**6. The role of neurons**

Hepatic encephalopathy has traditionally been assumed to be a metabolic disorder that affects glial cells but maintains the neuronal architecture preserved. However, this belief is easily contradicted by the presence of neuronal loss in its most extreme form: hepatocerebral degeneration. Such disorder is characterized by chronic manifestations (ataxia, dysarthria, apraxia, and parkinsonian symptoms), often associated with repeated and prolonged episodes of hepatic encephalopathy. Its anatomopathological study demonstrates not only astrocytic changes, but also neuronal loss in the basal ganglia, cerebral cortex, and cerebellum [15].

Most patients who develop episodes of hepatic encephalopathy demonstrate some degree of brain injury. Studies have shown that previous episodes of hepatic encephalopathy are risk factors for the development of cognitive impairment, which persists even after hepatic transplantation. In MRI, these findings are related to the fall of N-acetylaspartate in spectroscopy, a marker of neuronal density. This loss may be greater in some brain areas, such as the basal ganglia, which are particularly sensitive to oxidative stress injury, which explains some of its more prominent clinical manifestations, such as movement disorders [15].

**43**

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

complex, in a process that culminates with learning [29].

equally counterproductive to neuronal activity [29].

ogy of hepatic encephalopathy [4, 12].

**7. Effects on neural networks**

It is known that in normal individuals, nitric oxide acts as a retrograde neurotransmitter to the neurons, activating the guanylate cyclase, with consequent increase of the cyclic GMP (cGMP) and decrease of the intracellular influx of chlorine in the glycine receptors. The resulting electrochemical imbalance decreases the threshold of neuronal depolarization, facilitating the generation of action potentials, with subsequent intracellular influx of calcium through ionotropic channels, which amplifies the phosphorylating cascade of the calcium-calmodulin

Hyperammonemia induces an increased expression of nitric oxide synthase in astrocytes, promoting the formation of excessive amounts of nitric oxide, which diffuses into the extracellular environment. Prolonged hyperexposure of neurons adjacent to nitric oxide depletes the formation of cGMP, but the activity of nitric oxide synthase remains unchanged. The result is a high intraneuronal calcium influx and subsequent activation of NADPH oxidase, leading to the formation of superoxide. Superoxide and nitric oxide then combine to form the free radical peroxynitrite, in another vicious cycle that results in apoptosis [12]. In addition, neuronal ATP depletion is observed because of low nucleotide synthesis and high degradation rate, although its levels do not appear to correlate linearly with the concentration of glutamine and ammonia in the brain parenchyma [21].

Cyclic GMP also plays an important role in the reduction of neuroinflammation and microglial activation. It is known that this reduction is associated with an increase in the concentration of IL-1b and TNFα receptors [28]. The fact that chronic hyperammonemia promotes decreased cGMP production has been explored as a potential target for drug-based experimental treatments that increase the concentration of cGMP by inhibiting its degradation (e.g., sildenafil and zaprinast). One of the major obstacles to this strategy, however, is the fact that cGMP seems to act within narrow concentration limits, above which its accumulation becomes

Under normal conditions, glutamine and glutamate synthesized by astrocytes

Cognitive functions—attention, executive functions, memory, visuospatial skills, language, and social cognition—are the emerging results of neurotransmission [26]. They depend on the cooperation of multiple cortical areas, connected to each other through the white matter by bundles and fascicles of axonal fibers, in circuits known as neural networks. Changes in the synchronization of the activity

are transferred to neurons, which internalize them via excitatory amino acid transporters 1 and 2 (EEAT1 and EEAT2). In neurons and astrocytes, the storage process of glutamate within presynaptic vesicles depends on the activity of vesicular glutamate transporters (VGLUT), which have three isoforms (VGLUT1–3). The VGLUT3 isoform, expressed mainly by astrocytes, is easier to release glutamate than the VGLUT1 and VGLUT2 isoforms found in neurons, which depend on intracellular calcium variations. That is the reason why astrocytes are more likely to release accumulated vesicular glutamate than neurons [4, 12]. Moreover, glutamate is able to donate amines for the synthesis of serine, a precursor amino acid of glycine, increasing its synthesis and, consequently, of ammonia in the brain parenchyma [19]. Hyperammonemia, on the other hand, reduces the expression of EEAT1 and 2 on the neuronal surface, impairing its capacity of uptake. The result is the extracellular accumulation of glutamate, with consequent hyperactivation of adjacent receptors. This sequence of events seems to be the key in the pathophysiol-

