Brain Edema In Fulminant Hepatic Failure Research Articles

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA–LPA6–G12/13–Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema.

Matrix metalloproteinase‐9 in fulminant hepatic failure†

We read with interest the article by Chen et al.,1 who found altered expression of several tight junction (TJ) proteins in cultured brain endothelial cells and brain from mice with acute liver failure (ALF) and relate these abnormalities to the activation of matrix metalloproteinase-9 (MMP-9). The results are in accordance to prior data in the same animal model of toxic liver injury (azoxymethane).2 Their findings led to the proposal that MMP-9 released by the necrotic liver could alter the expression of TJ proteins and cause blood-brain barrier (BBB) leakage and brain swelling.3 The hypothesis is interesting because it could result in new treatments for this severe complication of fulminant hepatic failure (FHF). To further explore the relevance of this hypothesis, we determined the plasmatic levels of MMP-9 (via enzyme-linked immunosorbent assay) in 32 patients with FHF and compared the values to those obtained in 11 patients with acute hepatitis A and 20 patients with advanced (Child class B/C) hepatic cirrhosis with or without hepatic encephalopathy. During the follow-up of patients with FHF, intracranial hypertension was diagnosed in 14 patients (confirmed by intracranial pressure monitor in 13). We found high levels of MMP-9 compared to normal reference values in all group of patients (Fig. 1). Patients with acute liver injury (FHF or acute hepatitis A) showed higher values than those with chronic liver failure (cirrhosis). There was no association between MMP-9 and intracranial hypertension. Plasmatic levels of MMP-9 as determined by an enzyme-linked immunosorbent assay. FHF, fulminant hepatic failure; HC, hepatic cirrhosis; HE, hepatic encephalopathy; ICH, intracranial hypertension. *Compared to controls. #Compared to HC with or without HE. The presence of high levels of MMP-9 in our patients can be explained by remodeling liver parenchyma during acute and chronic liver injury.4 The lack of relationship with intracranial hypertension does not invalidate that MMP-9 can cause disturbances of TJ proteins. However, our results are in accordance with a series of data indicating that brain edema in FHF is mostly secondary to cytotoxic mechanisms. In vasogenic edema, brain swelling relates to leakage of the BBB and develops in the extracellular compartment. In comparison, cytotoxic edema develops secondary to osmotic differences across the BBB or energy failure and causes accumulation of water in the intracellular compartment. Most available data indicate that the BBB is grossly intact in FHF.5 Magnetic resonance shows a decrease in the apparent diffusion coefficient in humans6 and in rats,7 which is in accordance with an increase of water in the intracellular compartment. Our findings indicate that MMP-9 is increased in the plasma of patients with FHF, but does not participate in the pathogenesis of brain edema. MMPs are big molecules that must cross the BBB to exert their function in brain tissue. In patients and experimental models of stroke, the effects of MMPs are associated with neutrophil infiltration that may carry some of those MMPs in their tertiary granules.8 In previous neuropathological studies, no signs of neutrophil infiltration were detected in patients with FHF.5 Recently, Bémeur et al.9 performed a study in mice with azoxymethane-induced ALF, where they strictly controlled the temperature (a factor that affects the outcome of this model) and could not detect immunoglobulin G extravasation, in accordance with BBB integrity. Lluis Palenzuela Ph.D.* , Antoni Mas M.D. §, Joan Montaner M.D. , Juan Cordoba M.D.* §, * Liver Unit and Institut de Recerca, Hospital Universitari Vall d'Hebron, Barcelona, Spain, Neurovascular Research Laboratory, Hospital Universitari Vall d'Hebron, Barcelona, Spain, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos III, Madrid, Spain, § Liver Unit and Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clinic i Provincial, Barcelona, Spain.

