On the presence and function of closed lymphatic stomata in the diaphragm of the golden hamster

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1 Okajimas Folia Anat. Jpn., 66 (2-3): 69-80, August, 1989 On the presence and function of closed lymphatic stomata in the diaphragm of the golden hamster By Harumichi SHINOHARA, Yoshifumi FUKUO and Takeshi MATSUDA Department of Anatomy I, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama Addressee for correspondence -Received for Publication, January 17, Key words: Lymphatic stomata, diaphragm, golden hamster Summary: The peritoneal surface of the golden hamster diaphragm was examined for closed lymphatic stomata by scanning electron microscopy (SEM) and serial sectioning. Closed stomata were absent on SEM but present on serial sectioning. Some closed stomata in the serial sections diameter and consisted of an outer mesothelial margin and an inner lymphatic wall. The lymphatic wall was formed by several endothelial cells adjoined with various junctional abutments. The discrepancy of results between SEM and serial sectioning, and the functional aspects of lymphatic stomata are discussed. It is more than a century since Recklinghausen first described lymphatic stomata of the diaphragm as devices for fluid drainage from the body cavity (Recklinghausen, 1863). His discovery led to a long-standing controversy on whether stomata structures really existed or were artifactually produced, and this controversy was settled only recently (Leak and Rahil, 1978). However, this does not mean that morphological explorations of lymphatic stomata are sufficient. Many investigators have postulated that lymphatic stomata are dynamic structures which may open in response to changes of environment (Bettendorf, 1978; Tsilibary and Wissig, 1983; Shinohara et al., 1985). If so, some lymphatic stomata should be 'closed' under certain conditions, although no investigator has ever presented a plausible morphology for such closed stomata. Histological sections often reveal structures in which the body cavity and lymphatic lumen are separated by the membranous wall of the lymphatics without overlying connective tissue and mesothelial layer (Bettendorf, 1979; Tsilibary and Wissig, 1979). We suggested previously that such membranous structures might represent the sectioned morphology of closed stomata (Shinohara et al., 1985). The present study was undertaken to test this hypothesis. Materials and Methods Adult golden hamsters of 645 weeks of age were used for the experiments. They were kept in an air-conditioned room with water and 69

2 70 H. Shinohara et al. laboratory chow available ad libitum. The animals were flushed with 0.1 M phosphate buffer solution (PBS) at ph 7.4 supplemented with Pio sucrose, and then perfused with 2% glutaraldehyde in PBS. The diaphragm was isolated, cut into small blocks approximately 1 x 1 mm wide, and immersed in fresh glutaraldehyde fixative for a few hours. They were then rinsed in PBS, postfixed in 2% 0s04, dehydrated in ethanol, placed in propylene oxide, and embedded in Quetol 812. The blocks were sectioned at a thickness of 0.3 pm with a diamond knife. After each cut, the semithin sections were stained with 0.507o toluidine blue and examined photomicroscopically to determine whether the membranous structure of the lymphatic wall, which was directly exposed to the peritoneal cavity, was intact or open to the cavity. Such sectioning was continued until the membranous structure disappeared from the sections. Special care was taken to obtain serial sections; if two succeeding sections were lost, the sectioning procedure was abandoned. The serial sections were placed in a computerized image analysis system (CIA 102, Olympus), and closed stomata were three dimensionally reconstructed. In addition, ultrathin sections of the membranous structure were obtained. These were stained with uranyl acetate and lead citrate, and viewed under a transmission electron microscope (200 CX; Hitachi). To obtain specimens for scanning electron microscopy, the diaphragm was cut into fragments approximately 5 x 5 mm wide after perfusion fixation with 2c7o glutaraldehyde. The tissues were rinsed, postfixed in 2% 0s04, dehydrated in ethanol, placed in isoamyl acetate, critical-point dried, gold coated, and viewed under a scanning electron microscope (X-650; Hitachi). Results On transmission electron microscopy, the membranous structures were found to consist of a layer of lymphatic endothelium (Fig. 1). This layer formed part of the lymphatic wall that was directly exposed to the peritoneal cavity without overlying connective tissue and peritoneal mesothelium. The adjacent endothelial cells comprising the wall had various junctional abutments: desmosomes, tight junctions and open junctions. The lymphatic endothelium and peritoneal mesothelium approached close to each other but did not adjoin. It was technically difficult to obtain serial sections which satisfied our condition that two succeeding sections should not be lost. However, membranous structures were not rare to encounter, and we sectioned as many as 30 blocks to obtain satisfactory serial sections. Many membranous structures did not allow communication between the peritoneal cavity and lymphatic lumen throughout their serial sections; i.e. the lymphatic wall was intact (Figs. 2-7). Computerized reconstructions of such serial sections revealed that the exposed abluminal surface of the lymphatic wall formed a circular floor in the gully created by several peritoneal mesothelial cells (Fig. 8). This part of the lymphatic wall may be termed a 'closed' stoma in the sense that it would be open only if dehiscence of junctional abutments occurred. The closed stomata were assumed to be generally 3-5 pm in diameter, and rarely less than 1 pm or more than 10 am in diameter; i.e. they continued to be present in at least 3 sections and usually disappeared when sections were cut. Scanning electron microscopy revealed that the peritoneal surface of the diaphragm was lined with flattened cells and cuboidal cells. The former cells were polygonal in shape and continuously lined the surface (Fig. 9). The latter cells were interconnected with numerous