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

*Liver Disease and Surgery*

they remain quiescent and have an aspect endowed with ramifications (resting phenotype). When an inflammatory stimulus occurs, they become reactive and acquire an ameboid aspect (active phenotype), migrating to the injured site, where they proliferate and produce neurotoxic and neurotrophic factors that control tissue damage and regeneration. In hepatic encephalopathy, molecules such as ammonia, glutamate, and some locally produced neuroactive steroids (neurosteroids) may trigger the transition

Neuroinflammation modulates glutamatergic activity. Studies have shown that microglial activation in the cerebellum of rats exposed to chronic hyperammonemia promotes an increase in the production of proinflammatory cytokines, such as TNFα and IL-1b, in addition to an increase in the expression of TNFα receptors. Of particular importance, TNFα receptors are also expressed on the surface of astrocytes and their stimulation induces increased glutaminase, contributing to the increase of glutamate synthesis [28]. There is also evidence that excess glutamate causes microglial activation, resulting in an intercellular vicious cycle [11].

Another important neurotransmission system affected by neuroinflammation includes a class of peripheral gamma-aminobutyric acid (GABA) receptor, known as translocator protein (TSPO), which is expressed in the outer mitochondrial membrane of neurons. Although poorly present under normal conditions, microglial activation strongly increases its concentration, which can be seen in cirrhotic patients through studies with positron emission tomography and carbon 11-labeled radiotracer that specifically bind to it [11]. It is known that TSPO mediates the synthesis of neurosteroids from cholesterol, and its increased expression provides an important link between neuroinflammation and increased GABAergic activity [12]. Like hyperammonemia, neuroinflammation is not sufficient to produce minimal hepatic encephalopathy: evidence of this is the fact that microglial proliferation can also be found in cirrhosis without encephalopathy, suggesting that it plays a role much more associated with neuroprotection than production of tissue damage [11]. Current knowledge supports the theory that there is the necessity of the coexistence of hyperammonemia and neuroinflammation, interacting synergistically, for the occurrence of neuropsychiatric disorders [10, 26]. In addition, at least one experimental study demonstrates that it is possible to produce cognitive deficits with the combination of

these two factors, even in the absence of underlying liver disease [11].

neuronal loss in the basal ganglia, cerebral cortex, and cerebellum [15].

clinical manifestations, such as movement disorders [15].

Hepatic encephalopathy has traditionally been assumed to be a metabolic disorder that affects glial cells but maintains the neuronal architecture preserved. However, this belief is easily contradicted by the presence of neuronal loss in its most extreme form: hepatocerebral degeneration. Such disorder is characterized by chronic manifestations (ataxia, dysarthria, apraxia, and parkinsonian symptoms), often associated with repeated and prolonged episodes of hepatic encephalopathy. Its anatomopathological study demonstrates not only astrocytic changes, but also

Most patients who develop episodes of hepatic encephalopathy demonstrate some degree of brain injury. Studies have shown that previous episodes of hepatic encephalopathy are risk factors for the development of cognitive impairment, which persists even after hepatic transplantation. In MRI, these findings are related to the fall of N-acetylaspartate in spectroscopy, a marker of neuronal density. This loss may be greater in some brain areas, such as the basal ganglia, which are particularly sensitive to oxidative stress injury, which explains some of its more prominent

from the resting phenotype to the active phenotype [11].

**42**

**6. The role of neurons**

It is known that in normal individuals, nitric oxide acts as a retrograde neurotransmitter to the neurons, activating the guanylate cyclase, with consequent increase of the cyclic GMP (cGMP) and decrease of the intracellular influx of chlorine in the glycine receptors. The resulting electrochemical imbalance decreases the threshold of neuronal depolarization, facilitating the generation of action potentials, with subsequent intracellular influx of calcium through ionotropic channels, which amplifies the phosphorylating cascade of the calcium-calmodulin complex, in a process that culminates with learning [29].

Hyperammonemia induces an increased expression of nitric oxide synthase in astrocytes, promoting the formation of excessive amounts of nitric oxide, which diffuses into the extracellular environment. Prolonged hyperexposure of neurons adjacent to nitric oxide depletes the formation of cGMP, but the activity of nitric oxide synthase remains unchanged. The result is a high intraneuronal calcium influx and subsequent activation of NADPH oxidase, leading to the formation of superoxide. Superoxide and nitric oxide then combine to form the free radical peroxynitrite, in another vicious cycle that results in apoptosis [12]. In addition, neuronal ATP depletion is observed because of low nucleotide synthesis and high degradation rate, although its levels do not appear to correlate linearly with the concentration of glutamine and ammonia in the brain parenchyma [21].

