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Ammonia and the Neutrophil in the Pathogenesis of Hepatic Encephalopathy in Cirrhosis

Debbie L. Shawcross, Shabnam S. Shabbir,* Nicholas J. Taylor,* and Robin D. Hughes

Hepatic encephalopathy (HE) constitutes a neuropsychiatric syndrome which remains a major clinical problem in patients with cirrhosis. In the severest form of HE, cirrhotic patients may develop varying degrees of confusion and coma. Ammonia has been regarded as the key precip- itating factor in HE, and astrocytes have been the most commonly affected cells neuropathologi- cally. Although the evidence base supporting a pivotal role of ammonia is robust, in everyday clinical practice a consistent correlation between the concentration of ammonia in the blood and the manifest symptoms of HE is not observed. More recently the synergistic role of inflammation and infection in modulating the cerebral effects of ammonia has been shown to be important. Furthermore, it has been recognized that infection impairs brain function both in the presence and absence of liver disease. Thus it could be postulated that in the presence of ammonia, the brain is sensitized to a systemic inflammatory stimulus and is able to elicit an inflammatory response involving both proinflammatory and neurotransmitter pathways. Ammonia is not only directly toxic to astrocytes but induces neutrophil dysfunction with the release of reactive oxygen species, which contribute to oxidative stress and systemic inflammation. This may further exac- erbate the cerebral effects of ammonia and potentially reduce the capacity of the neutrophil to fight microbial attack, thus inducing a vicious circle. This evidence supports the neutrophil in addition to ammonia as being culpable in the pathogenesis of HE, making the neutrophil a target for future anti-inflammatory therapeutic strategies in addition to ammonia lowering therapies.

(HEPATOLOGY 2010;51:1062-1069.)

H epatic encephalopathy (HE) constitutes a neu- ropsychiatric syndrome associated with both acute and chronic liver dysfunction. In acute

liver failure, 25% of patients may develop significant brain swelling and increased intracranial pressure, but in subacute liver failure only 9% may be affected. 1 In cirrho-

sis, HE causes a range of neuropsychiatric disturbances spanning a spectrum of subtle abnormalities apparent only by performing psychometric testing (minimal HE)

Abbreviations: BDL, bile duct–ligated; HE, hepatic encephalopathy; LPS, lipo- polysaccharide; OB, oxidative burst; ROS, reactive oxygen species; SIRS, systemic inflammatory response syndrome; TLR, Toll-like receptor. From the Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, King’s College Hospital, London, UK. Received August 11, 2009; accepted October 1, 2009. D. L. Shawcross is funded by a 5-year UK Department of Health HEFCE Clinical Senior Lectureship and a Young Investigator Award (2008) from the Intensive Care Society. *These authors contributed equally to this work. Address reprint requests to: D. L. Shawcross, Institute of Liver Studies, King’s College London School of Medicine at King’s College Hospital, 3rd Floor Cheyne Wing, King’s College Hospital, Denmark Hill, London SE5 9RS, UK. E-mail:

debbie.shawcross@kcl.ac.uk; fax: (44)-020-3299-3167. Copyright © 2009 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.23367 Potential conflict of interest: Nothing to report.

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through to more clinically apparent neurological signs and symptoms. In the most severe form of HE, patients may develop varying degrees of confusion, stupor and coma. 2 Minimal HE is thought to be a disorder of exec- utive functioning primarily leading to impairments in se- lective attention, response inhibition, and working memory. This frequently affects quality of life and been shown to impair the ability to drive a motor vehicle. 3

The Ammonia Hypothesis

Ammonia has been regarded as the key precipitating factor in HE since the first description of the development of a neurobehavioural syndrome (meat intoxication syn- drome) in portocaval shunted dogs by Nencki et al. in their seminal paper of 1896. 4 When ammonium salts were administered to the dogs, they rapidly fell into coma and died. Ammonia was later confirmed as the main caus- ative factor of the meat intoxification syndrome by Mat- thews in 1922. 5 The role of ammonia became increasingly recognized as being important when Gabuzda, et al. 6 dis- covered that a cation exchange resin given to patients with ascites that absorbed sodium and released ammonium ions led to significant reversible neurological dysfunction that was indistinguishable from the syndrome we now

