Br. J. exp. Path. (1976) 57, 604

EFFECT OF HEPATIC VENOUS OUTFLOW OBSTRUCTION ON PORES AND FENESTRATIONS IN SINUSOIDAL ENDOTHELIUM* W. NOPANITAYA, J. C. LAMB, J. W. GRISHAMI AND J. L. CARSON Fromn the Department of Pathology, School of MIedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514

Received( for publication Junie 15, 1976

Summary.-The ultrastructure of pores and fenestrations in hepatic sinusoidal endothelial cells was examined following partial surgical occlusion of the suprahepatic portion of the inferior vena cava. Within 12 h after partial obstruction of hepatic venous outflow, endothelial pores ( < 0-1 [km in diameter) and sieve plates in the distal halves of sinusoids were greatly reduced in number or were totally absent, and they were replaced by large fenestrations ( < 1 0 um in diameter). These results suggest that pores forming sieve plates may fuse to form large fenestrations. The findings also indicate that sinusoidal hypertension and hypoxia associated with obstruction of hepatic venous outflow alter the distribution of pores and fenestrations in sinusoidal endothelium. THE ultrastructure of hepatic sinusoidal MATERIALS AND METHODS endothelial cells of several species recently Experiments were carried out oni female has been established by studies employing Wlistar albino rats. The animals were retired transmission (TEM) and scanning (SEM) breeders weighing approximately 325 g. They electron microscopy (Fawcett, 1955; Bruni were kept in wire bottom cages and fed Purina chow and tap water ad libitum for at and Porter, 1965; Wisse, 1970; Orci, laboratory least 3 weeks before being used for this study. Matter and Rouiller, 1971; Ogawa et al., All animals were anaesthetized by injecting 1973; Brooks and Haggis, 1973; Motta and sodium pentobarbital (5 mg/100 g body weight) Porter, 1974; Motta, 1975; Grisham et al., i.p. and laparotomy was performed to expose the cava between the liver and the 1975; Nopanitaya and Grisham, 1975). inferior vena To diaphragm. produice a consistent partial The cytoplasm of sinusoidal endothelial occluision of the vena cava in the experimental cells is penetrated by a large number of group of rats, a probe 2 mm in diameter was pores and fenestrations, allowing direct placed parallel to the vena cava and a 4-0 suture access of the fluid contents of the blood to was slipped around both the probe and vena juist above the entrance of the hepatic the sinusoidal surface of underlying cava veins. The suture was tied and the hepatocytes. The differential physiologic probe was removed to securely partially restore the functions of endothelial pores and fenes- lumen- of the vena cava. Sham-operated rats trations, if any, is not clear. This study (controls) underwent laparotomy and the vena was manipulated buit the vessel was not represents an attempt to assess the effect cava of experimentally altering the character- occluided. Thiree animals from both control and istics of blood flow and the contents of experimental grouips were killed 12, 24, 48 and blood on the ultrastructure of sinusoidal 72 h after operation. At each interval, the endothelium. The effects on the ultra- intrahepatic sinusoidal blood pressure was by the measurement of splenic structure of endothelial pores and fenes- estimated sinuisoidal pressure throuigh a 20-gauge needle trations when hepatic venous outflow is iniserted iinto the splenic pulp under direct experimentally impeded are described. vision. The splenic sinusoidal pressure was * Supporte(d by Grant VC595 from the UNC Res('arch Council, training grant GM\ 92 from the National

Institute of General Meolical Sciences, an(d research grant AML 17595 from the National Institute of Arthritis, Metabolic and Digestive Diseases.

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605

FIG. 1. Liver of a sham-operated rat showing normal appearance of sinusoids and terminal hepatic vein (V). Haematoxylin and eosin. x 125. FIG. 2. Liver from rat in which the vena cava was partially occluded for 48 h. The terminal hepatic veini (V) and its surrounding sinusoids are greatly dilated. Haematoxylin and eosin. x 125.

recorded on a Grass Model 7C polygraph employing a Statham Model P23DC transducer. Livers from all animals were fixed by perfusion with 4% phosphate-buffered paraformaldehyde at pH 7-3. After fixation, pieces of liver were manually fractured and treated for SEM by a technique described by Grisham et al. (1975). Specimens of the liver were also prepared for light microscopy and transmission electron microscopy by conventional techniques. SEM and TEM study of endothelium concentrated on the distal one-half of sinusoids, since this region is normally populated by pores alone (Grisham et al., 1975). RESULTS

The intrasplenic pressure of 3 sham-

operated rats was 20-0 ± 1-0 mm Hg, while that of 12 rats whose suprahepatic vena cavae had been partially obstructed was 248 ± 0-1 mm Hg. This difference was statistically significant (P < 0-001). The intrasplenic pressure did not vary between 12 and 72 h following partial occlusion of the vena cava.

