NF-κB in the mechanism of brain edema in acute liver failure: Studies in transgenic mice
Research highlights
►Tg mice with inactive astrocytic NF-κB are resistant to ALF-induced brain edema. ►Brain sections from transgenic (Tg) mice showed no astrocyte swelling in ALF. ►Cultured astrocytes from Tg mice treated with ammonia showed a less cell swelling. ►Factors activated by NF-κB and induce cell swelling are reduced in Tg mice astrocytes. ►NF-kB is critical in the mechanism of brain edema in acute liver failure (ALF).
Introduction
Hepatic encephalopathy (HE) is a major neuropsychiatric disorder that occurs in patients with severe liver failure. The acute form of HE (acute liver failure; ALF) is a potentially lethal condition as it may lead to cerebral edema, increased intracranial pressure and brain herniation. While the pathogenesis of HE is incompletely understood, ammonia has been strongly implicated as an important factor (for reviews, see Albrecht and Jones, 1999, Hazell and Butterworth, 1999), and astrocytes appear to be the primary target of its neurotoxicity (Norenberg, 1996). Astrocyte swelling is the major neuropathologic finding in ALF (Norenberg, 1977, Traber et al., 1987, Swain et al., 1991), and both in vivo (Voorhies et al., 1983, Willard-Mack et al., 1996) and in vitro (Norenberg et al., 1991, Zwingmann et al., 2000) studies have shown astrocyte swelling after exposure to a pathophysiological concentration of ammonia. The precise mechanism by which ammonia brings about such swelling remains unclear.
We recently documented that exposure of astrocyte cultures to ammonia stimulated the activation (nuclear translocation) of the transcription factor nuclear factor-κB (NF-κB) (Sinke et al., 2008). Ammonia-treated astrocyte cultures have also been shown to increase the activity of inducible nitric oxide synthase (iNOS) and to promote NO generation (Schliess et al., 2002, Sinke et al., 2008), factors known to be associated with cell swelling/brain edema (for review, see Norenberg et al., 2007), while treatment of cultures with the NF-κB inhibitor BAY 11-7082, diminished iNOS protein expression and NO generation (Sinke et al., 2008). Additionally, inhibition of NF-κB activation by BAY 11-7082 reduced astrocyte swelling in culture (Sinke et al., 2008).
Another consequence of NF-κB activation is induction of oxidative stress (OS) (for review, see Bowie and O'Neill, 2000). Regulation of NADPH oxidase (NOX) by NF-κB represents a major mechanism for the induction of OS, since NOX is an important source of superoxide generation in most cells (Manea et al., 2007, Luengo-Blanco et al., 2008). OS is known to induce cell swelling/brain edema in different neurological conditions (for review, see Norenberg et al., 2009). Ammonia-treated astrocyte cultures have been shown to increase NOX activity, and treatment of cultures with the NOX inhibitor, apocyanin, reduced ammonia-induced astrocyte swelling (Reinehr et al., 2007, Jayakumar et al., 2009). These studies suggested that activation of NF-κB is involved in the mechanism of ammonia-induced astrocyte swelling, in part through enhanced NOX activation. While these studies implicate NF-κB in cell swelling mechanisms in cultured astrocytes as a consequence of oxidative stress, the role of NF-κB in the brain edema associated with ALF is not known.
Since pharmacological inhibitors often have non-specific effects, the present study examined transgenic (Tg) mice that possess a functional inactivation of astrocytic NF-κB, and determined whether these mice are resistant to thioacetamide (TAA)-induced brain edema. Additionally, we examined whether astrocyte cultures derived from Tg mice leads to a reduction in cell swelling in cultured astrocytes following ammonia treatment, and whether a reduction in iNOS and NOX activities (both are known to be activated by NF-κB) occur in astrocytes derived from Tg mice. Our study demonstrates that Tg mice treated with the hepatotoxin TAA are resistant to the development of brain edema as compared to WT mice. Similarly, cultured astrocytes derived from Tg mice when treated with ammonia showed no significant cell swelling. These findings invoke a critical role of NF-κB in the mechanism of the astrocyte swelling/brain edema associated with ALF.
Section snippets
ALF model
The mouse model used in this study was developed by Brambilla et al., 2005, in which the astroglial NF-κB is inactivated by expressing a mutated form of the I-κB-alpha under the control of GFAP promoter (glial fibrillary acidic protein [GFAP]-dn mice). This mutation prevents its phosphorylation and thereby inhibits the activation of NF-κB. These transgenic (Tg) mice survive normally to adulthood and do not exhibit any apparent abnormal sensori-motor phenotype. Such astroglial specific NF-κB
Treatment of mice with TAA
Clinical grading of encephalopathy in mice (7/group) treated with TAA was as follows: Day 1: All mice from both groups (WT and Tg) were in Grade 1. Day 2: In WT mice, 1 was in Grade I, and 6 were in Grade II, whereas in Tg mice 5 were in Grade I, and 2 in Grade II. Day 3: In WT mice, 5 were in Grade IV, and 2 in Grade V, whereas in Tg mice 4 were in Grade III, and 3 in Grade IV. This data is presented in graphical form in Fig. 1.
Light microscopic examination of livers taken from WT mice treated
Discussion
Our study demonstrates that Tg mice in which the astrocytic NF-κB had been inactivated, displayed no brain edema after TAA-induced ALF, in contrast to its WT control. Tg mice treated with TAA also displayed a delay in the development of encephalopathy when compared to WT mice treated with TAA. Additionally, Tg mice treated with TAA did not exhibit an increase in iNOS immunoreactivity. By contrast a marked increase in iNOS immunoreactivity was observed in astrocytes from WT mice treated with
Acknowledgments
This work was supported by a Merit Review from the Department of Veterans Affairs and by National Institutes of Health grants DK063311. ARJ is supported by the American Association for the Study of Liver Disease/American Liver Foundation grants. We thank Alina Fernandez-Revuelta for the preparation of cell cultures.
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