show any (Kato et al. , 1993). Similar valve-like structures in differencesamong the four zones. Furthermore, fewer the dermal lymphaticshave been found to be variable valves were found in the muscularis externa than in or to disappear with changes in the local environment the submucosa in our study. The abundant 'flap- (Daroczy, 1984). Unlike the valve-like structures, valves' preventing backflow into the interstitium the ultrastructural details revealed that the valves appear to be necessary in the submucosa (Figs. 10a, with dense granules aligned in a row of punctate 10b), while the combined contraction of the muscu- densities were located in the large lymphatic vessels laris externa and lamina muscularis mucosa seems to of both the superficial and deep submucosa. The be essential to regulate and propel lymph into the junctions in the valvular endothelium are relatively subserosal vessels. Thus, the strong contraction of tight and marginal folds are almost absent. This the muscularis externa caused by direct or indirect suggest that the valvular endothelium is not involved factors during the fluorescent stream investigation in active transport even though the vesicles of the experiment process in vivo might have caused the valvular endothelium are as numerous as in the walls lymphatic capillary network in this layer to have of lymphatic vessels. They may mainly serve to been missed (Nagata and Guth, 1984). prevent retrograde lymph flow. In this context, it is The pattern of subserosal lymphatic capillaries important to identify the structural and functional differed among the zones and the differences are differences between the valve-like structures and summarized in Table 1. The subserosal lymphatic valves. capillaries in the stomach were mainly arranged in Functionally, lymphatic capillaries are found in three patterns: irregular, lobular and fan-shaped. different states of contraction and dilation, especially Similar features were present in the submucosa, but in the C-A and A-D zones. Significantcontraction of two contrasting observations willbe mentioned here. the outermost annular lymphatic capillaries in the First, only the corpus and antrum of the E-C and antrum of the A-D zone indicated that lymph flowed E-A zones exhibited typical lymphatic islands having from the antrum to the proximal portion of the morphological and 5'-Nase-enzyme reactive charac- duodenum progressively. Short-circuit lymphatic teristics similar to those of true lymphatics. As 44 R.C. Ji et al.