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know as HE. Blood ammonia concentration was subse- quently noted to be elevated in patients with liver disease and hepatic coma, the highest values being found in those patients who were comatosed. 7 In the presence of chronic liver dysfunction, urea syn- thesis is impaired and the brain acts as an alternative ma- jor ammonia detoxification pathway. Astrocytes have the ability to eliminate ammonia by the synthesis of glu- tamine through amidation of glutamate by the enzyme glutamine synthetase. Hyperammonemia leads to the ac- cumulation of glutamine within astrocytes, which exerts an osmotic stress that causes astrocytes to take in water and swell. 8 Low-grade brain edema has been demon- strated in patients with minimal HE undergoing liver transplantation using magnetic resonance imaging. A de- crease in magnetization transfer ratio indicative of in- creased brain water correlated with abnormalities in neuropsychological function and was reversed by liver transplantation. 9 Further support for the ammonia– glu- tamine– brain water hypothesis has been provided by in- ducing hyperammonemia in patients with cirrhosis through the oral administration of an amino acid solution mimicking the composition of hemoglobin (upper gas- trointestinal bleeding being a common precipitant of HE). An increase in brain glutamine, reduction in mag- netization transfer ratio, and significant deterioration in neuropsychological function was suggestive of an increase in brain water. 10

The Blood–Brain Barrier in Cirrhosis and Hepatic Encephalopathy

The blood– brain barrier is a dynamic structure con- sisting of vascular endothelial cells and pericytes, with astrocytes and neurons closely juxtaposed. Astrocytes pro- vide physical and nutritional support for neurons. Cere- bral blood flow is modulated by contact and communication between these cells which ultimately in- fluence the permeability of the blood– brain barrier. The blood– brain barrier remains anatomically intact in HE 11 but positron emission tomography studies have demon- strated an increased permeability surface area to ammonia with increasing severity of liver disease. 12 A recent positron emission tomography study compar- ing whole brain and regional brain oxygen consumption and blood perfusion in patients with cirrhosis and an acute episode of HE demonstrated that whole brain oxy- gen consumption and cerebral blood flow were signifi- cantly reduced. This was not the case in patients with cirrhosis and no evidence of overt HE or in healthy con- trols. Oxygen delivery was not the rate- limiting factor for oxygen consumption, suggesting that the reduced cere- bral blood flow results from a diminished brain oxygen

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requirement in patients with HE. 13 This may arise from an ammonia-induced reduction in brain metabolism 14 or increased GABAergic tone 15 leading to an inhibition of neuronal activity. Alzheimer type II astrocytosis is characteristically seen in patients with cirrhosis. Protoplasmic astrocytes are found in increased numbers in patients with cirrhosis dying of hepatic coma and are typically deformed. Characteristically they ex- hibit a large swollen pale nucleus, prominent nucleolus, mar- gination of the chromatin pattern, and expansion of the cytoplasm with proliferation of cytoplasmic organelles. These neuropathological findings have been replicated in the brains of patients with congenital abnormalities of the urea cycle enzymes resulting in hyperammonemia, 16 in experi- mental animal models 17,18 and astrocyte cultures exposed chronically to ammonia. 19 Unfortunately, we lack a good animal model of cirrho- sis and HE in which to study the changes in the blood– brain barrier. 20 Ammonia-fed bile duct–ligated (BDL) rats have increased cerebral ammonia and demonstrate the presence of type II Alzheimer astrocytosis analogous to patients with cirrhosis but are not representative of a model of overt HE. 21

Infection Versus Inflammation in Cirrhosis

Patients with cirrhosis are prone to developing infection, which complicates their clinical course and frequently leads to the development of organ failure and death. Patients with cirrhosis are functionally immunosuppressed and have im- pairment of host defense mechanisms. The hemodynamic derangement of cirrhosis resembles that produced by endo- toxin, and bacteremia can greatly exacerbate this state. In- deed, patients with cirrhosis may display a sepsis-like immune paralysis 22 and a reduction in monocyte human leukocyte antigen DR expression. 23 Neutrophils are the most abundant white blood cells within the body and are rapidly recruited to sites of acute infection/inflammation. Neutrophils engulf invading mi- crobes and cell debris (phagocytosis); they then proceed to eliminate them by generating reactive oxygen species (ROS) through a process termed oxidative burst (OB). The products of OB not only eradicate invading micro- organisms, but may also damage innocent bystanders leading to tissue destruction and organ failure (Fig. 1). Neutrophils and macrophages have a reduced capacity to phagocytose and eliminate engulfed microbes and cell debris in patients with cirrhosis. Mookerjee et al. 24 have recently demonstrated neutrophil dysfunction with high spontaneous OB and reduced phagocytosis in patients with alcoholic hepatitis and cirrhosis. This was associated with a significantly greater risk of infection, organ failure and mortality.

