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 supra- hepatic 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 laboratory chow and tap water ad libitum for at and Porter, 1965; Wisse, 1970; Orci, 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 1975; Nopanitaya and Grisham, 1975). inferior vena cava between the liver and the diaphragm. To 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 cava juist above the entrance of the hepatic the sinusoidal surface of underlying veins. The suture was securely tied and the hepatocytes. The differential physiologic probe was removed to 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 represents an attempt to assess the effect cava was manipulated buit the vessel was not occluided. of experimentally altering the character- 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 and estimated by the measurement of splenic structure of endothelial pores fenes- 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. EFFECT OF HEPATIC VENOUS OUTFLOW OBSTRUCTION 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 Prior to perfusion, livers of all experi- employing a Statham Model P23DC transducer. mental animals, in contrast to livers of Livers from all animals were fixed by perfusion with 4% phosphate-buffered para- sham-operated rats, appeared finely formaldehyde at pH 7-3. After fixation, pieces reticulated through the capsule with of liver were manually fractured and treated for alternating dark red and pale areas. SEM by a technique described by Grisham et al. Spleens of treated animals were enlarged (1975). Specimens of the liver were also Light microscopic prepared for light microscopy and transmission and congested. electron microscopy by conventional tech- examination of histologic sections of niques. SEM and TEM study of endothelium livers stained with haematoxylin and concentrated on the distal one-half of sinusoids, eosin demonstrated that terminal hepatic since this region is normally populated by pores veins and the surrounding sinusoids were alone (Grisham et al., 1975). greatly dilated (Fig. 1, 2). Hepatic plates in the region of terminal hepatic veins RESULTS were markedly thinned, and focally some The intrasplenic pressure of 3 sham- hepatocytes in these areas appeared to be operated rats was 20-0 ± 1-0 mm Hg, dead. Occasional focal accumulations of while that of 12 rats whose suprahepatic polymorphonuclear leucocytes signalled vena cavae had been partially obstructed the location of necrotic hepatocytes. was 248 ± 0-1 mm Hg. This difference These pathological alterations were some- was statistically significant (P < 0-001). what less pronounced in rats killed 12 h The intrasplenic pressure did not vary after partial occlusion of the vena cava between 12 and 72 h following partial than in the animals killed at later intervals, occlusion of the vena cava. but no difference could be discerned 606 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 greatly dilated, measuring up to 150 ,um of the later time intervals. in diameter (normal: 20-40 1tm) (Fig. 3, 4) Regardless of the postoperative inter- Sieve plates composed of 10 to 50 pores, val, SEM demonstrated that hepatic each less than 0.1 ,pm in diameter, were sinusoids of experimental animals were prominent in endothelium in efferent EFFECT OF HEPATIC VENOUS OUTFLOW OBSTRUCTION 607

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 micro- graph. x 15,000. 608 W. NOPANITAYA, J. C. LAMB, J. W. GRISHAM AND J. L. CARSON halves of sinusoids of sham-operated rats, interrelated. Because sieve plates and but large fenestrations (about 1-0 jtm) large fenestrations occupy about the same were sparse (Fig. 5). Obstruction of the area, we have suggested that fenestrations vena cava, even as soon as 12 h after may be formed when all of the pores in a surgery, was associated with the occur- sieve plate coalesce (Nopanitaya and rence of large fenestrations 2 to 5 ,tm in Grisham, 1975). The study reported in diameter in endothelium in the efferent this paper represents an initial attempt to halves of sinusoids, with a marked investigate this potential relationship and, reduction or absence of pores and sieve more importantly, to develop insights into plates (Fig. 6). Through the large the pathophysiologic functions of pores fenestrae the sinusoidal surfaces of under- and fenestrations. lying hepatocytes were visible (Fig. 6). This study indicates that obstruction During the next 60 h after surgery these to hepatic venous outflow is associated ultrastructural alterations did not vary with a marked change in the distribution greatly. TEM of thin sections corro- of pores and fenestrations in sinusoidal borated these changes in sinusoidal endothelium of rat liver. The degree of endothelium. Wide gaps separated occlusion of the suprahepatic inferior vena adjacent profiles of sectioned endothelium cava obtained was sufficient to cause (Fig. 7). Additionally, the underlying severe hepatic sinusoidal congestion, intra- hepatocytes showed deep invaginations of hepatic and portal hypertension (as the surface membrane, which in cross- reflected by a significant elevation of section appeared as intracellular vacuoles intrasplenic pressure) (Yamamoto et al., (Fig. 7). Hepatocellular organelles did 1968; Iber, 1970) and, most likely, not appear to be altered markedly, but intrahepatic hypoxia caused by stagnation our examination of them was not detailed. of hepatic blood flow. This