Treatment of encephalopathy varies depending on the primary cause of the symptoms. Consequently, not all cases of encephalopathy are treated in the same way. The best treatments are designed by the treating physician once the patient's primary diagnosis is made. Treatments are highly variable because the causes are so different (Bajaj, 2010). Examples can show how different "encephalopathy treatment" may change depending on the cause: a) short-term anoxia (usually less than two minutes): oxygen therapy; b) longterm anoxia: rehabilitation; c) short- term alcohol toxicity: IV fluids or no therapy; d) longterm alcohol status (cirrhosis or chronic liver failure): oral lactulose, low-protein diet, antibiotics; e) uremic encephalopathy (due to kidney failure): correct the underlying physiological cause, dialysis, kidney transplant; f) diabetic encephalopathy: glucose for hypoglycemia, removal of blood glucose to treat hyperglycemia; g) hypo- or hypertensive encephalopathy: raise (for hypotension) or reduce (for hypertension) blood pressure

The key to treatment of any encephalopathy is to understand the basic cause and thus design a treatment scheme to reduce or eliminate the cause(s). Static encephalopathy (an altered mental state or brain damage that is permanent) is one type of encephalopathy difficult or impossible to treat. The best that can be done in this case if possible is to prevent further damage and implement rehabilitation to allow the individual to live at his or her

Complications of encephalopathy vary from none to profound mental impairments that could lead to death. The complications can be similar in some cases. Also, many investigators consider encephalopathy to be a complication that arises from a primary health problem or primary diagnosis. Complications depend on the primary cause of encephalopathy and can be illustrated by citing a few examples from the wide variety of causes: a) hepatic (liver) encephalopathy (brain swelling with herniation, coma, death); b) metabolic encephalopathy (irritability, lethargy, depression, tremors; occasionally coma or death); c) anoxic encephalopathy (wide range of complications, from none in short-term anoxia to personality changes, severe brain damage and death in long- term anoxic events); d) uremic encephalopathy (lethargy, hallucinations, stupor, muscle twitching, seizures, and death); e) Hashimoto's encephalopathy (confusion, heat intolerance, and dementia); f) Wernicke's encephalopathy (mental confusion, memory loss, and decreased ability for eye movement); g) bovine spongiform encephalopathy or "Mad Cow disease" (ataxia, dementia and myoclonus or muscle twitching without any rhythm or pattern); h) Shigella encephalopathy (headache, stiff neck, delirium, seizures, coma); i) infectious causes of pediatric encephalopathy (irritability, poor feeding, hypotonia or floppy baby syndrome,

waves); and l) gradual recovery to begin treatment (Randolph *et al*., 1998).

**3.4 Treatment**

(Sundaram & Shaikh, 2009).

**3.5 Complications**

seizures, death; Nabbout *et al*., 2011).

highest possible functional level (Bismuth *et al*., 2011).

The prognosis of a patient with encephalopathy depends on the initial cause and, in general, the length of time it takes to reverse, stop, or inhibit those causes. Consequently, the prognosis varies from patient to patient and ranges from complete recovery to a poor prognosis that often leads to permanent brain damage or death. One good example of this highly variable prognosis is a patient with hypoglycemic encephalopathy.

When patients with hypoglycemia are given glucose at first signs of encephalopathy (for example, irritability, mild confusion), most of them completely recover from the problem. Delays in correcting hypoglycemia (hours to days) may lead to seizures or coma which may be halted by treatment with complete or partial recovery (minimal permanent brain damage). A long delay or multiple delays in treatment can lead to a poor prognosis with extensive brain damage, coma, or death (Munoz, 2008).

Although the symptoms and time frame vary widely from patient to patient and according to the initial causes of encephalopathy (see above), the prognosis of each case usually follows the pattern described in hypoglycemic cause and depends upon the extent and rapidity with which the underlying cause is treated. The doctor or team of doctors treating the underlying cause of encephalopathy can offer the best information on the individual's prognosis (Häussinger, 2010).

#### **3.7 Can encephalopathy be prevented?**

Many cases of encephalopathy can be prevented. The key to prevention is to stop or limit the chance of developing any of the multitudes of causes of encephalopathy. If encephalopathy develops, the quicker the underlying cause is treated, the more likely it is to prevent the eruption of severe encephalopathy (Montgomery & Bajaj, 2011). Examples of prevention and avoidable situations are listed below: a) diabetic encephalopathy: daily check of glucose level and assurance of correct dosage of insulin; b) hepatic encephalopathy: avoidance of alcohol intoxication, drug overdose, and IV injections of illegal drugs; c) anoxic encephalopathy: prevention of food choking, avoidance of risky behavior that could lead to head and neck trauma, and exposure to carbon monoxide; d) hypertensive encephalopathy: monitoring of blood pressure; use of antihypertensive medication as directed and not quitting medications or changing medication without consulting the doctor; e) infectious encephalopathy: eluding physical contact with individuals known to be infected with organisms that may cause encephalopathy such as *N. meningitidis* or *Shigella*; f) uremic encephalopathy: do not skip or avoid scheduled dialysis, taking all medications as directed and having frequent assessment of mental status. Methods for prevention of encephalopathy are almost as numerous as the underlying causes. However, some cases of encephalopathy may not be preventable especially those originating from congenital cause or traumatic accident.