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1064 SHAWCROSS ET AL. HEPATOLOGY, March 2010 Fig. 1. An activated neutrophil undergoing oxidative burst. Neutro-

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Fig. 1. An activated neutrophil undergoing oxidative burst. Neutro- phils engulf invading microbes and cell debris (phagocytosis); they then proceed to eliminate them by gen- erating ROS through a process termed oxidative burst. The products of oxidative burst not only eradicate invading micro-organisms, but may also damage innocent bystanders, leading to tissue destruction and or- gan failure.

The terms infection and inflammation are frequently used interchangeably. Systemic inflammatory response syndrome (SIRS) 25 results from the release and circula- tion of proinflammatory cytokines and mediators. This may result directly from liver injury and damage to hepa- tocytes (e.g., acetaminophen hepatotoxicity or alcoholic hepatitis) or can arise peripherally from the production of ROS and tissue destruction (e.g., pancreatitis or burns). Alternatively, it arises as a sequelae from local and sys- temic infection. Although they are functionally related, they should be regarded as separate entities. However, it is often difficult to delineate the two in patients with cirrho- sis and low-grade endotoxemia, who are prone to devel- oping infection and where microbial cultures often have a low yield. Furthermore, the extent of inflammation is also dependent on the cause of the liver injury and the severity of liver disease. Needless to say, infection is a frequent precipitating factor for HE and it is not unusual for changes in mental state to be the sole clinical manifesta- tion of infection in this population.

Infection and the Blood–Brain Barrier

The recognition that infection causes brain dysfunc- tion dates back to the rudimentary observations of Hip- pocrates over 2,500 years ago. In 200 A.D., Galen went on to describe delirium as a condition that emanated from the effect of inflammation on the mind. These early the- ories were later consolidated by Osler, and it is now rou- tinely accepted that sepsis is a cause of agitation and delirium.

Sepsis-associated encephalopathy is characterized by changes in mental status and motor activity, ranging from delirium to coma. Indeed, one-third of patients with sep- sis have a reduced level of consciousness, which is an in- dependent prognostic factor for death. Agitation and somnolence may occur alternately. Furthermore, para- tonic rigidity, asterixis, tremor, and myoclonus are not infrequently observed. It occurs due to alterations in ce- rebral blood flow, brain metabolites, and the release of inflammatory mediators; importantly, this happens with- out direct infection of brain tissue. 26 Although sepsis- associated and hepatic encephalopathy have distinct pathophysiological mechanisms, it is not inconceivable that infection may influence changes in mental status in patients with and without underlying liver disease. During an episode of sepsis, cytokines (15-20 kDa) can- not diffuse across the blood– brain barrier and are unable to have a direct effect. Nevertheless, the peripheral immune system can signal the brain to elicit a response through the expression of proinflammatory cytokines, both in the pe- riphery and in the brain. Brain signaling may occur through direct transport of the cytokine across the blood– brain bar- rier by way of an active transport mechanism, the interaction of the cytokine with circumventricular organs and activation of afferent neurons of the vagus nerve. 27 The circumven- tricular organs express components of innate and adaptive immune systems and are strategically located close to neu- roendocrine nuclei. Furthermore, the endothelial cell, along with the astrocyte, is a major constituent of the blood– brain barrier (Fig. 2). Endothelial cells are activated during sepsis,

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HEPATOLOGY, Vol. 51, No. 3, 2010 SHAWCROSS ET AL. 1065 Fig. 2. A pictorial representation of

Fig. 2.

