Additive Effect of Resveratrol on Astrocyte Swelling Post-exposure to Ammonia, Ischemia and Trauma In Vitro.

Swelling of astrocytes represents a major component of the brain edema associated with many neurological conditions, including acute hepatic encephalopathy (AHE), traumatic brain injury (TBI) and ischemia. It has previously been reported that exposure of cultured astrocytes to ammonia (a factor strongly implicated in the pathogenesis of AHE), oxygen/glucose deprivation, or to direct mechanical trauma results in an increase in cell swelling. Since dietary polyphenols have been shown to exert a protective effect against cell injury, we examined whether resveratrol (RSV, 3,5,4'-trihydroxy-trans-stilbene, a stilbenoid phenol), has a protective effect on astrocyte swelling following its exposure to ammonia, oxygen-glucose deprivation (OGD), or trauma in vitro. Ammonia increased astrocyte swelling, and pre- or post-treatment of astrocytes with 10 and 25 µM RSV displayed an additive effect, while 5 µM did not prevent the effect of ammonia. However, pre-treatment of astrocytes with 25 µM RSV slightly, but significantly, reduced the trauma-induced astrocyte swelling at earlier time points (3 h), while post-treatment had no significant effect on the trauma-induced cell swelling at the 3 h time point. Instead, pre- or post-treatment of astrocytes with 25 µM RSV had an additive effect on trauma-induced astrocyte swelling. Further, pre- or post-treatment of astrocytes with 5 or 10 µM RSV had no significant effect on trauma-induced astrocyte swelling. When 5 or 10 µM RSV were added prior to, or during the process of OGD, as well as post-OGD, it caused a slight, but not statistically significant decline in cell swelling. However, when 25 µM RSV was added during the process of OGD, as well as after the cells were returned to normal condition (90 min period), such treatment showed an additive effect on the OGD-induced astrocyte swelling. Noteworthy, a higher concentration of RSV (25 µM) exhibited an additive effect on levels of phosphorylated forms of ERK1/2, and p38MAPK, as well as an increased activity of the Na+-K+-Cl- co-transporter-1 (NKCC1), factors known to induce astrocytes swelling, when the cells were treated with ammonia or after trauma or ischemia. Further, inhibition of ERK1/2, and p38MAPK diminished the RSV-induced exacerbation of cell swelling post-ammonia, trauma and OGD treatment. These findings strongly suggest that treatment of cultured astrocytes with RSV enhanced the ammonia, ischemia and trauma-induced cell swelling, likely through the exacerbation of intercellular signaling kinases and ion transporters. Accordingly, caution should be exercised when using RSV for the treatment of these neurological conditions, especially when brain edema is also suspected.

Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in defects in neuronal integrity associated with chronic traumatic encephalopathy: in vitro studies.

Transactivating DNA-binding protein-43 (TDP-43) inclusions and the accumulation of phosphorylated and ubiquitinated tau proteins (p-tau) have been identified in postmortem brain specimens from patients with chronic traumatic encephalopathy (CTE). To examine whether these proteins contribute to the development of CTE, we utilized an in vitro trauma system known to reproduce many of the findings observed in humans and experimental animals with traumatic brain injury. Accordingly, we examined the role of TDP-43 and Tau in an in vitro model of trauma, and determined whether these proteins contribute to the defective neuronal integrity associated with CNS trauma. Single or multiple episodes of trauma to cultured neurons resulted in a time-dependent increase in cytosolic levels of phosphorylated TDP-43 (p-TDP-43). Trauma to cultured neurons also caused an increase in levels of casein kinase 1 epsilon (CK1ε), and ubiquitinated p-TDP-43, along with a decrease in importin-ß (all factors known to mediate the "TDP-43 proteinopathy"). Defective neuronal integrity, as evidenced by a reduction in levels of the NR1 subunit of the NMDA receptor, and in PSD95, along with increased levels of phosphorylated tau were also observed. Additionally, increased levels of intra- and extracellular thrombospondin-1 (TSP-1) (a factor known to regulate neuronal integrity) were observed in cultured astrocytes at early stages of trauma, while at later stages decreased levels were identified. The addition of recombinant TSP-1, conditioned media from cultured astrocytes at early stages of trauma, or the CK1ε inhibitor PF4800567 hydrochloride to traumatized cultured neurons reduced levels of p-TDP-43, and reversed the trauma-induced decline in NR1 subunit of the NMDA receptor and PSD95 levels. These findings suggest that a trauma-induced increase in TDP-43 phosphorylation contributes to defective neuronal integrity, and that increasing TSP-1 levels may represent a useful therapeutic approach for the prevention of the neuronal TDP-43 proteinopathy associated with CTE. Read the Editorial Highlight for this article on page 531.

