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The development of hepatic microvascular heterogeneity after birth, and its temporal relationship to the development of parenchymal cell plates have received little attention. As a result, the morphogenesis of some of the parameters contributing to this heterogeneity in suckling and weaned rats was studied as a function of time between postpartum days 4 and 30 using in vivo light microscopic, electron microscopic, and immunocytochemical methods. During the early suckling period, the sinusoid network is highly anastomotic, with little evidence of zonation, and the parenchymal cell plates contain multiple cells and are irregularly arranged throughout the lobule. Sinusoidal endothelial fenestration is sparse at 4 days, but phagocytic Kupffer cell (KC) function already exists and exhibits zonal heterogeneity, with more cells located in the periportal zone. With increasing age, endothelial fenestrae increase and organize as sieve plates. Widened centrilobular radial sinusoids form through a loss (“drop-out”) of intersinusoidal sinusoids (ISS). Concomitantly, the associated cell plates straighten and become one cell thick. Hepatocyte DNA synthesis and mitosis are higher in the periportal zone, which retains thickened cell plates and anastomotic sinusoids. The centrilobular sinusoids may widen to accommodate the increased volume of blood that results from the loss of ISS as well as the increased numbers of periportal sinusoids containing flow that feed these vessels. KC phagocytic activity increases during the suckling period concomitant with an increase of gut-derived endotoxin in the portal blood, which suggests that the KCs may be releasing mediators that affect sinusoid diameter, blood flow, endothelial fenestration, and perhaps parenchymal growth either directly or through the stimulation of growth factors. Anat Rec Part A 275A:1019–1030, 2003. © 2003 Wiley-Liss, Inc.

This study examined the role of decreased nitric oxide (NO) in the microcirculatory obstruction of hepatic sinusoidal obstruction syndrome (SOS). SOS was induced in rats with monocrotaline. Monocrotaline caused hepatic vein NO to decrease by 30% at 24 hours and by 70% at 72 hours; this decrease persisted throughout late SOS. N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, exacerbated monocrotaline toxicity, whereas V-PYRRO/NO, a liver-selective NO donor prodrug, restored NO levels, preserved sinusoidal endothelial cell (SEC) integrity and sinusoidal perfusion as assessed by in vivo microscopy and electron microscopy, and prevented clinical and histologic evidence of SOS. NO production in vitro by SEC and Kupffer cells, the 2 major liver cell sources of NO, decreases largely in parallel with loss of cell viability after exposure to monocrotaline. Increased matrix metalloproteinase (MMP) activity increases early on in SOS and this increase in activity has been implicated in initiating SOS. Infusion of V-PYRRO-NO prevented the monocrotaline-induced increase in MMP-9. In conclusion, decreased hepatic NO production contributes to the development of SOS. Infusion of an NO donor preserves SEC integrity and prevents development of SOS. These findings show that a decrease in NO contributes to SOS by allowing up-regulation of MMP activity, loss of sinusoidal integrity, and subsequent disruption of sinusoidal perfusion.

The present study was conducted to elucidate the sequential alterations in the hepatic microvascular inflammatory response to extrahepatic biliary obstruction.: The hepatic microvasculature in anesthetized Sprague-Dawley rats was studied by in vivo microscopy 3, 7, and 14 days after bile duct ligation (BDL) or sham operation.: The numbers of adhering leukocytes and swollen sinusoidal endothelial cells were significantly increased at 3, 7, and 14 days after BDL when compared with sham-operated controls. Concomitantly, the numbers of sinusoids containing blood flow were significantly and progressively decreased by up to 30%. The phagocytic activity of hepatic macrophages was significantly elevated during the development of biliary cholestasis. In particular, centrilobular phagocytosis at 14 days after BDL was significantly increased 1.4- to 2.0-fold when compared with that at 3 and 7 days after BDL. Electron microscopy also revealed evidence of activated Kupffer cells reflected by numerous filopodia and ruffles.: These results suggest that hepatic microcirculatory dysfunction subsequent to BDL contributes to cholestatic liver injury. Microcirculation (2003) 10, 421-432. doi:10.1038/sj.mn.7800208

Mechanisms leading to the obstruction of the microcirculation in sinusoidal obstruction syndrome (SOS) have been unclear. Because this occurs at the onset of disease, this is a potential key target for therapeutic intervention. Rats were treated with monocrotaline with or without continuous intraportal infusion of glutathione and were studied at 0.5, 1, 2, 4, 6, and 10 days after monocrotaline treatment with the use of in vivo microscopy and transmission electron microscopy. Sinusoidal perfusion decreased from days 1 through 10 with a nadir on day 4. At 12 h, numerous swollen sinusoidal endothelial cells (SECs) were observed. Subsequently, red blood cells penetrated into the space of Disse through gaps between and through swollen SEC and dissected the sinusoidal lining away from the parenchymal cells. Sinusoidal blood flow was obstructed by an embolism of aggregates of sinusoidal lining cells, red blood cells, and adherent monocytes. All changes were prevented by glutathione infusion, notably the initial swelling of SEC. SOS is initiated by changes in SEC. Microcirculatory obstruction is due to dissection of the sinusoidal lining, followed by embolization of the sinusoid by sinusoidal lining cells, compounded by aggregates of monocytes adherent in the sinusoids. Glutathione prevents SOS by preserving an intact sinusoidal barrier.