Altered Expression of Zonula Occludens-2 Precedes Increased Blood–Brain Barrier Permeability in a Murine Model of Fulminant Hepatic Failure

Brain edema secondary to increased blood–brain barrier (BBB) permeability is a lethal complication in fulminant hepatic failure (FHF). Intact tight junctions (TJ) between brain capillary endothelial cells are critical for normal BBB function. However, the role of TJ in FHF has not been explored. We hypothesized that alterations in the composition of TJ proteins would result in increased BBB permeability in FHF. In this study, FHF was induced in C57BL/6J mice by using azoxymethane. BBB permeability was assessed with sodium fluorescein. Expression of TJ proteins was determined by Western blot, and their cellular distribution was examined using immunofluorescent microscopy. Comatose FHF mice had significant cerebral sodium fluorescein extravasation compared with control and precoma FHF mice, indicating increased BBB permeability. Western blot analysis showed a significant decrease in zonula occludens (ZO)-2 expression starting in the precoma stage. Immunofluorescent microscopy showed a significantly altered distribution pattern of ZO-2 in isolated microvessels from precoma FHF mice. These changes were more prominent in comatose FHF animals. Significant alterations in ZO-2 expression and distribution in the tight junctions preceded the increased BBB permeability in FHF mice. These results suggest that ZO-2 may play an important role in the pathogenesis of brain edema in FHF.

Glutamine in the mechanism of ammonia-induced astrocyte swelling

Brain edema and the subsequent increase in intracranial pressure are the major neurological complications in fulminant hepatic failure (FHF). Brain edema in FHF is predominantly “cytotoxic” due principally to astrocyte swelling. It is generally believed that ammonia plays a key role in this process, although the mechanism by which ammonia brings about such swelling is yet to be defined. It has been postulated that glutamine accumulation in astrocytes subsequent to ammonia detoxification results in increased osmotic forces leading to cell swelling. While the hypothesis is plausible and has gained support, it has never been critically tested. In this study, we examined whether a correlation exists between cellular glutamine levels and the degree of cell swelling in cultured astrocytes exposed to ammonia. Cultured astrocytes derived from rat brain cortices were exposed to ammonia (5 mM) for different time periods and cell swelling was measured. Cultures treated with ammonia for 1–3 days showed a progressive increase in astrocyte cell volume (59–127%). Parallel treatment of astrocyte cultures with ammonia showed a significant increase in cellular glutamine content (60–80%) only at 1–4 h, a time when swelling was absent, while glutamine levels were normal at 1–3 days, a time when peak cell swelling was observed. Thus no direct correlation between cell swelling and glutamine levels was detected. Additionally, acute increase in intracellular levels of glutamine by treatment with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine (DON) after ammonia exposure also did not result in swelling. On the contrary, DON treatment significantly blocked (66%) ammonia-induced astrocyte swelling at a later time point (24 h), suggesting that some process resulting from glutamine metabolism is responsible for astrocyte swelling. Additionally, ammonia-induced free radical production and induction of the mitochondrial permeability transition (MPT) were significantly blocked by treatment with DON, suggesting a key role of glutamine in the ammonia-induced free radical generation and the MPT. In summary, our findings indicate a lack of direct correlation between the extent of cell swelling and cellular levels of glutamine. While glutamine may not be acting as an osmolyte, we propose that glutamine-mediated oxidative stress and/or the MPT may be responsible for the astrocyte swelling by ammonia.

TIMP-1/MMP-9 Imbalance in Brain Edema in Rats With Fulminant Hepatic Failure

Fulminant hepatic failure (FHF) is a devastating disease. When coma sets in, brain edema develops, changing FHF into a lethal condition. Liver transplantation is the definitive treatment. However, a third of these patients die as the result of brain edema before a donor becomes available. Tissue inhibitor of matrix metalloproteinase (MMP), or TIMP, and MMP-9 are implicated in ischemic brain edema. We thus hypothesized that an imbalance in TIMP-1/MMP-9 relationship plays a role in the development of increased brain extravasation and edema in FHF. FHF was induced with a single intraperitoneal injection of D-galactosamine (250 mg/kg). Control rats received saline. GM6001, a synthetic MMP inhibitor, was administered (30 mg/kg) every 12 h for 3 doses starting at 12 h after D-galactosamine injection. MMP-9 was assayed with standard gelatin zymography. Brain extravasation, a measurement of the blood-brain barrier permeability, was determined with Evans blue. Brain edema was determined using specific gravity method. The active MMP-9 in the systemic circulation was significantly increased in the comatose FHF as compared to the precoma FHF and control animals (6.5 +/- 0.7 versus 4.6 +/- 0.4 versus 2.6 +/- 0.5 pg/microg, respectively; P < 0.05). Conversely, TIMP-1 was steadily decreased in precoma and coma FHF rats by 35% and 45%, respectively. Blocking MMP-9 activity with GM6001 significantly attenuated brain extravasation and edema in rats with FHF. Our study strongly supports that the perturbation of decreased TIMP-1 and increased MMP-9 contributes to the pathogenesis of brain edema in FHF. Our findings present a potential therapeutic approach to effectively increase the window of opportunity for life-saving liver transplantation.