Expression of Gls and Gls2 glutaminase isoforms in astrocytes.

The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes.

Increased toll-like receptor 4 in cerebral endothelial cells contributes to the astrocyte swelling and brain edema in acute hepatic encephalopathy.

Astrocyte swelling and the subsequent increase in intracranial pressure and brain herniation are major clinical consequences in patients with acute hepatic encephalopathy. We recently reported that conditioned media from brain endothelial cells (ECs) exposed to ammonia, a mixture of cytokines (CKs) or lipopolysaccharide (LPS), when added to astrocytes caused cell swelling. In this study, we investigated the possibility that ammonia and inflammatory agents activate the toll-like receptor 4 (TLR4) in ECs, resulting in the release of factors that ultimately cause astrocyte swelling. We found a significant increase in TLR4 protein expression when ECs were exposed to ammonia, CKs or LPS alone, while exposure of ECs to a combination of these agents potentiate such effects. In addition, astrocytes exposed to conditioned media from TLR4-silenced ECs that were treated with ammonia, CKs or LPS, resulted in a significant reduction in astrocyte swelling. TLR4 protein up-regulation was also detected in rat brain ECs after treatment with the liver toxin thioacetamide, and that thioacetamide-treated TLR4 knock-out mice exhibited a reduction in brain edema. These studies strongly suggest that ECs significantly contribute to the astrocyte swelling/brain edema in acute hepatic encephalopathy, likely as a consequence of increased TLR4 protein expression by blood-borne noxious agents.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.

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

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.

Glutamine in the pathogenesis of hepatic encephalopathy: the trojan horse hypothesis revisited.

Hepatic encephalopathy (HE) is major neuropsychiatric disorder occurring in patients with severe liver disease and ammonia is generally considered to represent the major toxin responsible for this condition. Ammonia in brain is chiefly metabolized ("detoxified") to glutamine in astrocytes due to predominant localization of glutamine synthetase in these cells. While glutamine has long been considered innocuous, a deleterious role more recently has been attributed to this amino acid. This article reviews the mechanisms by which glutamine contributes to the pathogenesis of HE, how glutamine is transported into mitochondria and subsequently hydrolyzed leading to high levels of ammonia, the latter triggering oxidative and nitrative stress, the mitochondrial permeability transition and mitochondrial injury, a sequence of events we have collectively termed as the Trojan horse hypothesis of hepatic encephalopathy.

Brain edema in acute liver failure: mechanisms and concepts.

Brain edema and associated increase in intracranial pressure continue to be lethal complications of acute liver failure (ALF). Abundant evidence suggests that the edema in ALF is largely cytotoxic brought about by swelling of astrocytes. Elevated blood and brain ammonia levels have been strongly implicated in the development of the brain edema. Additionally, inflammation and sepsis have been shown to contribute to the astrocyte swelling/brain edema in the setting of ALF. We posit that ammonia initiates a number of signaling events, including oxidative/nitrative stress (ONS), the mitochondrial permeability transition (mPT), activation of the transcription factor (NF-κB) and signaling kinases, all of which have been shown to contribute to the mechanism of astrocyte swelling. All of these factors also impact ion-transporters, including Na(+), K(+), Cl(-) cotransporter and the sulfonylurea receptor 1, as well as the water channel protein aquaporin-4 resulting in a perturbation of cellular ion and water homeostasis, ultimately resulting in astrocyte swelling/brain edema. All of these events are also potentiated by inflammation. This article reviews contemporary knowledge regarding mechanisms of astrocyte swelling/brain edema formation which hopefully will facilitate the identification of therapeutic targets capable of mitigating the brain edema associated with ALF.

Age and Sex in the Development of Hepatic Encephalopathy: Role of Alcohol.