demonstrated in the silver-stained mesentery of the 65:391-404. guinea pig by Castenholz et al. (1991), the lymphatic 10) Kato S. Enzyme-histochemical demonstration of intralobular islands may represent proliferating vascular units lymphatic vessels in the mouse thymus. Arch Histol Cytol, Suppl 1990a; 53:87-94. which later fuse to form a final continuous lymphatic 11) Kato S. Histochemical localization of 5'-nucleotidase in the capillary (Kato et al., 1993). The present findings lymphatic endothelium. Acta Histochem Cytochem 1990b; demonstrated that much of the lymph from the rat 23(5):613-620. stomach drains into the lymphatic vessels accompany- 12) Kato S and Gotoh M. Application of backscattered electron imaging to enzyme histochemistry of lymphatic capillaries. ing the left gastric artery. This suggests that active J Electron Microsc 1990; 39:186-190. proliferating vascular units corresponds to the im- 13) Kato S, Yasunaga A and Uchida Y. Enzyme-histochemical portance for lymph drainage in the region. Second, method for identification of lymphatic capillaries. Lymphology the fan-shaped distribution of the lymphatic vessels 1991; 24:125-129. in the antrum of the A-D transitional zone showed 14) Kato S, Miura M and Miyauchi R. Structural organization of the absence of a lymphatic network. The terminal the initial lymphatics in the monkey mesentery and intestinal wall as revealed by an enzyme-histochemical method. Arch end (initial part) of the lymphatic capillary seemed Histol Cytol 1993; 56(2):149 - 160. to merge directly into the interstitium. The relatively 15) Kinmoth JB and Taylor GW. Spontaneous rhythmic contrac- independent distribution of lymphatic capillaries in tivity in human lymphatics. (abstr.) J Physiol (London) the A-D zone indicated that the lymphatic capillaries 1956; 133:3-4. 16) Koelliker A. Von den Lymphefassen. In: Koelliker A, ed. in the submucosa are much more important for Koellikers Handbuch der Gewebslehre des Menschen. lymph drainage than the lymphatic capillaries of the Mikroskopische Anatomie. Vol. III/2a. Leipzig: Englemann- subserosa in this zone. Verlag, 1854. 17) Kunstyr I, Peters K and Gartner K. Investigations on the function of the rat forestomach. Lab Animal Sci 1976; Acknowledgements 26:166-170. 18) Lehnert T, Erlandson RA and Decosse JJ. Lymph and blood capillaries of the human gastric mucosa: A morphologic The authors are grateful to Mr. T. Kajiwara basis for metastasis in early gastric carcinoma. Gastro- (Department of Anatomy), Mr. H. Kawazato and enterology 1985; 89:939 - 950. Miss A. Yasuda (Electron Microscopy Unit, Re- 19) Listrom MB and Fenoglio-Preiser CM. Lymphatic distri- bution of the stomach in normal, inflammatory, hyperplastic, search Laboratory Center) for their skilled technical and neoplastic tissue. Gastroenterology 1987; 93:506-514. assistance. 20) Lowden S and Heath T. Lymphatic drainage from the distal small intestine in sheep. J Anat 1993; 183:13-20. 21) More J, Fioramonti J and Bueno L. Aluminium hydroxide References uptake in the stomach and in the intestine of the rat: A histochemical study. Acta Anat 1992; 145:50-54. 1) BurstoneMS. Phosphatase.Enzyme histochemistry and its 22) Nagata H and Guth PH. In vivo observation of the lymphatic applicationin the studyof neoplasma.New York and London: system in the rat stomach. Gastroenterology 1984; 86:1443- AcademicPress pp. 160-292,1962. 1450. 2) Casley-SmithJR. The structural basis for the conservative 23) Noguchi T, Kato S, Shimada T and Uchida Y. Demon- treatmentof lymphedema.In lymphedema.Edit.: L. Clodius, stration of lymphatic capillaries in the rat stomach using histochemical and electron microscopic techniques. Elsevier pp. 15-25, GeorgeThieme, Stuttgart, 1977. 3) CastenholzA, Hauk G and RettbergU. Lightand electron Science Publishers B.V. (Biomedical Division) Progress in microscopyof the structural organizationof the tissue- Lymphology-XII pp. 543-544, 1990. lymphaticfluid drainage system in the mesentery:An experi- 24) Ohhashi T, Azuma T and Sakaguchi M. Active and passive mentalstudy. Lymphology 1991; 24:82-89. mechanical characteristics of bovine mesentery lymphatics. 4) DaroczyJ. Newstructural details of dermallymphatic valves Am J Physiol 1980; 239:88-95. and its functionalinterpretation. Lymphology 1984; 17 :54- 25) Ohtani 0 and Murakami T. Lymphatics and myenteric 60. plexus in the muscular coat in the rat stomach: A scanning 5) De Pierre JW and KarnovskyML. Plasmamembranes of electron microscopic study of corrosion cast made by intra- mammaliancells. A reviewof methodsfor theircharacteriza- arterial injection. Arch Histol Jap 1987; 50(1):87-93. tion and isolation.J Cell Biol 1973; 56:275-303. 26) Robert A. Proposed terminology for the anatomy of the rat 6) Eker R and EfskindJ. Investigations on the intramural stomach. Gastroenterology 1971; 60(2):344-345. spreadof gastriccarcinoma. Acta PatholMicrobiol Scand 27) Sakuma A, Ouchi A, Sugawara T and Sato T. Histologic 1952;30:371-381. infiltrating pattern of gastric microcarcinoma by means of 7) Isomaki AM. A newcell type (tuft cell) in the gastrointestinal serial sections. Cancer 1985; 55:1087-1092. mucosaof the rat. ActaPath MicrobiolScand, Sect A, Suppl 28) Uusitalo RJ and Karnovsky MJ. Surface localization of 5'-nucleotidase on the mouse lymphocytes. J Histochem pp. 240, 1973. 8) Kamei Y. The distributionand relative locationof the Cytochem 1977; 25:87-96. lymphaticand blood vesselsin the mucosaof the rabbit 29) Wachstein M and Meisel E. Histochemistry of hepatic phos- colon. NagoyaMed J 1969;15:223-243. phatase at physiologic pH. Am J Clin Pathol 1957; 130:153-176. 9) Kato S and MiyauchiR. Enzyme-histochemical visualization 30) Wang XY, Wong WC and Ling EA. Studies of the lymphatic of lymphaticcapillaries in the mouse tongue: Light and vessel-associated neurons in the intestine,of the guinea pig. J electronmicroscopic study. Okajimas Folia Anat Jpn 1989; Lymphatic Vessels in the Rat Gastric Wall 45 Plate I

Explanations of Figures

Plate I

Figs. 1. Anatomy of the rat stomach (E: esophagus, F: forestomach, C: corpus, A: anti-urn, D: duodenum) la: Survey of the rat stomach. lb: The scheme is that of a stomach opened along the greater curvature. lc: Diagram illustrating the lymph drainage in the gastric wall, the distal end of esophagus and the proximal portion of duodenum. 46 R.C. Ji et al.