occurrence of hepatocytic vacuoles, produced by invaginations of the sinusoidal plasma DISCUSSION membranes of these cells, suggests that Pores (or small fenestrations) that hepatocytes were hypoxic (Brewer and measure uniformly less than 0-1 ,tm in Heath, 1965; Schaffner, 1970). Within diameter are present in endothelial cells as short a period as 12 h following partial throughout the entire length of sinusoids occlusion of the vena cava, pores had of normal rat liver; although some pores almost completely disappeared from sinu- occur singly, they are more characteristic- soidal endothelial cells and they were ally aggregated into groups of 10 to 50 or replaced by large fenestrations. These more. Aggregates of pores have been observations are consistent with our termed sieve plates (Wisse, 1970) and each previous hypothesis that fenestrations sieve plate measures about 0 5 to 2 0 ,tm may represent sieve plates in which the in diameter. Large fenestrations measur- pores have fused or coalesced (Nopanitaya ing from 1-0 to 2 0 pm in diameter have and Grisham, 1975). also been described in sinusoidal endo- The physiologic role of endothelial thelium of normal rat liver (Orci et al., pores is unclear. Wisse (1970) has hypo- 1971). Under normal circumstances these thesized that pores filter particles sus- large fenestrations appear to be concen- pended in blood and determine their trated in the periportal region of hepatic ability to enter or leave the sinusoidal sinusoids (Grisham et al., 1975; Nopanitaya lumen. Specifically, he speculates that and Grisham, 1975). Neither pores nor they may control the exchange of fenestrations are closed by a membrane, chylomicrons and lipo-proteins, for and the sinusoidal surfaces of underlying example, between blood and hepatocyte hepatocytes are visible through them. and vice versa. The function of large Pores and large fenestrations may be fenestrations is even more obscure. Their EFFECT OF HEPATIC VENOUS OUTFLOW OBSTRUCTION 609 size is such that they allow free access to REFERENCES the sinusoidal surfaces of hepatocytes of BROOKS, S. E. H. & HAGGIS, G. H. (1973) Scanning Electron Microscopy of Rat's Liver. Application anything in blood other than whole of Freeze-fracture and Freeze-drying Techniques. leucocytes and erythrocytes. The con- Lab. Invest., 29, 60. at the BREWER, D. B. & HEATH, D. (1965) Electron centration of large fenestrations Microscopy of Anoxic Vacuolation in the Liver portal ends of sinusoids under normal and its Comparison with Sucrose Vacuolation. conditions suggests that their distribution J. Path. Bact., 90, 437. BRUNI, C. & PORTER, K. R. (1965) The Fine Struc- may be related to some property deter- ture of the Parenchymal Cells of the Normal Rat mined by blood flow. This opinion is Liver. I. General Observations. Am. J. Path., reinforced by our findings that patho- 56, 691. FAWCETT, D. W. (1955) Observation of the Cytology logical modification of hepatic blood flow and Electron Microscopy of Hepatic Cells. J. led to drastic changes in the distribution natn. Cancer Inst., 15, 1457. pores and GRISHAM, J. W., NOPANITAYA, W., COMPAGNO, J. & and character of sinusoidal NAGEL, A. E. H. (1975) Scanning Electron fenestrations. Microscopy of Normal Rat Liver. The Surface These results did not allow us to Structure of its Cells and Tissue Components. Am. J. Anat., 144, 295. determine whether hypertension or IBER, F. (1970) Portal Hypertension in the Presence hypoxia, singly or together, were res- of Normal Morphology. Ann. N. Y. Acad. Sci., ponsible for the alterations to endothelial 170, 115. MOTTA, P. (1975) A Scanning Electron Microscopic cells. The prominent occurrence of large Study of the Rat Liver Sinusoid. Endothelial fenestrations at portal ends of sinusoids and Kupffer Cells. Cell Tiss. Res., 164, 371. under normal conditions, where the blood MOTTA, P. & PORTER, K. R. (1974) Structure of Rat Liver Sinusoids and Associated Tissue Spaces as is both better oxygenated and under Revealed by Scanning Electron Microscopy. higher pressure, suggests that hypoxia is Cell Tiss. Res., 148, 111. NOPANITAYA, W. & GRISHAM, J. W. (1975) Scanning not essential; similarly the near absence of Electron Microscopy of Mouse Intrahepatic large fenestrations at efferent ends of Structures. Exp. mol. Path., 23, 441. sinusoids where oxygenation of blood is OGAWA, K., MINASE, T., ENOMOTO, K. & ONOE, T. (1973) Ultrastructure of Fenestrated Cells in the poorer supports this opinion. Further Sinusoidal Wall of Rat Liver after Perfusion experimental studies will be required to Fixation. Tohoku J. exp. Med., 110, 89. elucidate the precise relationship between ORCI, L., MATTER, A. & ROUILLER, C. (1971) A Comparative Study of Freeze-etch Replicas and hypertension, hypoxia, and other factors Thin Sections of Rat Liver. J. Ultrastruc. Res., and the character and distribution of 35, 1. endothelial and fenes- SCHAFFNER, F. (1970) Oxygen Supply and Hepato- hepatic pores cyte. Ann. N. Y. Acad. Sci., 170, 67. trations. At least, this study demon- WISSE, E. (1970) An Electron Microscopic Study of strates the dynamic nature of pores and the Fenestrated Lining of Rat Liver Sinusoids. J. Ultrastruct. Res., 31, 125. fenestrations and indicates that their YAMAMOTO, S., YOKOYAMA, Y., TAKESHIGE, K. & occurrence and frequency are subject to IWATSUKI, K. (1968) Budd-Chiari Syndrome with environmental modification. Obstruction of the Inferior Vena Cava. G(astro- enterol., 54, 1070.