Hepatic encephalopathy (HE) is a neurological condition linked to liver failure. Acute HE (Type A) occurs with acute liver failure, while chronic HE (Type C) is tied to cirrhosis and portal hypertension. HE treatments lag due to gaps in understanding its development by gender and age. We studied how sex and age impact HE and its severity with combined liver toxins. Our findings indicate that drug-induced (thioacetamide, TAA) brain edema was more severe in aged males than in young males or young/aged female rats. However, adding alcohol (ethanol, EtOH) worsens TAA's brain edema in both young and aged females, with females experiencing a more severe effect than males. These patterns also apply to Type A HE induced by azoxymethane (AZO) in mice. Similarly, TAA-induced behavioral deficits in Type C HE were milder in young and aged females than in males. Conversely, EtOH and TAA in young/aged males led to severe brain edema and fatality without noticeable behavioral changes. TAA metabolism was slower in aged males than in young or middle-aged rats. When TAA-treated aged male rats received EtOH, there was a slow and sustained plasma level of thioacetamide sulfoxide (TASO). This suggests that with EtOH, TAA-induced HE is more severe in aged males. TAA metabolism was similar in young, middle-aged, and aged female rats. However, with EtOH, young and aged females experience more severe drug-induced HE as compared to middle-aged adult rats. These findings strongly suggest that gender and age play a role in the severity of HE development and that the presence of one or more liver toxins may aggravate the severity of the disease progression.

Mechanism of Alzheimer type II astrocyte development in hepatic encephalopathy.

Type C hepatic encephalopathy (Type C HE) is a major and complex neurological condition that occurs following chronic liver failure. The molecular basis of Type C HE remains elusive. Type C HE is characterized by mental confusion, cognitive and motor disturbances. The presence of Alzheimer type II astrocytes (AT2A) is the key histopathological finding observed in Type C HE. However, nothing is currently known regarding AT2A development and its involvement in cognitive, and motor deficits in Type C HE. We, therefore, examined in rats the mechanisms by which liver failure contributes to the progression of AT2A, and its role in the development of cognitive and motor deficits in thioacetamide (TAA) model of Type C HE. We and others earlier reported increased oxidative/nitrosative stress (ONS), JNK1/2, and cMyc activation in ammonia-treated astrocyte cultures, as well as in brains from chronic liver failure. We now found increased levels of astrocytic glia maturation factor (GMF, a factor strongly implicated in neuroinflammation), as well as various inflammatory factors (IL-1ß, TNF-α, IL-6, MMP-3, COX2, CXCL1, and PGE2), and reduced levels of GFAP and increased levels of aggregated nuclear protein Lamin A/C in rat brain cortex post-chronic liver failure. We also found increased levels of GMF and inflammatory factors (MMP-3, COX2, CXCL1, and PGE2) in astrocytes post-ammonia treatment in vitro. Additionally, pharmacological inhibition of upstream signaling of GMF (ONS, JNK1/2, and cMyc) or GMF inhibitors W-7 and trifluoperazine significantly reduced the levels of inflammatory factors, the number of AT2A cells, as well as the cognitive and motor deficits in TAA-treated rats. Increased levels of GMF were also identified in human post-mortem brain sections. These findings strongly suggest that increased levels of astrocytic GMF due to elevated levels of ONS, JNK1/2, and cMyc and the subsequent inflammation contribute to the development of AT2A and the consequent cognitive, and motor deficits in chronic liver failure.

Reduced extracellular zinc levels facilitate glutamate-mediated oligodendrocyte death after trauma.

Spinal cord injury results in irreversible paralysis, axonal injury, widespread oligodendrocyte death, and white matter damage. Although the mechanisms underlying these phenomena are poorly understood, previous studies from our laboratory indicate that inhibiting activation of the nuclear factor-κB transcription factor in astrocytes reduces white matter damage and improves functional recovery following spinal cord injury. In the current study, we demonstrate that activation of the nuclear factor-κB transcription factor within astrocytes results in a significant increase in oligodendrocyte death following trauma by reducing extracellular zinc levels and inducing glutamate excitotoxicity. By using an ionotropic glutamate receptor antagonist (CNQX), we show that astroglial nuclear factor-κB-mediated oligodendrocyte death is dependent on glutamate signaling despite no change in extracellular glutamate concentrations. Further analysis demonstrated a reduction in levels of extracellular zinc in astrocyte cultures with functional nuclear factor-κB signaling following trauma. Cotreatment of oligodendrocytes with glutamate and zinc showed a significant increase in oligodendrocyte toxicity under low-zinc conditions, suggesting that the presence of zinc at specific concentrations can prevent glutamate excitotoxicity. These studies demonstrate a novel role for zinc in regulating oligodendrocyte excitotoxicity and identify new therapeutic targets to prevent oligodendrocyte cell death in central nervous system trauma and disease.

Microglia contribute to ammonia-induced astrocyte swelling in culture.