Anat 1994; 185:65-74. 32) Werner JA, Schtinke M and Tillmann B. Histochemical 31) Wattel W and Geuze JJ. Ultrastructural and carbohydrate visualization of lymphatic capillaries in the rat: A comparison histochemical studies on the differentiate and renewal of of methods demonstrated at the posterior pharyngeal surface. mucous cells in the rat gastric fundus. Cell Tiss Res 1977; Arch Histol Jap 1987; 50(5):505-514. 176:445-462.

Plate II

Figs. 2.3. Light micrographs of the whole mount preparations of the subserosa (Figs. 2a, 2b) and the submucosa peeled off the muscle (Figs. 3a, 3b) in the rat stomach (F-C zone) with enzyme-histochemical demonstration of the lymphatics and blood vessels. 2a, 3a: 5'-Nase activity. Lymphatics are colored dark-brown due to positive 5P-Nase activity, whereas blood vessels reveal lower or no activity. x 10, x25. 2b, 3b: 5'-Nase and ALPase activity of the same preparations as in Figs. 2a, 3a respectively followed by ALPase staining, but Fig. 2b shows further magnification of Fig. 2a indicated by asterisk. The blood capillaries and arterioles are blue to ALPase activity. x 25, x 25. Lymphatic Vessels in the Rat Gastric Wall 47 Plate II 48 R.C. Ji et al.

Plate III

Figs. 4. 5'-Nase-stained whole mount preparations showing the subserosal lymphatics in the stomach. 4a: 5'-Nase-positive lymphatic networks are significantly developed in the corpus and antrum of E-C and E-A zones. x25. 4b: 5'-Nase-positive lymphatic capillaries are distributed fan-shapedly in the antrum side of A-D zone. They centralize towards the median line and the distal part of the antrum with a distinctive draining area respectively. x25.

Figs. 5. Light micrographs of plastic (JB-4) sections in F-C and E-S transitional zones of the stomach with 5'-Nase staining. 5a: There are many 5'-Nase-positive lymphatics (arrows) in the deep lamina propria close to the lamina muscularis mucosa and the submucosa in F-C zone. x40. 5b: A cluster of 51-Nase-positive lymphatics are seen in the subserosa and muscularis externa at E-C and E-A zones. Note the cellular edges of typical lymphatic endothelium (arrows) exhibiting a 5'-Nase-positive irregular pattern of serrate borders. x80. Lymphatic Vessels in the Rat Gastric Wail 49 Plate HI 50 R.C. Ji et al.

Plate IV

Figs. 6. Light micrographs of 5'-Nase-positive lymphatic network in the submucosa after peeling off the muscle. 6a: More rod- or knob-like blind endings are visualized in the esophageal side of E-C and E-A zones. 5'-Nase only. x 10. 6b: Further magnification of the same area (1 as in Fig. 6a. Note the valve-like structures located in the obviously constrictive point of the plexus and ALPase-positive blood capillary networks. x45.

Fig. 7. Several annularly paralleled lymphatic capillaries with short-circuit connections are detected to surround the pylorus adjacent to the duodenum in A-D zone. x25.

Fig. 8. Light micrograph of the subserosal lymph drainage in E-C and E-A zones revealed by interstitial injection with ultra-fine carbon particle suspension. x 15. Lymphatic Vessels in the Rat Gastric Wall 51

Plate IV 52 R.C. Ji et al.

Plate V

Figs. 9. TEM views of 51-Nase-positive lymphatic in the stomach. 9a: Note 51-Nase-positive lymphatic (LV) and 5'-Nase-negative blood vessel (BV) in the submucosa. x800. 9b—d: The 51-Nase-positive end-to-end (9b), overlapping (9c) and interdigitating (9d) intercellular junctions with granular precipitates on the surface of the lymphatic endothelial cells. x20,000.

Figs. 10. Lymphatic with valves in the submucosa of C-A zone. 10a: Light micrograph (semithin resin section) stained with toluidine blue. x70. 10b: High magnification of the valves by TEM. It arises from the region adjacent to Fig. 10a (arrow). Note the surface of the endothelial cells of the valve labelled with 5'-Nase reactive precipitates. x5400. Lymphatic Vessels in the Rat Gastric Wall 53

Plate V