Mammalian females are born with a finite number of ovarian oocytes, the vast majority of which ultimately undergo degeneration by atresia. The overall process of ovarian follicular atresia has been morphologically well described only in large antral follicles. Additionally, little attention has been focused on ultrastructural changes in the oocyte. Furthermore, most such morphological studies were performed prior to identification of apoptosis as a mechanism of physiological cell death. Therefore, the purpose of this study was to use electron microscopy to compare the process of atretic oocyte degradation in ovarian follicles of female Fischer 344 rats (38 days old) with ultrastructural characteristics of apoptosis. Examination of ovarian follicles revealed that nucleolar segregation, cytoplasmic or nuclear condensation, apoptotic body formation, and chromatin margination along the nuclear membrane are never observed in atretic oocytes during the degenerative process. Instead, early morphological changes in atretic oocytes include retraction of granulosa cell- and oocyte-derived microvilli and condensation of mitochondria and loss of cristae. These occurrences coincide with initiation of granulosa cell apoptosis. After most granulosa cells are lost, more severe changes occur, including segmentation of the oocyte and cytoplasmic vacuolization as atresia progresses. Thus, these results suggest that, during atresia, oocytes are removed by physiological oocyte cell death, a method that does not involve classically described apoptosis.

Lack of a reproducible animal model has hampered progress in understanding hepatic veno-occlusive disease (HVOD). This article characterizes a reproducible model of HVOD. Rats gavaged with monocrotaline, 160 mg/kg, were killed between days 1 and 10. Sections were evaluated by light microscopy with a standardized scoring system, by immunoperoxidase staining with ED-1 (monocytes, macrophages) and ED-2 (Kupffer cells) antibodies, and by transmission (TEM) and scanning electron microscopy (SEM). On days 1 and 2, the earliest manifestations were progressive injury to the sinusoidal wall with loss of sinusoidal lining cells, sinusoidal hemorrhage, and mild damage to central vein (CV) endothelium. On days 3 through 5 ("early HVOD"), there was centrilobular coagulative necrosis, severe injury to sinusoids, severe sinusoidal hemorrhage, and severe CV endothelial damage; inflammation with ED-1-positive cells was most marked on these days. Days 6 and 7 ("late HVOD") were characterized by subendothelial and advential fibrosis of CVs, damage of the CV endothelium with subendothelial hemorrhage, and some restoration of the sinusoidal wall. Between days 8 and 10, sections showed interindividual variation ranging from mild, residual fibrosis to severe, late HVOD. From days 1 through 10, ED-2-positive cells were decreased in number, and the number of ED-1-positive cells was increased. Sinusoidal damage is the earliest change in HVOD. Coagulative necrosis follows sinusoidal injury and resolves with improvement in sinusoidal endothelial cell (SEC) morphology. Moderate-to-severe CV fibrosis occurs after reappearance of sinusoidal lining cells and resolution of hepatocyte necrosis. The inflammatory response within the lobule and CVs is a result of recruitment of monocytes, whereas Kupffer cells are decreased in number.

Interleukin-1 (IL-1), which is produced by Kupffer cells and sinusoidal endothelial cells, may play an important role in immunological and microvascular responses to a variety of stimuli in the liver. The responses of the hepatic microvasculature including phagocytic activity of sinusoidal lining cells to IL-1 alpha were examined in C57Bl/6 mice in vivo and using electron microscopy. One hour after recombinant mouse IL-1 alpha was injected at doses of 80 U, the low dose group, and 800 U, the high dose group, the phagocytic activity of sinusoidal lining cells showed significant differences between the two treated groups and between the two groups and the controls. In the low dose group, the numbers of sinusoids containing blood flow and of leukocytes adhering to the sinusoidal lining remained unchanged, but the former decreased and the latter increased significantly in the high dose group. Ultrastructurally, Kupffer cells that phagocytosed latex particles appeared to have decreased in number while the sinusoidal endothelial cells became phagocytic. A considerable number of leukocytes were seen adhering to the sinusoidal endothelium. These findings demonstrate that IL-1 alpha not only elicited sticking and plugging by leukocytes in sinusoids but also activated phagocytic functions in the hepatic sinusoidal endothelial cells. These endothelial responses are similar to those seen following FV3 virus infection, chronic administration of ethanol, or a combination of cocaine and ethanol, or during extracorporal perfusion, suggesting that IL-1 may participate in these responses.