Brain edema, a lethal complication of acute liver failure (ALF), is believed to be largely cytotoxic due to the swelling of astrocytes. Ammonia, a principal neurotoxin in ALF, has been strongly implicated in the development of the brain edema. It was previously shown that treatment of cultured astrocytes with ammonia (5 mM NH4Cl) results in cell swelling. While ammonia continues to exert a direct effect on astrocytes, it is possible that ammonia can affect other neural cells, particularly microglia. Microglia are capable of evoking an inflammatory response, a process known to contribute to the brain edema. We therefore examined the potential role of microglia in the mechanism of ammonia-induced astrocyte swelling. Conditioned media (CM) derived from ammonia-treated cultured microglia when added to cultured astrocytes resulted in significant cell swelling. Such swelling was synergistically increased when astrocytes were additionally treated with 5 mM ammonia. CM from ammonia-treated microglia also showed significant release of oxy-radicals and nitric oxide into the CM. CM from ammonia-treated microglia containing Tempol (a superoxide scavenger) or uric acid (a peroxynitrite scavenger) when added to astrocytes resulted in marked reduction in the cell swelling. Together, these studies indicate that microglia contribute to the ammonia-induced astrocyte swelling by a mechanism involving oxidative/nitrosative stress.

Primary cultures of astrocytes: their value in understanding astrocytes in health and disease.

During the past few decades of astrocyte research it has become increasingly clear that astrocytes have taken a central position in all central nervous system activities. Much of our new understanding of astrocytes has been derived from studies conducted with primary cultures of astrocytes. Such cultures have been an invaluable tool for studying roles of astrocytes in physiological and pathological states. Many central astrocytic functions in metabolism, amino acid neurotransmission and calcium signaling were discovered using this tissue culture preparation and most of these observations were subsequently found in vivo. Nevertheless, primary cultures of astrocytes are an in vitro model that does not fully mimic the complex events occurring in vivo. Here we present an overview of the numerous contributions generated by the use of primary astrocyte cultures to uncover the diverse functions of astrocytes. Many of these discoveries would not have been possible to achieve without the use of astrocyte cultures. Additionally, we address and discuss the concerns that have been raised regarding the use of primary cultures of astrocytes as an experimental model system.

Na-K-Cl cotransporter-1 in the mechanism of cell swelling in cultured astrocytes after fluid percussion injury.

Brain edema and associated increased intracranial pressure are major consequences of traumatic brain injury (TBI). An important early component of the edema associated with TBI is astrocyte swelling (cytotoxic edema). Mechanisms for such swelling, however, are poorly understood. Ion channels/transporters/exchangers play a major role in cell volume regulation, and a disturbance in one or more of these systems may result in cell swelling. To examine potential mechanisms in TBI-mediated brain edema, we employed a fluid percussion model of in vitro barotrauma and examined the role of the ion transporter Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) in trauma-induced astrocyte swelling as this transporter has been strongly implicated in the mechanism of cell swelling in various neurological conditions. Cultures exposed to trauma (3, 4, 5 atm pressure) caused a significant increase in NKCC1 activity (21%, 42%, 110%, respectively) at 3 h. At 5 atm pressure, trauma significantly increased NKCC1 activity at 1 h and it remained increased for up to 3 h. Trauma also increased the phosphorylation (activation) of NKCC1 at 1 and 3 h. Inhibition of MAPKs and oxidative/nitrosative stress diminished the trauma-induced NKCC1 phosphorylation as well as its activity. Bumetanide, an inhibitor of NKCC1, significantly reduced the trauma-induced astrocyte swelling (61%). Silencing NKCC1 with siRNA led to a reduction in trauma-induced NKCC1 activity as well as in cell swelling. These findings demonstrate the critical involvement of NKCC1 in the astrocyte swelling following in vitro trauma, and suggest that blocking NKCC1 activity may represent a useful therapeutic strategy for the cytotoxic brain edema associated with the early phase of TBI.

The Na-K-Cl cotransporter in the brain edema of acute liver failure.

