Elsevier

Neurobiology of Disease

Volume 41, Issue 2, February 2011, Pages 498-507
Neurobiology of Disease

NF-κB in the mechanism of brain edema in acute liver failure: Studies in transgenic mice

https://doi.org/10.1016/j.nbd.2010.10.021Get rights and content

Abstract

Astrocyte swelling and brain edema are major complications of the acute form of hepatic encephalopathy (acute liver failure, ALF). While elevated brain ammonia level is a well-known etiological factor in ALF, the mechanism by which ammonia brings about astrocyte swelling is not well understood. We recently found that astrocyte cultures exposed to ammonia activated nuclear factor-κB (NF-κB), and that pharmacological inhibition of such activation led to a reduction in astrocyte swelling. Although these findings suggest the involvement of NF-κB in astrocyte swelling in vitro, it is not known whether NF-κB contributes to the development of brain edema in ALF in vivo. Furthermore, pharmacological agents used to inhibit NF-κB may have non-specific effects. Accordingly, we used transgenic (Tg) mice that have a functional inactivation of astrocytic NF-κB and examined whether these mice are resistant to ALF-associated brain edema. ALF was induced in mice by treatment with the hepatotoxin thioacetamide (TAA). Wild type (WT) mice treated with TAA showed a significant increase in brain water content (1.65%) along with prominent astrocyte swelling and spongiosis of the neuropil, consistent with the presence of cytotoxic edema. These changes were not observed in Tg mice treated with TAA. Additionally, WT mice with ALF showed an increase in inducible nitric oxide synthase (iNOS) immunoreactivity in astrocytes from WT mice treated with TAA (iNOS is known to be activated by NF-κB and to contribute to cell swelling). By contrast, Tg mice treated with TAA did not exhibit brain edema, histological changes nor an increase in iNOS immunoreactivity. We also examined astrocytes cultures derived from Tg mice to determine whether these cells exhibit a lesser degree of swelling and cytopathological changes following exposure to ammonia. Astrocyte cultures derived from Tg mice showed no cell swelling nor morphological abnormalities when exposed to ammonia for 24 h. By contrast, ammonia significantly increased cell swelling (31.7%) in cultured astrocytes from WT mice and displayed cytological abnormalities. Moreover, we observed a lesser increment in iNOS and NADPH oxidase activity (the latter is also known to be activated by NF-κB and to contribute to astrocyte swelling) in astrocyte cultures from Tg mice treated with ammonia, as compared to ammonia-treated WT mice astrocytes. These findings strongly suggest that activation of NF-κB is a critical factor in the development of astrocyte swelling/brain edema in ALF.

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.

References (66)

  • K.V. Rama Rao

    Brain edema in acute liver failure: inhibition by L-histidine

    Am. J. Pathol.

    (2010)
  • I. Stancovski et al.

    NF-kappaB activation: the I kappaB kinase revealed?

    Cell

    (1997)
  • R. Stanislaus

    Amelioration of experimental allergic encephalomyelitis in Lewis rats by lovastatin

    Neurosci. Lett.

    (1999)
  • C.L. Willard-Mack

    Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat

    Neuroscience

    (1996)
  • Q.W. Xie

    Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase

    J. Biol. Chem.

    (1994)
  • M. Zielinska

    Excitotoxic mechanism of cell swelling in rat cerebral cortical slices treated acutely with ammonia

    Neurochem. Int.

    (2003)
  • J. Albrecht et al.

    Hepatic encephalopathy: molecular mechanisms underlying the clinical syndrome

    J. Neurol. Sci.

    (1999)
  • BaldwinA.S.

    The NF-kappa B and I kappa B proteins: new discoveries and insights

    Annu. Rev. Immunol.

    (1996)
  • J.S. Beckman

    The double-edged role of nitric oxide in brain function and superoxide-mediated injury

    J. Dev. Physiol.

    (1991)
  • V. Bracchi-Ricard

    Astroglial nuclear factor-kappaB regulates learning and memory and synaptic plasticity in female mice

    J. Neurochem.

    (2008)
  • R. Brambilla

    Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury

    J. Exp. Med.

    (2005)
  • R. Brambilla

    Transgenic inhibition of astroglial NF-kappa B leads to increased axonal sparing and sprouting following spinal cord injury

    J. Neurochem.

    (2009)
  • R. Brambilla

    Transgenic inhibition of astroglial NF-kappa B improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation

    J. Immunol.

    (2009)
  • O. Detry

    Brain edema and intracranial hypertension in fulminant hepatic failure: pathophysiology and management

    World. J. Gastroenterol.