The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

Neutrophil extravasation from sinusoids is a critical step for acute inflammatory tissue injury. However, the role of sinusoidal endothelial cells (SECs) in this process remains unclear. Matrix metalloproteinases (MMPs) have been shown to involve gap formation in SECs in several liver diseases. Therefore, the present study examined SEC modifications elicited by galactosamine (Gal)/endotoxin (ET). Treatment of male C3Heb/FeJ mice with Gal/ET or Gal/TNF caused the formation of numerous gaps in SECs at 4 h when no neutrophil extravasation occurred. Six hours after Gal/ET or Gal/TNF treatment, blood elements started to penetrate to the extrasinusoidal space through large gaps. Treatment with ET alone caused sinusoidal neutrophil accumulation but no gap formation, neutrophil extravasation, or hemorrhage. Gal/ET treatment increased hepatic MMP-2 and MMP-9 mRNA expression (6.7- and 11-fold, respectively). Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (5 mg/kg), minimized gap formation after Gal/ET and Gal/TNF treatment. The MMP inhibitor reduced injury only in the Gal/ET model mainly due to reduced TNF formation. The MMP inhibitor attenuated sinusoidal neutrophil accumulation at 6 h but failed to attenuate Gal/TNF-induced liver injury at 7 h due to excessive apoptosis. These results suggest that Gal/ET or Gal/TNF activates MMPs, which are responsible for SEC gap formation. Although the initial appearance of gap formation is independent of neutrophils, the gaps allow initial contact of neutrophils with damaged hepatocytes. In addition, MMP activation promotes neutrophil accumulation in sinusoids.

Objective: To determine whether hepatic steatosis is susceptible to acetaminophen (APAP) hepatotoxicity. Methods: Male C57Bl/6 mice were fed a “Western-style’’ diet (high fat and high carbohydrate) for 4 months to develop severe hepatic steatosis with mild increases in alanine aminotransferase (ALT) levels. These were compared to mice fed a standard chow diet. Results: Treatment with APAP (300 mg/kg, orally) to mice fed a regular chow increased ALT levels (519-fold) and caused hepatic centrilobular injury at 6 h. APAP increased hepatic cytochrome-P (CYP)-2E1 mRNA levels (17-fold). In vivo microscopic studies showed that APAP caused a 30% decrease in sinusoidal perfusion and the infiltration of red blood cells into the space of Disse. Electron microscopy demonstrated that numerous gaps were formed in sinusoidal endothelial cells. Mice fed the “Western-style’’ diet were protected from APAP hepatotoxicity as evidenced by 89% decrease in ALT levels and less centrilobular injury, which was associated with 42% decrease in CYP2E1 mRNA levels. The APAP-induced liver microcirculatory dysfunction was minimized in mice fed the “Western-style’’ diet. Conclusions: These results suggest that hepatic steatosis elicited by the “Western-style’’ diet attenuated APAP-induced hepatotoxicity by inhibiting CYP2E1 induction and by minimizing sinusoidal endothelial cell injury, leading to protection of liver microcirculation. Microcirculation (2006) 13, 19‐27. doi:10.1080/10739680500383423

In alcoholic steatohepatitis, hepatic microvascular changes have pathogenic significance for hepatocellular function, perisinusoidal fibrosis, and portal hypertension. It is unclear whether similar changes occur in other forms of steatohepatitis. We therefore examined whether hepatic microvascular dysfunction occurs in fibrosing steatohepatitis induced by feeding mice a high-fat methionine- and choline-deficient (MCD) diet. Using in vivo microscopic--as well as histological and electron microscopic--methods, together with measurements of alanine aminotransferase (ALT), lipid content, and oxidative stress, hepatic microvascular structure and function were studied in relation to inflammatory and fibrotic changes during evolution of steatohepatitis. At 3 weeks of MCD diet intake, serum ALT was elevated and hepatic steatosis was pronounced. By 5 weeks, necroinflammatory change was noteworthy, and by 8 weeks perisinusoidal fibrosis was established. Compared with mice receiving the high-fat diet supplemented with methionine and choline (controls), levels of hepatic lipid and lipoperoxides were elevated at 3 weeks and beyond. The numbers of perfused sinusoids were significantly reduced at each time point. Enlarged, fat-laden hepatocytes together with perivascular fibrosis narrowed sinusoidal lumens, making vessels tortuous and impairing sinusoidal perfusion. At 3 and 5 weeks, MCD diet caused significant increases in phagocytic activity of macrophages in centrilobular regions. By 8 weeks, macrophage activity was less striking, but the number of leukocytes adherent to the sinusoidal lining had increased 5-fold compared with controls. In conclusion, these results are consistent with a dysfunctional hepatic microvasculature. Thus, microvascular changes may contribute to progressive liver injury in metabolic and toxic forms of steatohepatitis.

