Aquaporin-4 deletion in mice reduces encephalopathy and brain edema in experimental acute liver failure

Abstract

Brain edema and associated astrocyte swelling leading to increased intracranial pressure are hallmarks of acute liver failure (ALF). Elevated blood and brain levels of ammonia have been implicated in the development of brain edema in ALF. Cultured astrocytes treated with ammonia have been shown to undergo cell swelling and such swelling was associated with an increase in the plasma membrane expression of aquaporin-4 (AQP4) protein. Further, silencing the AQP4 gene in cultured astrocytes was shown to prevent the ammonia-induced cell swelling. Here, we examined the evolution of brain edema in AQP4-null mice and their wild type counterparts (WT-mice) in different models of ALF induced by thioacetamide (TAA) or acetaminophen (APAP). Induction of ALF with TAA or APAP significantly increased brain water content in WT mice (by 1.6% ± 0.3 and 2.3 ± 0.4%, respectively). AQP4 protein was significantly increased in brain plasma membranes of WT mice with ALF induced by either TAA or APAP. In contrast to WT-mice, brain water content did not increase in AQP4-null mice. Additionally, AQP4-null mice treated with either TAA or APAP showed a remarkably lesser degree of neurological deficits as compared to WT mice; the latter displayed an inability to maintain proper gait, and demonstrated a markedly reduced exploratory behavior, with the mice remaining in one corner of the cage with its head tilted downwards. These results support a central role of AQP4 in the brain edema associated with ALF.

Keywords: Acetaminophen; Acute liver failure; Aquaporin-4; Astrocyte swelling; Brain edema; Thioacetamide; Transgenic mice.

Figure 1

A. Grades of encephalopathy in WT and AQP4-null mice with ALF induced by thioacetamide (TAA). AQP4 gene deletion significantly delays the onset of encephalopathy in mice. Values are mean ± S.E.M. of 6–7 animals in each experimental group. * vs. AQP4-null mice, p<0.05. B. Grades of encephalopathy in WT and AQP4-null mice with ALF induced by acetaminophen (APAP). AQP4 gene deletion significantly delayed the development of encephalopathy. Values are mean ± S.E.M. of 6–7 animals in each experimental group. * vs. AQP4-null mice, p<0.05.

Figure 2

Brain water content in wild-type (WT) and AQP4-null mice treated with thioacetamide (TAA). WT-mice treated with TAA showed a 1.64 ± 0.3% increase in brain water content as compared to WT-control mice. Values are mean ± S.E.M. of 6–8 animals in each experimental group. * vs. control, p<0.01; ** vs. WT-TAA, p<0.05. CON, control.

Figure 3

Brain water content in WT and AQP4-null mice treated with acetaminophen (APAP). WT-mice treated with APAP displayed a 2.36 ± 0.39% rise in brain water content as compared to WT-control mice. Values are mean ± S.E.M. of 5–7 animals in each experimental group. * vs. control, p<0.01; ** vs. WT-APAP, p<0.05. CON, control.

Figure 4

A. AQP4 protein expression in brain plasma membranes of WT mice treated with TAA or APAP show a significant increase in AQP4 content. B. Quantitation of AQP4 protein content in plasma membrane fraction. Plasma membrane AQP4 protein content was normalized to protein content of Na⁺,K⁺ ATPase Values are mean ± S.E.M. of 5 animals in each experimental group. * vs. control, p<0.01. CON, control.

Figure 5

Immunohistochemistry of AQP4 (A, B, C), and the capillary endothelial marker GLUT 1 (D, E, F) in cerebral cortex of control (C), thioacetamide (TAA)-and acetaminophen (APAP)-treated WT-mice. Merged immunofluorescent images (G, H, I) display a co-localization of AQP4 with GLUT 1, consistent with the perivascular localization of AQP4 on astrocytic plasma membranes. Immunofluorescence shows an increase in AQP4 (arrows) in WT-mice treated with TAA or APAP. Scale bar = 50 μm.