**3. The role of hepatocytes and endothelial cells**

Ammonia reaches the liver through the portal circulation and is purified by periportal hepatocytes, which incorporate it into urea synthesis, or by perivenular hepatocytes, which catalyze the condensation of glutamate and ammonia into glutamine by the action of glutamine synthetase [9]. The ammonia concentration in the portal vein ranges from 300 to 600 μmol, dropping to 20–60 μmol in the hepatic veins [12]. The liver, thus, plays a central role in the regulation of its levels and, in healthy individuals, removes it almost completely: small amounts of escaping ammonia are metabolized in the skeletal muscle (which also expresses glutamine synthetase), and in the kidneys (where more than 70% of it is reabsorbed). In case of hepatic failure and portosystemic shunt, ammonia escapes this detoxification process, increasing its serum concentration [9]. This leads the skeletal muscle to play an important role

in its clearance, but this metabolic pathway is not sufficient to eliminate it from the body and there is a loss of muscle mass in about 40–76% of those with cirrhosis [17]. Moreover, it is common for such patients to have concomitant zinc deficiency, an important cofactor for glutamine synthetase, which may aggravate its elimination [9].

In cirrhosis, hepatic gluconeogenesis is impaired. The amino acid precursors of glucose synthesis, such as alanine, threonine, glycine, and aspartate, are increased, whereas peripheral anaerobic glycolysis increases lactate and pyruvate levels [18]. Of particular importance, studies demonstrate that glycine may be an ammoniagenic amino acid, causing increased ammonia synthesis in the gut and brain through induction of a reaction mediated by glycine oxidase [19]. This has been explored as a potential therapeutic target, since the reaction is bi-directional and the removal of glycine can lead to the use of ammonia to replenish its stocks, lowering its levels [20].

On the other hand, the low systemic availability of glucose causes hepatocytes to produce more ketone bodies from fatty acids, for the energetic metabolism of nervous and muscular tissues. However, it is hypothesized that in situations like this, hepatocytes prioritize the production of energy for its own subsistence rather than synthesizing products destined for exportation to other tissues [18]. Thus, ketogenesis would also be impaired, which is corroborated by significantly decreased betahydroxybutyrate and acetoacetate levels, resulting in a precarious energy metabolism in the central nervous system in the advanced stages of the disease [18, 21].

Given its location and abundant vascular supply, with immense exposure to antigens absorbed by the intestine, the liver regulates important immune functions [9]. In cirrhosis, intestinal bacterial overgrowth associated with hepatocellular failure triggers a systemic immune reaction, bypassing endotoxins such as membrane lipopolysaccharides, flagellins, and peptidoglycans for arterial circulation [15, 22]. Circulating cytokines, such as tumor necrosis factor alpha (TNFα), interleukin 1b (IL-1b) and interleukin 6, induce the synthesis of nitric oxide and prostanoids in endothelial cells, triggering a state of inflammatory hyperemia that facilitates the uptake of ammonia by the central nervous system [9]. In addition, the proinflammatory cytokines generated by the vascular endothelium activate the cells of the immune system in the brain parenchyma and the microglia, contributing indirectly to neuroinflammation [11].

### **4. The role of astrocytes**

Astrocytes are part of the blood-brain barrier and protect neurons from the toxic effects of ammonia [12]. Its perivascular extensions are rich in aquaporin 4, a protein constituent of water channels. Astrocytes are among the cells with the highest glycolytic activity of the central nervous system and are estimated to be responsible for 30% of its metabolism. They are believed to be particularly susceptible to the development of edema because they are part of the glymphatic system [23], a paravascular system discovered in 2012, which receives continuous influx of periarterial cerebrospinal fluid and has a leakage network through the perivascular spaces into the cerebral veins [24].

Liver failure can result in an uncontrollable rise in ammonia levels, which penetrate virtually all organs. Although the central nervous system is partially protected by the blood-brain barrier, which remains relatively intact until advanced stages of the disease, excessive amounts of ammonia can overtake it [12, 25]. Therefore, concentrations that normally range from 0.2 to 0.3 μmol in normal subjects can reach the mark of 3 to 5 mmol in patients with hepatic encephalopathy [4]. However, along with perivenular hepatocytes and skeletal muscle, astrocytes express glutamine synthetase and have the ability to convert ammonia into glutamine [4, 9].