A pictorial representation of the blood– brain barrier in the pathogenesis of HE. The relationship between the astrocyte, endothelial cell,

neuron, microglial cell, neutrophil, ammonia (NH 3 ), microbial pathogen/LPS, and proinflammatory cytokines is depicted.

resulting in the release of various mediators into the brain. Moreover, activated microglial cells and astrocytes have the ability to produce a full repertoire of cytokines in response to inflammation and injury. Interleukin-1 and TNF- are released early in sepsis and can also influence the permeabil- ity of the blood– brain barrier. 28,29 Endothelial cells have re- ceptors for interleukin-1 and TNF- that can transduce signals that ultimately culminate in the intracerebral synthe- sis of NO and prostanoids. Perivascular cells of macrophage origin are also targets for these cytokine effects.

Inflammation and Infection in Modulating the Manifestation of Hepatic Encephalopathy in Cirrhosis

Although the evidence base supporting a pivotal role of ammonia is robust, in clinical practice a consistent correlation between the concentration of ammonia in the blood and the manifest symptoms of HE is not always seen. In patients with cirrhosis, there is mount-

ing evidence for the role of SIRS in exacerbating the symptoms of HE. Studies have now shown this to be the case in patients with minimal HE and across the whole spectrum of patients with varying degrees of overt HE. A recent study has confirmed that the pres- ence and severity of minimal HE in cirrhosis are inde- pendent of the severity of liver disease and plasma ammonia concentration, but markers of systemic in- flammation are significantly higher in those with min- imal HE compared with those without. 30 In a further study, significant deterioration of neuropsychological test scores in patients with cirrhosis following induced hyperammonemia during infection, but not after its resolution, suggested that infection may be important in modulating the cerebral effect of ammonia in liver disease, supporting an inflammatory hypothesis. 31 In severe HE (grade 3/4) in cirrhosis, a prospective study found 46% of patients to have positive cultures, and a further 20% had evidence of sterile SIRS. Increasing

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grades of HE were associated with SIRS and neutro- philia, but not arterial ammonia concentration. 32 Lipopolysaccharide (LPS) injected into a healthy rat hippocampus results in long-term microglial activation and a decrease in glutamatergic transmission that leads to learning and memory deficits without inducing neuronal death. 33 In a chronic neuroinflammation rat model in- duced by LPS this was reversed by the administration of the glutamatergic antagonist memantine and an inhibitor of cyclo-oxygenase 2. 34 In a portocaval shunted rat model that is more akin to a model of minimal HE, Cauli et al. 35 have demonstrated an improved learning ability follow- ing the administration of 5 to 6 times the normal thera- peutic dose of the nonsteroidal anti-inflammatory drug, ibuprofen. 35

Infection and Inflammation Act Synergistically with Ammonia in Cirrhosis

As inflammation, infection, and ammonia have all been shown to be important in HE, the question has to be raised as to whether infection and inflammation have a synergistic relationship with ammonia. Ammonia-fed BDL rats have increased brain water compared with BDL controls, alluding to a potential synergistic relationship between ammonia and systemic inflammation. 21 LPS ad- ministration increased brain water in ammonia-fed, BDL, and sham-operated animals significantly, but this was as- sociated with the progression to pre-coma only in the BDL animals. LPS induced cytotoxic brain swelling, but the anatomical integrity of the blood– brain barrier was maintained. Nitrosation of proteins in the frontal cortex of BDL and LPS-treated animals was demonstrated. However, ammonia cannot be responsible alone because protein nitrosation was not demonstrated in ammonia- fed sham-operated and ammonia-fed BDL rats in the ab- sence of an inflammatory stimulus. Therefore, both ammonia and an additional inflammatory insult may need to be present for nitrosation of brain proteins to occur in animals with subliminal inflammation such as that which has been observed in the BDL model. 11

Are Neutrophils Culpable in HE?