Background/Aims The pathophysiology of binge drinking of ethanol and its potentiation of acetaminophen (APAP) toxicity has received very little attention. To evaluate if ethanol binging sensitizes hepatic sinusoidal endothelial cells (SEC) and liver to APAP toxicity. Methods The histopathological responses to APAP were evaluated in the livers of mice gavaged with APAP alone, following a single, week-end type ethanol binge (4g/kg every 12h×5 doses) or three weekly binges. Results Six hours after APAP, 600mg/kg elicited severe centrilobular necrosis together with hemorrhagic congestion and infiltration of erythrocytes into the Space of Disse through large gaps that had formed in SEC. There was no evidence of parenchymal injury at 2h, but gaps already were formed through the cytoplasm of the SEC by coalescence of fenestrae. A single binge followed by 300mg/kg APAP elicited SEC and parenchymal injury equivalent to 600mg/kg APAP alone at 2 and 6h. The responses were exacerbated following three binges. Lower glutathione levels in the liver were shown in ethanol-binged animals. Conclusions Ethanol binging increases APAP hepatotoxicity. SEC are an early target for APAP-induced injury and ethanol binging enhances the SEC injury prior to evidence of parenchymal cell injury.

The phenotypic features of liver sinusoidal endothelial cells (SEC), open fenestrae in sieve plates and lack of a basement membrane, are lost with capillarization. The current study examines localization of CD31 as a marker for the dedifferentiated, nonfenestrated SEC and examines regulation of SEC phenotype in vitro. CD31 localization in SEC was examined by confocal microscopy and immunogold-scanning electron microscopy. SEC cultured for 1 day express CD31 in the cytoplasm, whereas after 3 days, CD31 is also expressed on cell-cell junctions. Immunogold-scanning electron microscopy confirmed the absence of CD31 surface expression on fenestrated SEC 1 day after isolation and demonstrated the appearance of CD31 surface expression on SEC that had lost fenestration after 3 days in culture. SEC isolated from fibrotic liver do show increased expression of CD31 on the cell surface. Coculture with either hepatocytes or stellate cells prevents CD31 surface expression, and this effect does not require heterotypic contact. The paracrine effect of hepatocytes or stellate cells on SEC phenotype is abolished with anti-VEGF antibody and is reproduced by addition of VEGF to SEC cultured alone. VEGF stimulates SEC production of nitric oxide. NG-nitro-l-arginine methyl ester blocked the paracrine effect of hepatocytes or stellate cells on SEC phenotype and blocked the ability of VEGF to preserve the phenotype of SEC cultured alone. In conclusion, surface expression of CD31 is a marker of a dedifferentiated, nonfenestrated SEC. The VEGF-mediated paracrine effect of hepatocytes or stellate cells on maintenance of SEC phenotype requires autocrine production of nitric oxide by SEC.

4-Vinylcyclohexene diepoxide (VCD) destroys preantral ovarian follicles in rats. Female 28-day Fisher 344 (F344) rats were dosed (30 days) with VCD (80 mg/kg per day, i.p.) or vehicle, and animals were evaluated for reproductive function at subsequent time points for up to 360 days. At each time point animals were killed, and ovaries and plasma collected. VCD reduced (P

Dimethyl sulfoxide (DMSO) protects against liver injury elicited by chloroform (CHCl(3)) in rats even when given 24 h after the toxicant. Aminobenzotriazole (ABT) is a cytochrome P-450 inhibitor as is DMSO. The present study was conducted to examine the effect of DMSO or ABT on the hepatic microvascular inflammatory response to CHCl(3) using in vivo microscopy. Rats received 0.75 ml/kg CHCl(3) by oral administration followed 24 h later by either 2.0 ml/kg DMSO, 30 mg/kg ABT, or saline injected intraperitoneally. Untreated animals served as controls. At 28 h after CHCl(3) administration, the number of leukocytes adhering to the sinusoidal wall was significantly increased associated with a reduction in the numbers of perfused sinusoids. DMSO, but not ABT, caused a restoration of perfused sinusoids to 85% of normal. At 32 h after, leukocyte adhesion and Kupffer cell phagocytic activity were significantly increased. The numbers of perfused sinusoids were decreased by 32% in periportal regions and 50% in centrilobular regions. Sinusoidal perfusion and Kupffer cell activity were modified significantly by either DMSO or ABT, while leukocyte adhesion was reduced by DMSO alone. The results suggest that attenuation of the hepatic microvascular response to CHCl(3) is a potential mechanism of post-treatment with DMSO.