3 Closed lymphatic stomata 71 cytoplasmic extensions. These cells often formed circular gullies, usually less than 20 pm in diameter, in which lymphatic stomata opened (Fig. 10). The orifice of the stomata was formed by microvillus-free lymphatic endothelial cells. Some orifices were circular, while others were irregular in shape (Figs. 10 and 11). The circular orifices were more than a few micrometers and less than 15 pm in diameter. The precise size of the irregularshaped orifices was difficult to estimate because of their shape, but the slits were rarely narrower than 1 pm and did not exceed 15 m. In spite of an extensive survey of the peritoneal surface of the diaphragm, we did not encounter structures compatible with the reconstructed closed stomata. Discussion Our findings showed that closed stomata were present in serial sections, but were absent in scanning electron micrographs. This discrepancy raises the question of why closed stomata detectable by serial sectioning could not be detected by SEM. There may be a technical reason. The lymphatic wall of closed stomata is located in a gully surrounded by peritoneal mesothelial cells of several micrometers in height. In SEM, it was often found that the gully was too deep and dark to decide whether the lymphatic wall was completely closed or open through stomata orifices. The possibility of some artifact must also be considered. Biological specimens become reduced in their surface width by as much as 2207o during the histological procedures of SEM, and critical-point drying is largely responsible for such shrinkage (Wheeler et al., 1975). Closed stomata are composed of parquets of lymphatic endothelial cells which are adjoined with various junctional abutments. According to recent observations on cell-to-cell connections, even desmosomes and tight junctions are not necessarily stable but separable under certain conditions (Pitelka et al., 1973; Decker and Friend, 1974), and this is especially true for intercellular junctions in the lymphatic endothelium (Casley-Smith, 1980). We are inclined to think that shrinkage of the tissue during critical-point drying may artifactually separate the intercellular junctions of closed stomata. This assumption appears to be reinforced by the difference of procedures between serial sectioning and SEM; namely, criticalpoint drying is employed only in the latter. Consideration should thus always be given to the possibility of artifacts in the interpretation of the lymphatic stomata with irregularshaped orifices that appear on SEM. Bettendorf (1978) suggested that lymphatic stomata of the diaphragm may function as valves which open during inspiration and close during expiration. Tsilibary and Wissig (1983) reported that dynamic alterations of actin filaments in the lymphatic endothelial cells may lead to shrinkage or elongation of the cells, thus opening or closing the stomata orifices. These functional aspects of the lymphatic endothelial cells may not be altogether negative; however, we consider that the roles of the lymphatic endothelium may be overestimated. Intra-abdominal pressure varies greatly according to physical exertion, and in vital capacity tests, it reaches as high as more than 10 times that in quiet breathing (Campbell and Green, 1953). Fenn (1963) estimated that the maximum shortening of the diaphragm muscle fibers of one side was almost 50% of the initial length, and the maximum tension of the fiber has been calculated to be higher than 0.5 kg per cm (Agostoni, 1964). It is questionable whether minute valves consisting of lymphatic endothelium could accommodate such dynamic changes of the diaphragm and remain efficient as valves. As the present study has demonstrated, lymphatic stomata include not only open but also closed categories.

