Pathological alterations in liver injury following congestive heart failure induced by volume overload in rats

Figures

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  Fig 1. Heart and lung morphological changes following ACF-induced congestive heart failure. Increased heart and lung weight indices (A, B, D) but not body weight (C) following ACF-induced congestive heart failure. Note, that heart and lung weight indices were significantly increased at 28 days of ACF rats (n = 10) compared to those of sham operated controls (n = 5) (heart index: ACF 6.5±1.6 versus Controls 3.9±0.2; lung index: ACF 6.9±1.4 versus Controls 3.8±0.3; p<0.01, Student t-test) (D). Data show means ± SD.

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  Fig 2. Light microscopic photographs of representative haematoxylin-eosin and toluidine blue stained liver sections. A) Note normal lobular structure with hepatocytes having prominent rounded nuclei. B-D) Liver sections of ACF rats show pyknotic nuclei (PN), cytoplasmic vacuolization, sinusoidal dilation (SD), leukocyte infiltration (Leu) massive breakdown of hepatocytes (*), and congestive blood vessels (double arrows) as well as a crescent-shaped condensation of nuclear chromatin (arrow). E) Semithin liver sections of control rats were stained in 1% toluidine blue showing normal branching and anastomosing hepatocyte cords separated by hepatic blood sinusoids. Note, hepatocytes contain prominent rounded nuclei (N). F) Semithin liver sections of ACF rats show hepatocytes with large vacuoles (arrow head) in the periportal area, heterogeneous parenchyma, which consists of dark and compact hepatocytes flanked by ballooned cells (also called apoptotic cells) (arrow) with nuclear degeneration (ND). Bars = 20 μm.

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  Fig 3. Light microscopic photographs of representative pan-leukocyte marker CD45 immunohistochemistry as well as Periodic acid-Schiff (PAS) and Oil Red O staining of liver sections. A-C) show leukocyte infiltrations as detected with pan-leukocyte marker CD45 (green fluorescence) with DAPI-counterstained nuclei (blue fluorescence) within the liver sinusoids. Quantification of CD45+ leukocyte positive cells/200 mm2 showed significantly more leukocytes in ACF rats (34.3±5.6) compared to controls (2.5±0.3)(p<0.05, Student t-test). D-F) show that the glycogen granules of the hepatic cells as examined in PAS-stained sections of liver showed a significant decrease in glycogen granules in ACF rats (78.8±3.8) compared to control (100±2.8)(p<0.05, Student t-test). G-I) Representative Oil Red O stain highlighting fat globules in a frozen section of the liver revealed the presence of large (macrovesicular) fat globules only in ACF rats (n = 5) compared to controls (n = 5)(PAS-optical density ACF: 198.3±4.9% versus controls: 100±12.4%) p<0.05, Student t-test). Bars = 20 μm. Data show means ± SD.

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  Fig 4. Light microscopic photographs of representative Sirius red staining of liver sections in control (A-C) and ACF (D-F). Note, representative Sirius red staining of liver sections shows extensive collagen deposition, indicating fibrosis progression in ACF rats (n = 5) compared to controls (n = 5) (G)(Sirius red-optical density ACF: 160±11.7% versus controls: 100±7.6%; p<0.05, Student t-test). Bars = 20 μm. Data show means ± SD.

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  Fig 5. Transmission electron micrograph of the rat liver in control (A-C) and ACF (D-F) rats. A-C) Electron microscopic demonstration of control liver tissue. Liver cells of controls consist of round nuclei (N) with intact double-layered nuclear envelopes (arrowhead) (A), well-developed rough endoplasmic reticulum (ER) close to the nucleus, mitochondria (M), Golgi apparatus (GA), lysosomes (L) and a well-organized cytoplasm. D-F) Electron microscopic demonstration of ACF liver tissue shows cytoplasmic organelles are no longer distinguishable within hepatocytes. Ultrastructure evaluation shows marked cytoplasmic vacuolization with large vacuoles (*) of hepatocytes with irregular shaped nuclei (nucleus lost its rounded shape) (D), and degenerated fragmented rough endoplasmic reticulum (DER). F) Also, many shrunken mitochondria (M), became condensed (also known as apoptotic mitochondria) (arrow) in some hepatocytes. x5000; Bar:1 μm.

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  Fig 6. Confocal microscopy of TUNEL staining (A-E) or proapoptotic Bax protein (F-J) in the liver of control and ACF rats. A-E) showed TUNEL-positive (green fluorescence) with DAPI-counterstained nuclei (blue fluorescence) immunofluorescence of the liver in control or ACF adult rats. Note, apoptotic hepatic cells were detected in liver following ACF-induced heart failure in rats (C and D), however, no staining was found in controls (A and B)(Tunel positive cells per 200 μm2 ACF rats 16.1±2.5 versus Controls: 1.5±0.4; p<0.05, Student t-test). (F-J) Confocal microscopy of proapoptotic Bax protein (red fluorescence) with DAPI-counterstained nuclei (blue fluorescence) of the liver in controls or ACF rats. Note absent or weak Bax immunostaining in hepatocyte cytoplasm of controls (n = 5). In contrast in hepatocytes of ACF rats (n = 5), Bax immunofluorescence staining within hepatocyte cytoplasm is very prominent (Bax-optical density ACF rats 388.7±61.7 versus Controls 100±18%; p<0.05, Student t-test). Bars = 20 μm. Data show means ± SD.

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  Fig 7. Confocal microscopy of cytochrome C (red fluorescence) with mitochondrial marker (green fluorescence) double immunofluorescence of liver sections of controls (A-D) or ACF (E-H) rats. Note that the cytochrome C immunostaining overlapped as indicated with yellow immunofluorescence) with the mitochondrial marker in the cytoplasm of hepatocytes of control animals (C-D). However, in hepatocytes of ACF rats (G-H), cytochrome C (red immunofluorescence) leaked from mitochondria and was distributed distinct from the mitochondrial marker (green immunofluorescence). J) Quantitative analysis of immunofluorescence microscopy of mitochondrial leakage of cytochrome C into the cytosol of liver cells following congestive heart failure. Note, the colocalization coefficient of cytochrome C and mitochondrial marker showed a significant reduction of their colocalization in the liver in ACF animals (n = 5) compared to controls (n = 5) (ACF rats 73.6±1.5 versus Controls: 100±1.9; p<0.05, Students t-test). Bars = 20 μm. Bars = 20 μm. Data show means ± SD.

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  Fig 8. Confocal immunofluorescence microscopy of caspase 3 (red fluorescence) using an antibody detecting pro-caspase 3 recombinant protein (A-D) or cleaved caspase 3 recombinant protein (F-I) with DAPI-counterstained nuclei (blue fluorescence) in liver sections of control or ACF adult rats. Note, caspase 3 immunoreactivity was confined primarily to the well defined subcellular organelle-like structures in hepatic cells of control rats (A, B). In contrast in ACF rats, caspase 3 immunofluorescence was transferred to the perinuclear area of cells or inside nuclei of hepatic cells indicating an activation of pro-apoptotic factor caspase 3 (C,D). Confocal immunofluorescence microscopy activated caspase 3 (red fluorescence) and DAPI-counterstained nuclei (blue fluorescence) in liver sections using an antibody which detects exclusively cleaved caspase 3 recombinant protein. H, I) showed that cleaved caspase 3 immunoreactivity was confined primarily to the perinuclear area of cells or nuclei within hepatic cells of ACF rats, however, no staining was found in controls (F, G). Quantitative analysis of immunofluorescence microscopy of nuclear transfer of caspase 3 as activated caspase 3 into the…