An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats

Abstract

Quantitative data defining the relationship between the hepatic microcirculation and the development of liver pathological changes could provide a basis for a better understanding of fibrogenic processes, such as cirrhosis. Therefore, we established the technique of intravital fluorescence microscopy and computer-assisted microcirculation analysis systems in developing cirrhosis in rats with the aim of quantitatively assessing the association of hepatic microvascular morphology with its disordered acinar architecture, and nonparenchymal cell transformation with collagen deposition, parenchymal cell loss, and liver dysfunction. In animals chronically exposed to carbon tetrachloride The pathological process of developing cirrhosis represents an almost inconceivably complex network of interacting cells, fibrogenic mediators, and extracellular matrix molecules (ECM). 1 Dramatic improvements in cell and molecular biological techniques now permit a detailed analysis of the composition, structure, immunology, and histological localization of the individual components of extracellular matrix. Dissection of the cellular and molecular pathobiology of ECM deposition includes the identification of the cellular sources of specific ECM components as well as the definition of humoral factors and regulatory loops underlying both the amplification and degradation pathways of ECM molecules during fibrogenesis. 2 Beside these molecular aspects of fibrogenesis, vascular modifications during the development of cirrhosis have fully been recognized and interpreted in that microvascular changes in the liver are not the mere consequence of fibrosis and nodule formation, but dominate the entire development of the pathological condition. 3 Microvascular corrosion casts observed by scanning electron microscopy have proved useful in demonstrating microvascular morphology in hepatic cirrhosis [4][5][6][7][8] ; however, these techniques are neither suited for the investigation of dynamic alterations in the microvascular function nor for the direct assessment of the close interrelation between the hepatic microcirculation and liver structure and function. Combining several applications of intravital fluorescence microscopy in a rat model of developing cirrhosis, the present study provides the first in vivo appreciation of the association of microvascular, cellular, and functional aspects during the process of hepatic organ remodeling. MATERIALS AND METHODSAnimal Model.

Real-time histology in liver disease using multiphoton microscopy with fluorescence lifetime imaging

“…In this study, we observed the stellate cell spatial distribution changed in CCl 4 induced fibrotic liver directly from MPM images, which is consistent with the observation of Vollmar et al using fluorescence microscopy [24]. It is reported that stellate cell accumulation in fibrous septa only occurred early after CCl 4 exposure (1 to 4 weeks) while significant loss of vitamin A storage in liver was found after prolonged periods of CCl 4 administration (8 to 12 weeks) [24]. We suggest that in pathological conditions such as liver fibrosis caused by chronic hepatic injury, the quiescent stellate cells first accumulate in centrilobular region around central veins and between postsinusoidal venules at the early liver injury stage.…”

“…However, little information has been revealed about the activity of these cells (such as distribution or proliferation) at the early liver injury stage. In this study, we observed the stellate cell spatial distribution changed in CCl 4 induced fibrotic liver directly from MPM images, which is consistent with the observation of Vollmar et al using fluorescence microscopy [24]. It is reported that stellate cell accumulation in fibrous septa only occurred early after CCl 4 exposure (1 to 4 weeks) while significant loss of vitamin A storage in liver was found after prolonged periods of CCl 4 administration (8 to 12 weeks) [24].…”

“…Spatial distribution of stellate cells (positive vitamin Aassociated autofluorescence) was assessed by densitometric recording of positive sites of fluorescence per frame (10 × magnification images) within the periportal, midzonal and centrilobular regions of liver lobules [24]. The area of stellate cells was calculated as percent of the whole liver area of the frame from twelve representative image fields.…”

Portal branch ligation induces a hepatic arterial buffer response, microvascular remodeling, normoxygenation, and cell proliferation in portal blood-deprived liver tissue

“…Data analysis was performed by examiners unaware of the treatment. Sinusoidal density (per millimeter) was determined within the midzonal region of the liver acinus as total number of visible sinusoids crossing a 500-m raster line (31). The functional sinusoidal density (per millimeter) was determined as the number of perfused sinusoids crossing the 500-m raster line.…”

Heparin and phentolamine combined, rather than heparin alone, improves hepatic microvascular procurement in a non-heart-beating donor rat-model

“…Sinusoidal density was determined by counting the number of HTK-solution perfused sinusoids crossing a 200 pm raster line [13]. Acinar perfusion was assessed quantitatively by planimetric analysis of the perfused area of liver tissue using a computer-assisted image analysis system (CapImage, Zeintl, Heidelberg, Germany).…”