    (2006)
  • G. Dvoriantchikova

    Inactivation of astroglial NF-kappa B promotes survival of retinal neurons following ischemic injury

    Eur. J. Neurosci.

    (2009)
  • E.S. Fu

    Transgenic glial nuclear factor-kappa B inhibition decreases formalin pain in mice

    Neuroreport

    (2007)
  • S. Gammal

    Reversal of the behavioral and electrophysiological abnormalities of an animal model of hepatic encephalopathy by benzodiazepine receptor ligands

    Hepatology

    (1990)
  • J.B. Gregorios

    Morphologic effects of ammonia on primary astrocyte cultures: I. Light microscopic studies

    J. Neuropathol. Exp. Neurol.

    (1985)
  • A.S. Hazell et al.

    Hepatic encephalopathy: an update of pathophysiologic mechanisms

    Proc. Soc. Exp. Biol. Med.

    (1999)
  • W. Hilgier et al.

    Brain ion and amino acid contents during edema development in hepatic encephalopathy

    J. Neurochem.

    (1994)
  • E.A. Jaimes

    Stable compounds of cigarette smoke induce endothelial superoxide anion production via NADPH oxidase activation

    Arterioscler. Thromb. Vasc. Biol.

    (2004)
  • A.R. Jayakumar

    Oxidative stress and mitogen-activated protein kinase phos-phorylation mediate ammonia-induced cell swelling and glutamate uptake inhibition in cultured astrocytes

    J. Neurosci.

    (2006)
  • A.R. Jayakumar

    Calcium in the mechanism of ammonia-induced astrocyte swelling

    J. Neurochem.

    (2009)
  • Cited by (36)

    • Adrenal histological and functional changes after hepatic encephalopathy: From mice model to an integrative bioinformatics analysis

      2022, Acta Histochemica
      Citation Excerpt :

      Animals weights and stages of HE were recorded every day until day 10. Stages of encephalopathy were graded according to the previously mentioned study (Jayakumar et al., 2011): Grade I: lethargy; Grade II: mild ataxia; Grade III: lack of spontaneous movement (intact righting reflex); Grade IV: loss of righting reflex (intact pain reflex); Grade V: coma. On day 10, all animals were decapitated under deep anesthesia and trunk blood was collected into the ethylenediaminetetraacetic acid (EDTA) coated tubes to measure blood parameters and to separate plasma.

    • The nuclear factor kappa B (NF-κB) signaling pathway is involved in ammonia-induced mitochondrial dysfunction

      2021, Mitochondrion
      Citation Excerpt :

      Several studies have demonstrated that hyperammonemia triggers neuroinflammation (Rodrigo et al., 2010; van Horssen et al., 2019; Tapper et al., 2015; Tranah et al., 2013; Shawcross et al., 2004). It has been shown that elevated ammonia levels in the brain is capable of affecting critical inflammatory processes causing modifications in the expression/activation of inflammatory proteins such as nuclear factor kappa B (NF-κB) (Sinke, 2008; Jayakumar, 2011). Activation of NF-κB is initiated through the phosphorylation and subsequent degradation of inhibitor kappa B (IκB).

    • Development of an experimental rat model of hyperammonemic encephalopathy and evaluation of the effects of rifaximin

      2016, European Journal of Pharmacology
      Citation Excerpt :

      Moreover, neuronal vacuolar degeneration was observed. These changes are similar to those observed in other HE models, in which astrocyte swelling, Alzheimer type II astrocytes, and perivascular vacuolization have been reported (Jayakumar et al., 2011; Matkowskyj et al., 1999; Pilbeam et al., 1983). It has previously been suggested that the primary pathogenesis of astrocyte swelling is an increase in blood ammonia concentration which triggers oxidative stress, enhanced mitochondrial permeability transition and activation of signaling kinases (Jayakumar et al., 2011; Norenberg et al., 2007).

    • Regulating effect of activated NF-κB on edema induced by traumatic brain injury of rats

      2016, Asian Pacific Journal of Tropical Medicine
      Citation Excerpt :

      The active oxygen free radical could stimulate the activation of different kinds of transcription factors, especially nuclear transcription factor-κB (NF-κB) [7,8]. The activated NF-κB might contribute to the swelling of astrocytes after being exposed to ammonia [9], and it also plays a role during cerebral edema after hepatic failure [10]. Meanwhile, the activated NF-κB is found after TBI [11].

    View all citing articles on Scopus
    View full text