Histopathological Analysis of Lipopolysaccharide-Induced Liver Inflammation and Thrombus Formation in Mice: The Protective Effects of Aspirin

Abstract

Hepatitis, a significant medical concern owing to its potential to cause acute and chronic liver disease, necessitates early intervention. In this study, we aimed to elucidate the histopathological features of lipopolysaccharide-induced hepatitis in mice, focusing on tissue alterations. The results demonstrated that hepatocytes exhibited decreased eosin staining, indicating cellular shrinkage, whereas sinusoids were swollen with blood cells. Detailed electron microscope analysis identified these blood cells as leukocytes and erythrocytes, which confirmed a thrombus formation within the liver. Pre-treatment with aspirin significantly attenuated these pathological changes, including reductions in inflammatory markers such as C-reactive protein, interleukin-1β, and tumor necrosis factor-alpha. These findings highlight aspirin's anti-inflammatory and antiplatelet effects in mitigating liver inflammation and thrombus formation. In this study, we highlighted the potential of aspirin as a therapeutic agent for liver inflammation, in addition to providing insights into hepatocyte alterations and sinusoidal blood cell aggregation in liver inflammation. Aspirin, through the protection of endothelial cells and reduction of cytokine levels, may have broader applications in managing liver disease and other systemic inflammatory conditions. This emphasizes its value in prevention and therapy.

Keywords: aspirin; blood clot; correlative light and electron microscopy; inflammation; lipopolysaccharide; liver; scanning electron microscopy; thrombus.

Figure 1

HE-stained images of livers from normal mice (A) and LPS-treated mice (B). Yellow arrows indicate hematoxylin-positive cells in the sinusoids. Black arrows indicate hematoxylin-negative cells within the sinusoids. Scale bars in (A,B) are 100 μm. (C) Average area in control (white bar) and LPS-treated mice (black bar) of hepatocytes (200 cells/animal, four animals from each group). (D) Number of hematoxylin-positive cells within sinusoids in control (white bar) and LPS-treated mice (black bar) of (numbers/200,000 μm², 10 pictures/animal, four animals from each group). Each graph shows the mean ± standard deviation. *; p < 0.05 compared to control value. HE, hematoxylin-eosin; LPS, liposaccharide. All sale bars are 100 μm.

Figure 2

Correlative light and electron microscopical (CLEM) images of livers from liposaccharide-treated mice. (A,A’) Hematoxylin-stained images. (B–B”) Scanning electron microscope images of the same tissue. (A’) is a magnified image of the red frame of A. (B’) is a magnified image of the red frame of (B). (B”) is a magnified image of the red frame of (B’). Yellow arrows indicate hematoxylin-positive cells in the sinusoids. Red arrows indicate hematoxylin-negative cells. The scale bar in (B”) is 10 μm.

Figure 3

HE-stained (A) and SEM (B,B’) images of liver from LPS-treated mice. (B’) is a magnified image of the red frame of (B). Black arrowheads indicate hematocytes in the blood vessel. The scale bar in (A) is 10 μm, in (B’) is 10 μm. HE, hematoxylin-eosin; LPS, liposaccharide; SEM, scanning electron microscope.

Figure 4

SEM images of livers from LPS-treated mice. (A) lower magnified image and (B) higher magnified image. The scale bar in (A) is 20 μm, (B) is 10 μm. LPS, liposaccharide; SEM, scanning electron microscope.

Figure 5

SEM images of livers from normal (A,C) and LPS-treated (B,D) mice. (A,B) The images of the inner surface of endothelial cells. (C,D) The images of capillary bile ducts. Yellow arrows in B indicate the rough inner surfaces of endothelial cells. Red arrows in (C,D) indicate the capillary bile ducts. LPS, liposaccharide; SEM, scanning electron microscope. Scale bars in A and B are 50 μm, and in C and D are 10 μm.

Figure 6

HE-stained image of hepatitis-induced mouse liver by LPS after pre-administration of aspirin. Yellow arrows indicate hematoxylin-positive cells in the sinusoids. Black arrows indicate hematoxylin-negative cells within the sinusoids. Black arrowheads indicate hematocytes in the blood vessel. Scale bars in (A,B) are 100 μm. HE, hematoxylin-eosin; LPS, liposaccharide; SEM.

Figure 7

Changes in each inflammatory marker over time after saline pre-treatment and aspirin pre-treatment. (A) CRP (B) IL-1β (C) TNF-α. The blue line represents the control group (saline pre-treatment). The red line represents the aspirin group. 0H indicates values without LPS. 2H, 6H, 12H, and 24H indicate values 2 h, 6 h, 12 h, and 24 h after LPS administration, respectively. Each graph shows the mean ± standard deviation. *; p < 0.05 compared to control value. CRP, C-reactive protein; IL-1β, interleukin 1β; TNF-α; tumor necrosis factor alpha; LPS, liposaccharide.