Severe Acute Liver Dysfunction Induces Delayed Hepatocyte Swelling and Cytoplasmic Vacuolization, and Delayed Cortical Neuronal Cell Death

Abstract

Liver dysfunction is the main cause of hepatic encephalopathy. However, histopathological changes in the brain associated with hepatic encephalopathy remain unclear. Therefore, we investigated pathological changes in the liver and brain using an acute hepatic encephalopathy mouse model. After administering ammonium acetate, a transient increase in the blood ammonia level was observed, which returned to normal levels after 24 h. Consciousness and motor levels also returned to normal. It was revealed that hepatocyte swelling, and cytoplasmic vacuolization progressed over time in the liver tissue. Blood biochemistry also suggested hepatocyte dysfunction. In the brain, histopathological changes, such as perivascular astrocyte swelling, were observed 3 h after ammonium acetate administration. Abnormalities in neuronal organelles, especially mitochondria and rough endoplasmic reticulum, were also observed. Additionally, neuronal cell death was observed 24 h post-ammonia treatment when blood ammonia levels had returned to normal. Activation of reactive microglia and increased expression of inducible nitric oxide synthase (iNOS) were also observed seven days after a transient increase in blood ammonia. These results suggest that delayed neuronal atrophy could be iNOS-mediated cell death due to activation of reactive microglia. The findings also suggest that severe acute hepatic encephalopathy causes continued delayed brain cytotoxicity even after consciousness recovery.

Keywords: astrocyte; brain edema; hepatic encephalopathy; iNOS; microglial cell; pathology; scanning electron microscope; transmission electron microscope.

Figure 1

Control, 3H, and 24H indicate the average blood ammonia concentration for the animals in the vehicle treatment control group and the treated animal at 3 and 24 h after ammonia treatment, respectively. Data are expressed as the mean ± standard error. *: p < 0.05 compared with the control value.

Figure 2

Control (A,D,G), 3H (B,E,H), and 24H (C,F,I) representative images of the hepatic tissues from the control animals and those from animals sacrificed at 3 and 24 h post-ammonia treatment, respectively. (A–C) are high-magnification views of the hematoxylin-eosin (HE) stained hepatic tissues, while (D–F) are high-magnification views of the Periodic acid–Schiff (PAS) stained hepatic tissues. (G–I) are high-magnification views of the Iron-stained hepatic tissues. Black arrows indicate the hepatocytes exhibiting swelling and/or cytoplasmic vacuolization. Red arrows indicate the iron-positive sites. All scale bars = 100 μm. (J) shows the number of hepatocytes exhibiting swelling and/or cytoplasmic vacuolization. (K) shows the number of iron-positive hepatocytes. Data are expressed as mean ± standard deviation. * p < 0.05, compared to the control.

Figure 3

Scanning electron microscopy micrographs of the sinusoidal endothelium are shown. The sinusoidal endothelium of the control is shown (A). (B,C) Sinusoidal endothelium of animals, 3 h and 24 h post-ammonia treatment, respectively. Yellow arrows show the swelling of sinusoidal fenestrations. All scale bars = 1 µm.

Figure 4

Control (A), 3H (B), and 24H (C) representative images of the cerebral cortical tissues from the control animals and those from animals sacrificed at 3 h and 24 h after ammonia treatment, respectively. All scale bars = 500 μm. (D) Control, 3H, and 24H indicate the brain water contents for the animals in the control group and the treated animal at 3 and 24 h after ammonia treatment, respectively. Data are expressed as the mean ± standard deviation. *: p < 0.05 compared with the control value.

Figure 5

Control (A,D), 3H (B,E), and 24H (C,F) representative optical images of the cerebral cortical tissues from the control animals and those from animals sacrificed at 3 h and 24 h after ammonia treatment, respectively. (A–C) are high-magnification views of the hematoxylin-eosin-stained cerebral cortical tissues. (D–F) are high-magnification views of the Nissl-stained cerebral cortical tissues. The black arrows indicate perivascular edema of the cerebral cortex layer II/III. Red arrowheads indicate the swollen fibers of the cerebral cortex layer II/III. Black arrowheads indicate the neuronal atrophy of the cerebral cortex layer II/III. All scale bars = 100 μm. (G) shows the relative intensity of Nissl-stained neurons. Data are shown as relative intensity (100 = the intensity of the control value). (H) shows the number of atrophying neurons. Data are expressed as mean ± standard deviation. *: p < 0.05 compared with the control value.

Figure 6

Control (A,D), 3H (B,E), and 24H (C,F) representative electron microscopy images of the cerebral cortical tissues from the control animals and those from animals sacrificed 3 h and 24 h after ammonia treatment, respectively. (A–C) show the blood vessel and perivascular astrocytes in the cerebral cortex (green color). (D–F) show the neurons in the cerebral cortex (green color). Black double arrowheads show the basal membrane. Blue arrowheads indicate the perivascular edema of the cerebral cortex layer II/III. Black arrowheads indicate the rough endoplasmic reticulum. Red arrowheads indicate mitochondria. All scale bars = 2 μm.

Figure 7

(A–F) Transmitted electron microscopic images of the cerebral cortical tissues from the ammonia-treated animals 24 h post-administration. Green-colored neurons in the cerebral cortex are shown. Black arrow in (A) indicates the intact perineuronal glial cell (astrocyte). Black arrowheads indicate the rough endoplasmic reticulum. Red arrowheads indicate degenerating mitochondria. Blue arrowheads indicate neuronal atrophy. Scale bars in (A–D) = 2 μm, and (E,F) = 5 μm.

Figure 8

(A–C) Western blot analysis revealed the timing of iNOS, GFAP, and Iba1 expression following hepatic encephalopathy. In addition, as an internal control, the expression level of β-tubulin is presented. (D–F) Densitometry analysis of the Western blot data (each number = 5). Data are expressed as mean ± standard deviation. Data are shown as relative intensity (100 = the intensity of the control value). C, 1H, 3H, 24H, 3D, and 7D indicate the various treatment groups post-ammonia treatment (control, 1 h, 3 h, 24 h, 3 days, and 7 days), respectively. *: p < 0.05 compared with the control.