Cyclic GMP also plays an important role in the reduction of neuroinflammation and microglial activation. It is known that this reduction is associated with an increase in the concentration of IL-1b and TNFα receptors [28]. The fact that chronic hyperammonemia promotes decreased cGMP production has been explored as a potential target for drug-based experimental treatments that increase the concentration of cGMP by inhibiting its degradation (e.g., sildenafil and zaprinast). One of the major obstacles to this strategy, however, is the fact that cGMP seems to act within narrow concentration limits, above which its accumulation becomes equally counterproductive to neuronal activity [29].

Under normal conditions, glutamine and glutamate synthesized by astrocytes are transferred to neurons, which internalize them via excitatory amino acid transporters 1 and 2 (EEAT1 and EEAT2). In neurons and astrocytes, the storage process of glutamate within presynaptic vesicles depends on the activity of vesicular glutamate transporters (VGLUT), which have three isoforms (VGLUT1–3). The VGLUT3 isoform, expressed mainly by astrocytes, is easier to release glutamate than the VGLUT1 and VGLUT2 isoforms found in neurons, which depend on intracellular calcium variations. That is the reason why astrocytes are more likely to release accumulated vesicular glutamate than neurons [4, 12]. Moreover, glutamate is able to donate amines for the synthesis of serine, a precursor amino acid of glycine, increasing its synthesis and, consequently, of ammonia in the brain parenchyma [19]. Hyperammonemia, on the other hand, reduces the expression of EEAT1 and 2 on the neuronal surface, impairing its capacity of uptake. The result is the extracellular accumulation of glutamate, with consequent hyperactivation of adjacent receptors. This sequence of events seems to be the key in the pathophysiology of hepatic encephalopathy [4, 12].

#### **7. Effects on neural networks**

Cognitive functions—attention, executive functions, memory, visuospatial skills, language, and social cognition—are the emerging results of neurotransmission [26]. They depend on the cooperation of multiple cortical areas, connected to each other through the white matter by bundles and fascicles of axonal fibers, in circuits known as neural networks. Changes in the synchronization of the activity

of these different regions contribute to the appearance of neurological deficits. This synchronization depends on the integrity of the white matter, which modulates the information processing speed [30].

During the progression of hepatic encephalopathy, the diffusion-weighted imaging on MRI demonstrates cumulative abnormalities in the white matter. In addition to interstitial edema, there may be macroscopic atrophy of the white matter and damage to the microstructural integrity of bundles and fascicles. Studies in patients with cirrhosis have shown that these changes correlate with the incidence of attention deficit, executive dysfunction, and increase in the number of falls [30]. The largest reductions appear to occur in the frontal white matter and in the globus pallidus [27]. In addition, cortical thickness decreases in several regions, such as the lateral superior temporal gyrus and the precuneus, which may also present correlations, respectively, with attention and visuospatial deficits [30].

The final result of the accumulation of toxic, metabolic, cellular, and immunological alterations produced by liver failure and portosystemic shunt is the occurrence of dysfunction in the main axes of neurotransmission [31]. It is important to emphasize, however, that a same system may be involved with more than one cognitive function and that the mechanisms that lead to cognitive impairment are different from those involved in motor impairment [26]. **Table 1** summarizes the main changes found in neurotransmission. The most known repercussions for each neural system will be discussed below.


**45**

**Figure 1.**

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

**8. Effects on the glutamatergic system**

Glutamate is the main excitatory neurotransmitter of the central nervous system [31]. Two glutamatergic circuits are particularly important in the pathophysiology of hepatic encephalopathy: (1) an yet unproven hypothetic pathway that would descend from the frontal lobe and (2) the perforant pathway originated in the entorhinal cortex. It is believed that the frontal descending pathway (**Figure 1**) originates in layer V pyramidal neurons and projects to the centers of other neurotransmitters in the brainstem. There, it performs synapses with dopaminergic neurons of the ventral tegmental area and the substantia nigra, the serotonergic neurons of raphe nuclei and noradrenergic neurons of the locus coeruleus, influencing their activity [37]. If this hypothesis is correct, glutamatergic hyperactivity would act as a final pathway common to the changes induced by hyperammonemia and neuroinflammation, disturbing other neurotransmission systems, in steps that would invoke neuropsychiatric symptoms, and, in more severe cases, cause coma [4]. In addition, the frontal descending pathway would act as a "brake" for the dopaminergic pathway that leaves the ventral tegmental area toward the accumbens nucleus (located between the putamen and the caudate nucleus), influencing its activity through inhibitory GABAergic interneurons in the brainstem. This would result in tonic inhibition of dopamine release, with important consequences for executive and motor functions [37].