If ammonia and inflammation/infection act synergis- tically, then it is logical to question whether ammonia itself may directly impair immunity and predispose to the development of inflammation/infection. Indeed, ammo- nia impairs neutrophil chemotaxis, 36 phagocytosis, de- granulation, and stimulated OB. 37 In a proof of concept study, 38 normal neutrophils incubated with 75 M am- monium chloride (typical of the concentrations seen in patients with cirrhosis) in vitro demonstrated swelling,

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reduced capacity to engulf opsonized Escherichia coli, and high spontaneous OB. These findings were replicated in ammonia-fed rats and ex vivo in patients with stable cir- rhosis given an amino acid solution inducing hyperam- monemia compared with controls. These observations were consistent with the development of neutrophil swell- ing. A similar reduction in phagocytosis following induc- tion of hyponatremia, which is a well-known stimulus for cell swelling, supports neutrophil swelling as a potential mechanism to explain this neutrophil dysfunction. In- deed, hyponatremia is an independent predictor of mor- tality and may predispose to infection in cirrhosis. 39 It is therefore perhaps not a surprise to find that the effects of hyponatremia and ammonia were additive, causing more pronounced neutrophil swelling and phagocytic dysfunc- tion. Shawcross et al. 38 were able to show evidence of p38 -MAPK activation in ammonia-exposed neutrophils— the p38 -MAPK pathway being an important regulator of cell volume, driver of transcription of inflammatory genes, and regulator of neutrophil apoptosis. A p38 -MAPK agonist abrogated the ammonia-induced swelling and im- pairment of phagocytosis. This was at the expense of in- ducing spontaneous OB in unstimulated neutrophils. The impact of ammonia on the p38 -MAPK pathway and cell volume regulation has been supported by the findings in primary astrocyte cultures exposed to supraphysiologi- cal concentrations of ammonia 40 and in hepatocytes. 41 These observations implicate ammonia in the pathogen- esis of neutrophil swelling and dysfunction, which may play an important role in the predisposition to infection and inflammation in cirrhosis (Fig. 3).

Cytokines, Chemokines and the Neutrophil in Cirrhosis

Bacterial translocation of organisms from the gut in patients with cirrhosis and portal hypertension results in chronic endotoxemia. 42 This culminates in a local milieu of proinflammatory cytokines/chemokines, which can up-regulate the adhesion receptor CD11b/CD18 (MAC-1 and complement 3b receptor), and activate neu- trophils through Toll-like receptors (TLR) and chemo- kine receptors (CXCR1 and CXCR2). Stadlbauer et al. 43 demonstrated increased expression of TLR-2, TLR-4, and TLR-9 and decreased expression of CXCR1 and CXCR2 in normal neutrophils incubated with plasma from patients with alcoholic hepatitis. This was associated with phagocytic dysfunction and increased spontaneous OB, endotoxemia, and energy depletion, which was pre- vented by incubation with albumin, an endotoxin scav- enger. Inhibition of TLR-2, TLR-4, and TLR-9 prevented an increase in spontaneous OB and CXCR1/

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HEPATOLOGY, Vol. 51, No. 3, 2010 SHAWCROSS ET AL. 1067 Fig. 3. A diagrammatic representation of

Fig. 3. A diagrammatic representation of the downstream events resulting from the interaction between the neutrophil and infection/LPS that results in neutrophil chemotaxis, adhesion, migration, phagocytosis, and the production of a full repertoire of chemokines, proinflammatory cytokines, proteases, ROS, and transcription of inflammatory target genes. Ammonia impairs neutrophil chemotaxis and phagocytosis while up-regulating myeloperoxidase activity and increasing spontaneous oxidative burst.

CXR2 expression but did not improve phagocytosis. Ex vivo removal of endotoxin from the plasma of patients with alcoholic hepatitis and cirrhosis decreased neutro- phil spontaneous OB and improved phagocytosis. 24 In chronic endotoxemia, the adaptive immune system plays a key role in limiting overzealous neutrophil activa- tion by decreasing survival mediated by T regulatory cells. 44 Therefore, the interaction of neutrophils and T regulatory cells in cirrhosis and hyperammonemia war- rants further investigation.

Neutrophils in the Pathogenesis of HE

The rapid recruitment of activated neutrophils to the liver in patients with alcoholic hepatitis/liver injury, and data that supports the development of organ failure in sepsis (both conditions commonly being associated with encephalopathy) results from an inappropriately vigorous response of neutrophils to an inflammatory stimulus. Therefore, in the context of a patient with cirrhosis, hy- perammonemia and chronic endotoxemia, where it has already been shown that neutrophils are pre-primed and have a reduced ability to eliminate bacteria, then it would

be logical to suppose that there will be enhanced endothe- lial–neutrophil interaction within the cerebral microcir- culation. This would result in neutrophil adhesion, migration across the blood– brain barrier and the produc- tion of chemokines, proinflammatory cytokines, pro- teases, ROS, and transcription of inflammatory target genes (Fig. 3). It is within this inflammatory milieu that the cerebral effects of ammonia (with or without super- imposed infection) will have their greatest impact. Fur- thermore, astroglial activation promotes neutrophil recruitment by the production of neutrophil-specific che- mokines. 45