**41**

*The Neurobiology of Hepatic Encephalopathy DOI: http://dx.doi.org/10.5772/intechopen.86320*

The accumulation of glutamine in astrocytes, although not directly toxic, drastically affects its functioning [26]. Firstly, glutamine has an osmotic action, inducing predominantly cytotoxic and slightly vasogenic edema [25]. Generally, any form of edema increases the distance for diffusion of oxygen and metabolites in the brain parenchyma, exposing microareas of borderline irrigation to hypoxia [23]. This phenomenon is more pronounced in acute hepatic failure, in which the counterregulatory mechanisms do not have time to act, but can also be detected in the magnetic resonance imaging (MRI) of patients with chronic liver failure [9, 26]. Secondly, exceeding glutamine is transported to the mitochondria, where, by glutaminase action, it is hydrolyzed back into glutamate and ammonia. The passage of the latter to the interior of the mitochondria causes oxidative stress and modifies the internal mitochondrial membrane diffusivity, through the opening of permeability transition pore, causing water accumulation in the mitochondrial matrix, low capacity of oxidative phosphorylation, and low adenosine triphosphate (ATP) production [11, 12]. This results in a vicious cycle of formation of reactive oxygen

Studies with cultures of astrocytes and neurons show that only the former increase the production of free radicals when exposed to glutamine [12] and that's why astrocytes can be considered the basic morphofunctional unit of hepatic encephalopathy: the histopathological milestone of the disease is the swelling of astrocytes, both in the cytoplasm and in the nucleus, with chromatin marginalization, prominent nucleoli and glycogen accumulation, accompanied by little neuronal alteration [4, 27].

The effects of chronic hyperammonemia and astrocytic edema can be verified in specific sequences of brain MRI. In the spectroscopy of the basal ganglia, the Glx/ creatine ratio is increased and myo-inositol/ creatine and choline/ creatine ratios are decreased [14, 22]. Creatine is a constitutive marker of neurons and astrocytes. The increase of Glx demonstrates the accumulation of glutamine and glutamate [22]. This increase, however, seems to present large interindividual variations, and within a same animal model, there are forms in which there is a gradual increase, a strong increase followed by a plateau or only by a late rise [21]. Choline is a marker for the turnover of membrane phospholipids, and its decrease reflects reduction of basal metabolism of neurons and glial cells [22]. Furthermore, due to the osmotic imbalance generated by the accumulation of glutamine and glutamate, astrocytes export choline and myo-inositol, its main osmolyte, to the extracellular space, which leads to a reduction in the levels of the later, in an attempt to counterbalance the intracellular edema. This mechanism is known as regulatory volume decrease [11, 21, 23]. The diffusion-weighted imaging, in turn, shows interstitial edema resulting from the exportation of osmolytes from the astrocytes into the extracellular space, both in the white and gray matters. Because of that, multiple sites in the brain have an increase in mean diffusivity, including the frontal, temporal, inferior parietal, and insular lobes, as well as the corpus callosum, putamen, thalamus, and pons [22, 27]. The diffusion of water molecules, however, is not free; it reflects interactions with macromolecules, fibers, and membranes. It is important to emphasize that the diffusion-weighted sequences only show changes in the intra- and extracellular volume, and do not allow a definitive conclusion about the

and nitrogen species (free radicals) with mitochondrial damage [9].

total amount of water present in the brain parenchyma [23].

The activity of astrocytes and neurons can be modulated by microglia. The microg-

lial cells are innate of the immune system, have phagocytic function and perform active surveillance of the brain parenchyma. In the absence of inflammatory stimuli,

**5. The role of microglial cells**

#### *The Neurobiology of Hepatic Encephalopathy DOI: http://dx.doi.org/10.5772/intechopen.86320*

*Liver Disease and Surgery*

to neuroinflammation [11].

**4. The role of astrocytes**

spaces into the cerebral veins [24].

in its clearance, but this metabolic pathway is not sufficient to eliminate it from the body and there is a loss of muscle mass in about 40–76% of those with cirrhosis [17]. Moreover, it is common for such patients to have concomitant zinc deficiency, an important cofactor for glutamine synthetase, which may aggravate its elimination [9]. In cirrhosis, hepatic gluconeogenesis is impaired. The amino acid precursors of glucose synthesis, such as alanine, threonine, glycine, and aspartate, are increased, whereas peripheral anaerobic glycolysis increases lactate and pyruvate levels [18]. Of particular importance, studies demonstrate that glycine may be an ammoniagenic amino acid, causing increased ammonia synthesis in the gut and brain through induction of a reaction mediated by glycine oxidase [19]. This has been explored as a potential therapeutic target, since the reaction is bi-directional and the removal of glycine can lead to the use of ammonia to replenish its stocks, lowering its levels [20]. On the other hand, the low systemic availability of glucose causes hepatocytes to produce more ketone bodies from fatty acids, for the energetic metabolism of nervous and muscular tissues. However, it is hypothesized that in situations like this, hepatocytes prioritize the production of energy for its own subsistence rather than synthesizing products destined for exportation to other tissues [18]. Thus, ketogenesis would also be impaired, which is corroborated by significantly decreased betahydroxybutyrate and acetoacetate levels, resulting in a precarious energy metabolism

in the central nervous system in the advanced stages of the disease [18, 21].