BACKGROUND & AIMS: Astrocyte swelling and brain edema associated with increased intracranial pressure are major complications of acute liver failure (ALF). The mechanism for such astrocyte swelling/brain edema, however, is not well understood. We recently found that ammonia, a key etiological factor in ALF, caused the activation of the Na-K-Cl cotransporter-1 (NKCC1) in cultured astrocytes, and that inhibition of such activation led to a reduction in astrocyte swelling, suggesting that NKCC1 activation may be an important factor in the mechanism of brain edema in ALF. To determine whether NKCC activation is also involved in brain edema in vivo, we examined whether NKCC activation occurs in the thioacetamide (TAA) rat model of ALF and determined whether treatment with the NKCC inhibitor bumetanide reduces the severity of brain edema in TAA-treated rats. METHODS: Brain water content was measured using the gravimetric method. NKCC1 phosphorylation and protein expression were measured by Western blots. NKCC activity was measured in brain cortical slices. RESULTS: NKCC activity was elevated in brain cortical slices of TAA-treated rats as compared to sham animals. Western blot analysis showed significant increases in total as well as phosphorylated (activated) NKCC1 protein expression in the cortical tissue. These findings were associated with a significant increase in brain water content which was attenuated by treatment with the NKCC inhibitor bumetanide. CONCLUSIONS: Our studies suggest the involvement of NKCC in the development of brain edema in experimental ALF, and that targeting NKCC may represent a useful therapeutic strategy in humans with ALF.

Interaction between cytokines and ammonia in the mitochondrial permeability transition in cultured astrocytes.

Hepatic encephalopathy (HE) is the major neurological complication occurring in patients with acute and chronic liver failure. Elevated levels of blood and brain ammonia are characteristic of HE, and astrocytes are the primary target of ammonia toxicity. In addition to ammonia, recent studies suggest that inflammation and associated cytokines (CKs) also contribute to the pathogenesis of HE. It was previously established that ammonia induces the mitochondrial permeability transition (mPT) in cultured astrocytes. As CKs have been shown to cause mitochondrial dysfunction in other conditions, we examined whether CKs induce the mPT in cultured astrocytes. Cultures treated with tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interferon-γ, individually or in a mixture, resulted in the induction of the mPT in a time-dependent manner. Simultaneous treatment of cultures with a mixture of CKs and ammonia showed a marked additive effect on the mPT. As oxidative stress (OS) is known to induce the mPT, so we examined the effect of CKs and ammonia on hemeoxygenase-1 (HO-1) protein expression, a marker of OS. Such treatment displayed a synergistic effect in the upregulation of HO-1. Antioxidants significantly blocked the additive effects on the mPT by CKs and ammonia, suggesting that OS represents a major mechanism in the induction of the mPT. Treatment of cultures with minocycline, an antiinflammatory agent, which is known to inhibit OS, also diminished the additive effects on the mPT caused by CKs and ammonia. Induction of the mPT in astrocytes appears to represent a major pathogenetic factor in HE, in which CKs and ammonia are critically involved.

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

Brain edema and the associated increase in intracranial pressure are potentially lethal complications of acute liver failure (ALF). Astrocyte swelling (cytotoxic edema) represents a significant component of the brain edema in ALF, and elevated blood and brain ammonia levels have been strongly implicated in its formation. We earlier showed in cultured astrocytes that oxidative stress (OS) and the mitochondrial permeability transition (mPT) play major roles in the mechanism of ammonia-induced astrocyte swelling. Glutamine, a byproduct of ammonia metabolism, has also been shown to induce OS, the mPT, and astrocyte swelling. Such effects of glutamine were suggested to be mediated by its hydrolysis in mitochondria, potentially yielding high levels of ammonia in this organelle and leading to OS and the mPT. L-histidine, an inhibitor of mitochondrial glutamine transport, was recently shown to mitigate OS, mPT, and cell swelling in cultured astrocytes treated with ammonia. The present study examined whether L-histidine similarly abolishes OS, the mPT, and brain edema in a rat model of ALF. Treatment of rats with thioacetamide caused a significant degree of brain edema, which was associated with induction of OS and the mPT. These changes were completely abolished by L-histidine, supporting a key role of mitochondrial glutamine transport and hydrolysis in the mechanism of the brain edema associated with ALF.

Multi-Organ Histopathological Changes in a Mouse Hepatitis Virus Model of COVID-19.