4 72 H. Shinohara et al. However, this does not necessarily mean that alterations from closed to open, or vice versa, have any functional significance. Acknowledgements The authors wish to thank Dr. T. Nakatani, Dr. S. Morisawa and Mr. T. Horii for their technical advice and assistance. We are also grateful to Miss H. Matsuda for typing References the manuscript. 1) Agostoni, E.: Action of respiratory muscles. Ed. by W. 0. Fenn and H. Rahn. In: Handbook of Physiology, section 3 Respiration, vol. 1, pp American Physiological Society, Washington, D. C., ) Bettendorf, U.: Lymph flow mechanism of the subperitoneal diaphragmatic lymphatics. Lymphology, 11: , ) Betendorf, U.: Electronmicroscopic studies on the peritoneal resorption of intraperitoneally injected latex particles via the diaphragmatic lymphatics. Lymphology, 12: 66-70, ) Campbell, E. J. M. and Green, J. H.: The variations in intra-abdominal pressure and the activity of the abdominal muscles during breathing; A study in man. J. Physiol., 122: , ) Casley-Smith, J. R. L.: The response of the microcirculation to inflammation. Ed. by G. Weissman. In: Handbook of Inflammation, vol. 2, pp Elsevier/North-Holland Biomedical Press, Amsterdam, ) Decker, R. S. and Friend, D. S.: Assembly of gap junctions during amphibian neurulation. J. Cell Biol., 62: 32-47, ) Fenn, W. 0.: A comparison of respiratory and skeletal muscles. Ed. By C. F. Cori. In: Perspectives of Biology (Houssay Memorial Volume). pp Elsevier, Amsterdam, ) Leak, L. V. and Rahil, K.: Permeability of the diaphragmatic mesothelium; The ultrastructural basis for "stomata". Am. J. Anat., 151: , ) Pitelka, D. R., Hamamoto, S. T., Duafala, J. G. and Nemanic, M. K.: Cell contacts in the mouse mammary gland. I. Normal gland in postnatal development and secretory cycle. J. Cell Biol., 56: , ) Shinohara, H., Nakatani, T. and Matsuda, T.: The presence of lymphatic stomata in the ovarian bursa of the golden hamster. Anat. Rec., 213: 44-52, ) Tsilibary, E. C. and Wissig, S. L.: Lymphatic absorption from the peritoneal cavity; Regulation of patency of mesothelial stomata. Microvasc. Res., 25: 22-39, ) Tsilibary, E. C. and Wissig, S. L.: Light and electron microscope observation of the lymphatic drainage units of the peritoneal cavity of rodents. Am. J. Anat., 180: , ) Recklinghausen, F. v.: Zur Fettresorption. Arch. f. Path. Anat. u. Physiol., 26: , ) Wheeler, E. E., Gavin, J. B. and Seelye, R. N.: Freeze-drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy. Stain Technol., 50: , 1975.

5 PLATES Closed lymphatic stomata 73

6 74 H. Shinohara et al. Explanation of Figures Plate I Fig. 1. The membranous structure (black arrows) is part of the lymphatic vessel, of which the abluminal surface is directly exposed to the peritoneal cavity (PC). The open arrow indicates an intercellular junction of lymphatic endothelial cells. Note that dehiscence of the intercellular junction may allow communication between the peritoneal cavity and lymphatic lumen (L). M; peritoneal mesothelial cell, CT; connective tissue. x

7 Closed lymphatic stomata 75 Plate I

8 Shinohara cv Plate II Figs Serial sections of a lymphatic stoma. The membranous structure (arrows) appears in Fig. 3 and disappears in Fig. 6. The squares in Figs. 2 and 7 indicate the part used for reconstruction. 104(). Fig. 8. A closed stoma reconstructed from 20 serial sections, 6 of which are presented in Figs The stoma (asterisk) is composed of a circular parquet of lymphatic endo.helia! cells (red) surrounded by peritoneal mesothelium (blue and white).

9 Closed lymphatic stomata 77 Plate H

10 78 H. Shinohara et al. Plate III Fig. 9. Surface topography of flattened cells in the diaphragmatic peritoneum. Microvilli tend to be more numerous in the cell border. x Fig. 10. Circular stomata orifices (asterisk). the stomata consist of outer mesothelial (M) and inner endothelial (E) margins. The former is studied with numerous microvilli, whereas the latter is microvillusfree. x Fig. 11. An irregular stomata orifice (asterisk). The orifice appears as if the lymphatic endothelium has been disrupted. x

11 Closed lymphatic stomata 79 Plate III