Matrix metalloproteinase-9 contributes to brain extravasation and edema in fulminant hepatic failure mice

Fulminant hepatic failure (FHF) can be dreadful. When coma sets in, brain edema develops taking FHF into a lethal course. Mechanisms of brain extravasation leading to brain edema remain incompletely understood. Matrix metalloproteinase (MMP)-9 is implicated in various brain injuries. We hypothesized that MMP-9 contributes to brain edema in FHF. MMP-9 and its proform were assayed using SDS-PAGE and in situ gelatin zymographies. Brain extravasation was assessed with Evans blue. Brain water was determined by specific gravity and astrocytic endfoot swelling by electron microscopy. FHF in mice was induced by azoxymethane. MMP inhibitor GM6001 and MMP-9 monoclonal antibody were used. Active MMP-9 was significantly increased at the onset of coma and brain extravasation in FHF mice. Blocking MMP-9 with either GM6001 or MMP-9 monoclonal antibody significantly attenuated brain extravasation, astrocytic endfoot swelling, and brain edema. Brains of FHF mice did not show MMP-9 activity. In contrast, livers of these animals showed marked up-regulation of MMP-9 activity. Our findings suggest that MMP-9 contributes to the pathogenesis of brain extravasation and edema in FHF. The necrotic liver is the source of MMP-9 in FHF. Inhibition of MMP-9 may protect against the development of brain edema in FHF.

Fulminant hepatic failure

Abstract Analysis of the first 150 cases collected from 73 centers reporting to the Fulminant Hepatic Failure Surveillance Study showed that 80 patients had presumable viral hepatitis, of whom 62 died. Of 41 patients who had recent surgery, 36 died. In 35 patients massive liver necrosis developed less than three weeks after halothane anesthesia, and 27 (77 per cent) of these had multiple exposures to halothane. The risk from halothane is small, but this complication was present in nearly one quarter of the cases reported in our study. When the use of halothane is desired, multiple exposure should be avoided, if possible. Moreover, postoperative fever after exposure to halothane, otherwise "unexplained," is an important warning sign, and should be thoroughly investigated and taken into consideration when further exposure to this anesthetic is contemplated. The physician prescribing halothane must balance need against risk.

Suppression of ammonia-induced astrocyte swelling by cyclosporin A.

Brain edema is a serious complication of hepatic encephalopathy associated with fulminant hepatic failure (FHF). A major component of the edema seems to be cytotoxic, involving astrocyte swelling. Although the mechanism of brain edema in FHF is incompletely understood, it is generally believed that ammonia is involved critically in this process. Recent studies have shown that exposure of cultured astrocytes to ammonia results in the mitochondrial permeability transition (MPT), a phenomenon associated with mitochondrial failure and subsequent cellular dysfunction. The present study examined the potential role of the MPT in the astrocyte swelling associated with ammonia toxicity. Treatment of cultured astrocytes with ammonia (5 mM) caused a time-dependent increase in astrocyte cell volume (swelling), which was completely inhibited by the MPT inhibitor cyclosporin A (CsA). In this study, CsA also inhibited the ammonia-induced aquaporin 4 (AQP4) upregulation, which had been shown previously to be increased in cultured astrocytes by ammonia treatment. These findings suggest that the MPT plays a significant role in the ammonia-induced astrocyte swelling and may contribute to the brain edema associated with FHF.

Ammonia-induced astrocyte swelling in primary culture

The effect of ammonia on water space of astrocytes in culture was determined as a means of studying the neurotoxicity of ammonia in fulminant hepatic failure (FHF). Treatment of primary astrocyte cultures obtained from neonatal rat cortices with 10 mM NH4Cl for 4 days resulted in a 29% increase in astrocytic water space, as measured by an isotopic method utilizing 3-O-methyl-[3H]-glucose. This effect was time- and dose-dependent. The ammonia-induced swelling was reversible as the water space in cultures treated with 10 mH NH4Cl for 3 days, and then returned to normal culture media for 1 day, was similar to control cultures. These findings suggest that elevated levels of ammonia lead to astrocyte swelling and may contribute to the brain edema in FHF.