Prior to perfusion, livers of all experimental animals, in contrast to livers of sham-operated rats, appeared finely reticulated through the capsule with alternating dark red and pale areas. Spleens of treated animals were enlarged and congested. Light microscopic examination of histologic sections of livers stained with haematoxylin and eosin demonstrated that terminal hepatic veins and the surrounding sinusoids were greatly dilated (Fig. 1, 2). Hepatic plates in the region of terminal hepatic veins were markedly thinned, and focally some hepatocytes in these areas appeared to be dead. Occasional focal accumulations of polymorphonuclear leucocytes signalled the location of necrotic hepatocytes. These pathological alterations were somewhat less pronounced in rats killed 12 h after partial occlusion of the vena cava than in the animals killed at later intervals, but no difference could be discerned

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W. NOPANITAYA, J. C. LAMB, J. W. GRISHAM AND J. L. CARSON

FIG. 3. Surface structure of hepatic parenchyma from a sham-operated rat illustrating normal features of sinusoids (S) bordering hepatic plates (HP) that are the width of single hepatocytes. Scaining electron photo-micrograph. x 1,857. FIG. 4. Following 48 h of hepatic venous obstruction sinusoids (S) are greatly dilated and hepatic plates (HP) are narrowed. Scanning electron micrograph. x 1,875.

between livers from animals killed at any of the later time intervals. Regardless of the postoperative interval, SEM demonstrated that hepatic sinusoids of experimental animals were

greatly dilated, measuring up to 150 ,um in diameter (normal: 20-40 1tm) (Fig. 3, 4) Sieve plates composed of 10 to 50 pores, each less than 0.1 ,pm in diameter, were prominent in endothelium in efferent

EFFECT OF HEPATIC VENOUS OUTFLOW OBSTRUCTION

FIG. 5.-Pores and fenestrations in a sinusoidal endothelial cell from the liver of a sham-operated rat. A few large fenestrations (F) are randomly interspersed among smaller pores which are grouped into sieve plates. Scanning electron micrograph. x 6,750. FIG. 6.-Enlarged fenestrations (F) in an endothelial cell from a rat liver 48 h following partial occlusion of the vena cava. Microvilli on the sinusoidal surface of the underlying hepatocyte are visible through these large fenestrations. Pores and sieve plates are not present. Scanning electron micrograph. x 6,750. FIG. 7.-Ultrastructure of portions of the adjacent sinusoidal endothelial cell and an hepatocyte 48 h after partial occlusion of the inferior vena cava. Large fenestrations (open arrow) in the sinusoidal endothelial cell are apparent. Invaginations of plasma membrane (*) into the cytoplasm of the hepatocyte produce the appearance of vacuoles on cross-section. Transmission electron micrograph. x 15,000.

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W. NOPANITAYA, J. C. LAMB, J. W. GRISHAM AND J. L. CARSON

halves of sinusoids of sham-operated rats, but large fenestrations (about 1-0 jtm) were sparse (Fig. 5). Obstruction of the vena cava, even as soon as 12 h after surgery, was associated with the occurrence of large fenestrations 2 to 5 ,tm in diameter in endothelium in the efferent halves of sinusoids, with a marked reduction or absence of pores and sieve plates (Fig. 6). Through the large fenestrae the sinusoidal surfaces of underlying hepatocytes were visible (Fig. 6). During the next 60 h after surgery these ultrastructural alterations did not vary greatly. TEM of thin sections corroborated these changes in sinusoidal endothelium. Wide gaps separated adjacent profiles of sectioned endothelium (Fig. 7). Additionally, the underlying hepatocytes showed deep invaginations of the surface membrane, which in crosssection appeared as intracellular vacuoles (Fig. 7). Hepatocellular organelles did not appear to be altered markedly, but our examination of them was not detailed. DISCUSSION

Pores (or small fenestrations) that measure uniformly less than 0-1 ,tm in diameter are present in endothelial cells throughout the entire length of sinusoids of normal rat liver; although some pores occur singly, they are more characteristically aggregated into groups of 10 to 50 or more. Aggregates of pores have been termed sieve plates (Wisse, 1970) and each sieve plate measures about 0 5 to 2 0 ,tm in diameter. Large fenestrations measuring from 1-0 to 2 0 pm in diameter have also been described in sinusoidal endothelium of normal rat liver (Orci et al., 1971). Under normal circumstances these large fenestrations appear to be concentrated in the periportal region of hepatic sinusoids (Grisham et al., 1975; Nopanitaya and Grisham, 1975). Neither pores nor fenestrations are closed by a membrane, and the sinusoidal surfaces of underlying hepatocytes are visible through them. Pores and large fenestrations may be

