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-