Given its location and abundant vascular supply, with immense exposure to antigens absorbed by the intestine, the liver regulates important immune functions [9]. In cirrhosis, intestinal bacterial overgrowth associated with hepatocellular failure triggers a systemic immune reaction, bypassing endotoxins such as membrane lipopolysaccharides, flagellins, and peptidoglycans for arterial circulation [15, 22]. Circulating cytokines, such as tumor necrosis factor alpha (TNFα), interleukin 1b (IL-1b) and interleukin 6, induce the synthesis of nitric oxide and prostanoids in endothelial cells, triggering a state of inflammatory hyperemia that facilitates the uptake of ammonia by the central nervous system [9]. In addition, the proinflammatory cytokines generated by the vascular endothelium activate the cells of the immune system in the brain parenchyma and the microglia, contributing indirectly

Astrocytes are part of the blood-brain barrier and protect neurons from the toxic effects of ammonia [12]. Its perivascular extensions are rich in aquaporin 4, a protein constituent of water channels. Astrocytes are among the cells with the highest glycolytic activity of the central nervous system and are estimated to be responsible for 30% of its metabolism. They are believed to be particularly susceptible to the development of edema because they are part of the glymphatic system [23], a paravascular system discovered in 2012, which receives continuous influx of periarterial cerebrospinal fluid and has a leakage network through the perivascular

Liver failure can result in an uncontrollable rise in ammonia levels, which penetrate virtually all organs. Although the central nervous system is partially protected by the blood-brain barrier, which remains relatively intact until advanced stages of the disease, excessive amounts of ammonia can overtake it [12, 25]. Therefore, concentrations that normally range from 0.2 to 0.3 μmol in normal subjects can reach the mark of 3 to 5 mmol in patients with hepatic encephalopathy [4]. However, along with perivenular hepatocytes and skeletal muscle, astrocytes express glutamine synthetase and have the ability to convert ammonia into glutamine [4, 9].

**40**

The accumulation of glutamine in astrocytes, although not directly toxic, drastically affects its functioning [26]. Firstly, glutamine has an osmotic action, inducing predominantly cytotoxic and slightly vasogenic edema [25]. Generally, any form of edema increases the distance for diffusion of oxygen and metabolites in the brain parenchyma, exposing microareas of borderline irrigation to hypoxia [23]. This phenomenon is more pronounced in acute hepatic failure, in which the counterregulatory mechanisms do not have time to act, but can also be detected in the magnetic resonance imaging (MRI) of patients with chronic liver failure [9, 26]. Secondly, exceeding glutamine is transported to the mitochondria, where, by glutaminase action, it is hydrolyzed back into glutamate and ammonia. The passage of the latter to the interior of the mitochondria causes oxidative stress and modifies the internal mitochondrial membrane diffusivity, through the opening of permeability transition pore, causing water accumulation in the mitochondrial matrix, low capacity of oxidative phosphorylation, and low adenosine triphosphate (ATP) production [11, 12]. This results in a vicious cycle of formation of reactive oxygen and nitrogen species (free radicals) with mitochondrial damage [9].

Studies with cultures of astrocytes and neurons show that only the former increase the production of free radicals when exposed to glutamine [12] and that's why astrocytes can be considered the basic morphofunctional unit of hepatic encephalopathy: the histopathological milestone of the disease is the swelling of astrocytes, both in the cytoplasm and in the nucleus, with chromatin marginalization, prominent nucleoli and glycogen accumulation, accompanied by little neuronal alteration [4, 27].

The effects of chronic hyperammonemia and astrocytic edema can be verified in specific sequences of brain MRI. In the spectroscopy of the basal ganglia, the Glx/ creatine ratio is increased and myo-inositol/ creatine and choline/ creatine ratios are decreased [14, 22]. Creatine is a constitutive marker of neurons and astrocytes. The increase of Glx demonstrates the accumulation of glutamine and glutamate [22]. This increase, however, seems to present large interindividual variations, and within a same animal model, there are forms in which there is a gradual increase, a strong increase followed by a plateau or only by a late rise [21]. Choline is a marker for the turnover of membrane phospholipids, and its decrease reflects reduction of basal metabolism of neurons and glial cells [22]. Furthermore, due to the osmotic imbalance generated by the accumulation of glutamine and glutamate, astrocytes export choline and myo-inositol, its main osmolyte, to the extracellular space, which leads to a reduction in the levels of the later, in an attempt to counterbalance the intracellular edema. This mechanism is known as regulatory volume decrease [11, 21, 23]. The diffusion-weighted imaging, in turn, shows interstitial edema resulting from the exportation of osmolytes from the astrocytes into the extracellular space, both in the white and gray matters. Because of that, multiple sites in the brain have an increase in mean diffusivity, including the frontal, temporal, inferior parietal, and insular lobes, as well as the corpus callosum, putamen, thalamus, and pons [22, 27]. The diffusion of water molecules, however, is not free; it reflects interactions with macromolecules, fibers, and membranes. It is important to emphasize that the diffusion-weighted sequences only show changes in the intra- and extracellular volume, and do not allow a definitive conclusion about the total amount of water present in the brain parenchyma [23].