interrelated. Because sieve plates and large fenestrations occupy about the same area, we have suggested that fenestrations may be formed when all of the pores in a sieve plate coalesce (Nopanitaya and Grisham, 1975). The study reported in this paper represents an initial attempt to investigate this potential relationship and, more importantly, to develop insights into the pathophysiologic functions of pores and fenestrations. This study indicates that obstruction to hepatic venous outflow is associated with a marked change in the distribution of pores and fenestrations in sinusoidal endothelium of rat liver. The degree of occlusion of the suprahepatic inferior vena cava obtained was sufficient to cause severe hepatic sinusoidal congestion, intrahepatic and portal hypertension (as reflected by a significant elevation of intrasplenic pressure) (Yamamoto et al., 1968; Iber, 1970) and, most likely, intrahepatic hypoxia caused by stagnation of hepatic blood flow. This occurrence of hepatocytic vacuoles, produced by invaginations of the sinusoidal plasma membranes of these cells, suggests that hepatocytes were hypoxic (Brewer and Heath, 1965; Schaffner, 1970). Within as short a period as 12 h following partial occlusion of the vena cava, pores had almost completely disappeared from sinusoidal endothelial cells and they were replaced by large fenestrations. These observations are consistent with our previous hypothesis that fenestrations may represent sieve plates in which the pores have fused or coalesced (Nopanitaya and Grisham, 1975). The physiologic role of endothelial pores is unclear. Wisse (1970) has hypothesized that pores filter particles suspended in blood and determine their ability to enter or leave the sinusoidal lumen. Specifically, he speculates that they may control the exchange of chylomicrons and lipo-proteins, for example, between blood and hepatocyte and vice versa. The function of large fenestrations is even more obscure. Their

EFFECT OF HEPATIC VENOUS OUTFLOW OBSTRUCTION

size is such that they allow free access to the sinusoidal surfaces of hepatocytes of anything in blood other than whole leucocytes and erythrocytes. The concentration of large fenestrations at the portal ends of sinusoids under normal conditions suggests that their distribution may be related to some property determined by blood flow. This opinion is reinforced by our findings that pathological modification of hepatic blood flow led to drastic changes in the distribution and character of sinusoidal pores and fenestrations. These results did not allow us to determine whether hypertension or hypoxia, singly or together, were responsible for the alterations to endothelial cells. The prominent occurrence of large fenestrations at portal ends of sinusoids under normal conditions, where the blood is both better oxygenated and under higher pressure, suggests that hypoxia is not essential; similarly the near absence of large fenestrations at efferent ends of sinusoids where oxygenation of blood is poorer supports this opinion. Further experimental studies will be required to elucidate the precise relationship between hypertension, hypoxia, and other factors and the character and distribution of hepatic endothelial pores and fenestrations. At least, this study demonstrates the dynamic nature of pores and fenestrations and indicates that their occurrence and frequency are subject to environmental modification.

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REFERENCES BROOKS, S. E. H. & HAGGIS, G. H. (1973) Scanning Electron Microscopy of Rat's Liver. Application of Freeze-fracture and Freeze-drying Techniques. Lab. Invest., 29, 60. BREWER, D. B. & HEATH, D. (1965) Electron Microscopy of Anoxic Vacuolation in the Liver and its Comparison with Sucrose Vacuolation. J. Path. Bact., 90, 437. BRUNI, C. & PORTER, K. R. (1965) The Fine Structure of the Parenchymal Cells of the Normal Rat Liver. I. General Observations. Am. J. Path., 56, 691. FAWCETT, D. W. (1955) Observation of the Cytology and Electron Microscopy of Hepatic Cells. J. natn. Cancer Inst., 15, 1457. GRISHAM, J. W., NOPANITAYA, W., COMPAGNO, J. & NAGEL, A. E. H. (1975) Scanning Electron Microscopy of Normal Rat Liver. The Surface Structure of its Cells and Tissue Components. Am. J. Anat., 144, 295. IBER, F. (1970) Portal Hypertension in the Presence of Normal Morphology. Ann. N. Y. Acad. Sci., 170, 115. MOTTA, P. (1975) A Scanning Electron Microscopic Study of the Rat Liver Sinusoid. Endothelial and Kupffer Cells. Cell Tiss. Res., 164, 371. MOTTA, P. & PORTER, K. R. (1974) Structure of Rat Liver Sinusoids and Associated Tissue Spaces as Revealed by Scanning Electron Microscopy. Cell Tiss. Res., 148, 111. NOPANITAYA, W. & GRISHAM, J. W. (1975) Scanning Electron Microscopy of Mouse Intrahepatic Structures. Exp. mol. Path., 23, 441. OGAWA, K., MINASE, T., ENOMOTO, K. & ONOE, T. (1973) Ultrastructure of Fenestrated Cells in the Sinusoidal Wall of Rat Liver after Perfusion Fixation. Tohoku J. exp. Med., 110, 89. ORCI, L., MATTER, A. & ROUILLER, C. (1971) A Comparative Study of Freeze-etch Replicas and Thin Sections of Rat Liver. J. Ultrastruc. Res., 35, 1. SCHAFFNER, F. (1970) Oxygen Supply and Hepatocyte. Ann. N. Y. Acad. Sci., 170, 67. WISSE, E. (1970) An Electron Microscopic Study of the Fenestrated Lining of Rat Liver Sinusoids. J. Ultrastruct. Res., 31, 125. YAMAMOTO, S., YOKOYAMA, Y., TAKESHIGE, K. & IWATSUKI, K. (1968) Budd-Chiari Syndrome with Obstruction of the Inferior Vena Cava. G(astroenterol., 54, 1070.

Effect of hepatic venous outflow obstruction on pores and fenestration in sinusoidal endothelium.

The ultrastructure of pores and fenestrations in hepatic sinusoidal endothelial cells was examined following partial surgical occlusion of the suprahe...
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