The perforant pathway (**Figure 2**) originates in the medial portion of the temporal cortex, called the entorhinal cortex, and projects to the granular cells of the dentate gyrus. The axons of these cells form a pathway of mossy fibers, which goes to the *Cornu Ammonis* (CA) or Ammon's horn, more precisely to the pyramidal cells of the CA3 region. Then, the pyramidal cells emit excitatory collaterals, the Schaffer collaterals, that go to the pyramidal cells of the CA1 region. A brief discharge of high-frequency stimuli in any of these three components of the perforant pathway increases the excitatory postsynaptic potentials in hippocampal neurons, which can last for hours, days, or even weeks. This facilitation is called long-term potentiation and, in addition to the hippocampus, also occurs in the amygdala, striatum (putamen and caudate nucleus), and cerebellar Purkinje cells, being essential for the

*The frontal descending pathway would originate in the frontal cortex and influence directly or indirectly (through inhibitory interneurons) the activity of the neurotransmitter centers of the brainstem. AN: accumbens nucleus, GP: globus pallidus, RN: raphe nucleus, S: striatum, SN: substantia nigra, and T: thalamus.*

formation of new traces of memory and learning [29, 32].

#### **Table 1.**

*Main alterations found in neurotransmission in hepatic encephalopathy.*

*Liver Disease and Surgery*

information processing speed [30].

neural system will be discussed below.

Increased synthesis of neurotransmitters at

Increased release of neurotransmitters at

Decreased reuptake of neurotransmitters at

Increased degradation of neurotransmitters

Modulation of receptor activity at

Changes in signal transduction cascade at

*Main alterations found in neurotransmission in hepatic encephalopathy.*

Increased synthesis of retrograde neurotransmitters at postsynaptic terminals

presynaptic terminals

presynaptic terminals

presynaptic terminals

in the synaptic cleft

postsynaptic terminals

postsynaptic terminals

of these different regions contribute to the appearance of neurological deficits. This synchronization depends on the integrity of the white matter, which modulates the

During the progression of hepatic encephalopathy, the diffusion-weighted imaging on MRI demonstrates cumulative abnormalities in the white matter. In addition to interstitial edema, there may be macroscopic atrophy of the white matter and damage to the microstructural integrity of bundles and fascicles. Studies in patients with cirrhosis have shown that these changes correlate with the incidence of attention deficit, executive dysfunction, and increase in the number of falls [30]. The largest reductions appear to occur in the frontal white matter and in the globus pallidus [27]. In addition, cortical thickness decreases in several regions, such as the lateral superior temporal gyrus and the precuneus, which may also present correla-

The final result of the accumulation of toxic, metabolic, cellular, and immunological alterations produced by liver failure and portosystemic shunt is the occurrence of dysfunction in the main axes of neurotransmission [31]. It is important to emphasize, however, that a same system may be involved with more than one cognitive function and that the mechanisms that lead to cognitive impairment are different from those involved in motor impairment [26]. **Table 1** summarizes the main changes found in neurotransmission. The most known repercussions for each

**Neurotransmission changes in hepatic encephalopathy**

↑ Glutamate [28]

↑ Glycine [19] ↑ Histamine [31]

↑ Glutamate (VGLUT3) [4]

↑ Glutamate (↓ EEAT1 and 2) [4]

↑ GABA (reversal of GAT3) [32]

↑ GABAergic modulatory neurosteroids [33]

↓ Serotonin (↑ MAO-A) [35]

↓ Activity of adenosinergic receptors [36]

↑ Intracellular calcium [12]

↑ cGMP [28]

↑ Nitric oxide [29]

↑ GABAergic modulatory neurosteroids [12, 26]

[34]

[26]

↓ Acetylcholine (↑ acetylcholinesterase and

↑ Activity of metabotropic and ionotropic glutamatergic receptors (AMPA)

butyrylcholinesterase)

tions, respectively, with attention and visuospatial deficits [30].

**44**

**Table 1.**