Infection with SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, causes a respiratory illness that can severely impact other organ systems and is possibly precipitated by cytokine storm, septic shock, thrombosis, and oxidative stress. SARS-CoV-2 infected individuals may be asymptomatic or may experience mild, moderate, or severe symptoms with or without pneumonia. The mechanisms by which SARS-CoV-2 infects humans are largely unknown. Mouse hepatitis virus 1 (MHV-1)-induced infection was used as a highly relevant surrogate animal model for this study. We further characterized this animal model and compared it with SARS-CoV-2 infection in humans. MHV-1 inoculated mice displayed death as well as weight loss, as reported earlier. We showed that MHV-1-infected mice at days 7-8 exhibit severe lung inflammation, peribronchiolar interstitial infiltration, bronchiolar epithelial cell necrosis and intra-alveolar necrotic debris, alveolar exudation (surrounding alveolar walls have capillaries that are dilated and filled with red blood cells), mononuclear cell infiltration, hyaline membrane formation, the presence of hemosiderin-laden macrophages, and interstitial edema. When compared to uninfected mice, the infected mice showed severe liver vascular congestion, luminal thrombosis of portal and sinusoidal vessels, hepatocyte degeneration, cell necrosis, and hemorrhagic changes. Proximal and distal tubular necrosis, hemorrhage in interstitial tissue, and the vacuolation of renal tubules were observed. The heart showed severe interstitial edema, vascular congestion, and dilation, as well as red blood cell extravasation into the interstitium. Upon examination of the MHV-1 infected mice brain, we observed congested blood vessels, perivascular cavitation, cortical pericellular halos, vacuolation of neuropils, darkly stained nuclei, pyknotic nuclei, and associated vacuolation of the neuropil in the cortex, as well as acute eosinophilic necrosis and necrotic neurons with fragmented nuclei and vacuolation in the hippocampus. Our findings suggest that the widespread thrombotic events observed in the surrogate animal model for SARS-CoV-2 mimic the reported findings in SARS-CoV-2 infected humans, representing a highly relevant and safe animal model for the study of the pathophysiologic mechanisms of SARS-CoV-2 for potential therapeutic interventions.

Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes.

BACKGROUND: Brain edema leading to high intracranial pressure is a lethal complication of acute liver failure (ALF), which is believed to be cytotoxic due to swelling of astrocytes. In addition to the traditional view that elevated levels of blood and brain ammonia are involved in the mechanism of brain edema in ALF, emerging evidence suggests that inflammatory cytokines also contribute to this process. We earlier reported that treatment of astrocyte cultures with a pathophysiological concentration of ammonia (5 mM NH4Cl) resulted in the activation of nuclear factor-kappaB (NF-κB) and that inhibition of such activation diminished astrocyte swelling, suggesting a key role of NF-κB in the mechanism of ammonia-induced astrocyte swelling. Since cytokines are also well-known to activate NF-κB, this study examined for additive/synergistic effects of ammonia and cytokines in the activation of NF-κB and their role in astrocyte swelling. METHODS: Primary cultures of astrocytes were treated with ammonia and cytokines (TNF-α, IL-1, IL-6, IFN-γ, each at 10 ng/ml), individually or in combination, and cell volume was determined by the [3H]-O-methylglucose equilibration method. The effect of ammonia and cytokines on the activation of NF-κB was determined by immunoblots. RESULTS: Cell swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h. Simultaneous co-treatment with cytokines and ammonia showed no additional swelling. By contrast, cultures pretreated with ammonia for 24 h and then exposed to cytokines for an additional 24 h, showed a marked increase in astrocyte swelling (129%). Treatment of cultures with ammonia or cytokines alone also activated NF-κB (80-130%), while co-treatment had no additive effect. However, in cultures pre-treated with ammonia for 24 h, cytokines induced a marked activation of NF-κB (428%). BAY 11-7082, an inhibitor of NF-κB, completely blocked the astrocyte swelling in cultures pre-treated with ammonia and followed by the addition of a mixture of cytokines. CONCLUSION: Our results indicate that ammonia and a mixture of cytokines each cause astrocyte swelling but when these agents are added simultaneously, no additive effects were found. On the other hand, when cells were initially treated with ammonia and 24 h later given a mixture of cytokines, a marked potentiation in cell swelling and NF-κB activation occurred. These data suggest that the potentiation in cell swelling is a consequence of the initial activation of NF-κB by ammonia. These findings provide a likely mechanism for the exacerbation of brain edema in patients with ALF in the setting of sepsis/inflammation.

Central pontine myelinolysis: historical and mechanistic considerations.

Central pontine myelinolysis (CPM) is a demyelinating condition affecting not only the pontine base, but also involving other brain areas. It usually occurs on a background of chronic systemic illness, and is commonly observed in individuals with alcoholism, malnutrition and liver disease. Studies carried out 25-30 years ago established that the principal etiological factor was the rapid correction of hyponatremia resulting in osmotic stress. This article reviews progress achieved since that time on its pathogenesis, focusing on the role of organic osmolytes, the blood-brain, barrier, endothelial cells, myelinotoxic factors triggered by osmotic stress, and the role of various factors that predispose to the development of CPM. These advances show great promise in providing novel therapeutic options for the management of patients afflicted with CPM.