The Neutrophil as a Therapeutic Target in HE

Historically, treatments for HE have been based upon the hypothesis that the colon is the primary source of ammonia and have included dietary protein restriction, the use of nonabsorbable disaccharides and nonabsorb- able antibiotics. These therapies have focused on lowering arterial ammonia and modulating interorgan ammonia metabolism but remain largely ineffective.

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Although ammonia could potentially be responsible for the development of neutrophil dysfunction, a patient with cirrhosis also represents a model of chronic endotox- emia that has direct implications on the innate and adap- tive immune systems. We must therefore also look to therapies that directly or indirectly target the proinflam- matory milieu and enhance neutrophil and immune func- tion. However, caution must be observed in this regard, because a paradox exists in terms of developing pharma- cotherapeutic agents that enhance neutrophil function but that might potentially exacerbate organ damage by increasing oxidative stress and bystander damage. Potential therapeutic strategies might include the use of granulocyte colony-stimulating factor, leukodepletion, antagonism of proinflammatory cytokines or their recep- tors, antioxidants, anti-inflammatories, probiotics, and hypothermia. Excitement surrounds the prospect of TLR-2, TLR-4, and TLR-9 inhibitors and small mole- cules that modulate TLR-4 signaling, which could poten- tially down-regulate neutrophil activation and other cellular responses. Patients with advanced cirrhosis have been shown to have alterations in the functional capacity of albumin. 46 This supports the exploration of the admin- istration of albumin as an endotoxin scavenger in large randomized clinical trials and may explain the beneficial role of albumin dialysis on HE. 47 Modulation of intestinal microbiota is an emerging strategy to reduce the bacterial translocation of LPS and other bacterial activators of TLRs. Probiotics have been shown to reduce bacterial translocation and were shown to improve liver function and prevent the development of infection and HE in patients with cirrhosis. 48 Further- more, probiotics have been shown to restore neutrophil phagocytic capacity in patients with alcoholic cirrhosis, possibly by reducing endogenous levels of interleukin-10 and TLR-4 expression. 49 Recent studies show that hypothermia is efficacious in patients with uncontrolled intracranial hypertension who are undergoing liver transplantation. Hypothermia dis- plays many beneficial effects on brain water and intracra- nial hypertension relating to decreased brain ammonia, cerebral blood flow, mediators of inflammation, and oxi- dative stress. 50 Moderate hypothermia (33°C) abolishes ammonia-induced neutrophil spontaneous OB without impairing phagocytic capacity, suggesting that hypother- mia could be a valuable tool not only in patients present- ing with acute liver failure, but in patients with cirrhosis and grade 3/4 HE. 51

Conclusions

The synergistic role of inflammation and infection in modulating the cerebral effects of ammonia has been

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highlighted, and it has been demonstrated that astrocytes and endothelial cells respond to a systemic inflammatory stimulus and play a role in eliciting an inflammatory re- sponse involving a number of proinflammatory and neu- rotransmitter pathways. Ammonia is not only directly toxic to astrocytes but induces neutrophil dysfunction and the release of ROS. It is therefore becoming increasingly recognized that neu- trophils play an important role in the pathogenesis of HE, and further work is merited to define their precise role. It is clear that neutrophil dysfunction predisposes to infec- tion but may also have a more direct pathogenic role in HE by promoting increased endothelial cell interaction in the cerebral microcirculation exacerbating astrocyte oxi- dative stress and endothelial disruption. This supports the neutrophil as being culpable in HE, making it a novel pharmacotherapeutic target in a condition where current therapies are inadequate.

Acknowledgment: Debbie Shawcross would like to acknowledge Professor Rajiv Jalan, Dr. Gavin Wright, Dr. Nathan Davies, Dr. Vanessa Stadlbauer, and other members of the Liver Failure Group at the Institute of Hepatology, University College London, for their signif- icant contributions to the area of research discussed in this review.

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