Therefore and as mentioned earlier, the key to therapeutic follow-up of any encephalopathy is to understand the underlying mechanism of the pathology and thus propose the best

The Use of Microdialysis in the Study of Encephalopathies 209

administration of local intracerebral ammonia infusion. They found that total brain ECF amino acid level was increased by the elevation of blood ammonia and that in rats with hyperammonemia, there was an increased permeability of the blood-brain barrier. Also, they observed that only glutamate levels showed a marked elevation suggesting that high ammonia levels may increase the excitability of the brain and this situation may serve as a

In addition, other experimental data showed a significant increase in extracellular hippocampal glutamate concentration in rats thioacetamide-induced liver failure (McArdle *et al*., 1996). Moreover, other studies suggest that the beneficial effect of hypothermia (35°C) in rats with hepatic devascularization that induces ALF is mediated via mechanism involving reduced blood-brain transfer of ammonia and/or reduction of extracellular brain glutamate concentrations (Rose *et al*., 2000). It was also found that extracellular glutamate increased in substantia nigra pars reticulate (SNr) of rats with chronic liver failure due to portacaval shunt (PCS), and that this provoked activation of mGluR1 in SNr which together

On the other hand, it was demonstrated that the involvement of glutamate-nitric oxidecyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in HE. Rodrigo *et al* (2007) analyzed glutamate-nitric oxide-cGMP pathway function and nitric oxide synthase (NOS) activation by NMDA in an in vivo microdialysis in cerebral cortex of portacaval shunt (PCS) as well as in control and hyperammonemia rats without liver failure. They demonstrated that basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These results are associated to increased inducible NOS expression. In both animal models and in neurons exposed to ammonia, impairment in NOS activation by NMDA was found while in chronic liver failure, basal NOS activity, nitric oxide and cGMP increased whereas activation of NOS induced by NMDA receptors activation decreased. Their results are evidence that hyperammonemia is responsible for both effects and these alterations could contribute to neurological alterations in HE (Rodrigo *et al*., 2007). Additionally, there is an evidence indicating that hyperammonemia increases tonic activation of NMDA receptors leading to reduced activity of nNOS and of glutamate--NO--cGMP pathway. This was demonstrated by ElMlili et al (2010), where the blockade of NMDA receptors with MK-801 increases cGMP and NO metabolites in cerebellum in *in vivo* and in slices from hyperammonemic rats. This was due to the reduction of the phosphorylation and activity of calcium-calmodulinindependent protein kinase II (CaMKII), leading to normalization of nNOS phosphorylation and activity. Also in these experiments, MK-801 increases nNOS in synaptic membranes and

There is an evidence suggesting that not only glutamatergic neurotransmission is altered. It has been proposed that alterations of excitatory and inhibitory amino acids play a role in the pathogenesis of hepatic encephalopathy in ALF. This was determined in vivo cerebral microdialysis using extracellular concentrations of amino acid samples in the frontal cortex of non-anaesthetized rats at various times during the progression of encephalopathy resulting from ALF (induced by portacaval anastomosis). The amino acids from dialysate were measured by high-performance liquid chromatography (HPLC) with fluorescence detection and the results showed that the deterioration of neurological status was accompanied by two to four-fold increases in extracellular glutamate, glutamine, and

were responsible for reduced motor activity in these rats (Cauli *et al*., 2006).

key to the onset of HE (Suzuki *et al*., 1992).

reduces it in cytosol (ElMlili *et al*., 2010).

treatment scheme. In this sense, microdialysis may contribute to understanding these basic mechanisms of encephalopathy, as well as to the possible application of pharmacological treatments that could restore or improve brain function of the patients, and in this way improve the quality of life of those suffering from this problem.

#### **4. Microdialysis: Contributions in the study of encephalopathy**

#### **4.1 Encephalopathy induction mechanisms**

#### **4.1.1 Hepatic encephalopathy**

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that often occurs as a consequence of acute or chronic liver failure where hyperammonemia plays a major role in the pathogenesis of the disease. Hyperammonemia leads to altered cerebral function and neurological alterations in patients with hepatic encephalopathy. Studies with pigs showed that there is an association between extracellular brain ammonia and intracranial pressure (ICP), given that the concentration of ammonia in the brain, when it gets to about 1,200 microM of arterial accumulated ammonia, increases ICP. This result suggests that ICP could serve as a sensitive marker for HE development (Zwirner *et al*., 2010). Also, Szerb and Redondo (1993) suggest that ammonia alters the function of astrocytes, facilitating their entry into the brain. This physiological event contributes to the development of HE. This was demonstrated with local administration of fluoroacetate in rats´ hippocampal dentate gyrus using microdialysis (Szerb & Redondo, 1993).

Other studies have permitted the elucidation of the role of glutamatergic system and its receptors in HE. In 1996, Bergqvist *et al* tried to explain the participation of quinolinic acid (QUIN; a potent N-methyl-D-aspartate, NMDA-receptor agonist) and L-tryptophan (L-TRP) in HE. However, they did not find evidence suggesting their participation on observing that QUIN level and QUIN/L-TRP ratio were significantly low in rats subjected to a portacaval shunt (PCS), and that elevated L-TRP availability increased QUIN levels in a similar degree in both sham and PCS rats (Bergqvist *et al*., 1996). Whereas in rats with ischemic liver failure induced by portacaval anastomosis followed by a 24-hr hepatic artery ligation, a significant threefold increase of extracellular glycine (a positive alosteric modulator of NMDA receptor) measured by *in vivo* cerebral microdialysis was found suggesting the participation of NMDA-mediated excitatory neurotransmission in this pathological condition (Zwingmann *et al*., 2002). In other studies carried out to determine the role of NMDA receptors, it was shown that blocking these receptors by continuous administration of MK-801 or memantine (NMDA receptor antagonist) induces protection against acute liver failure (ALF) caused by galactosamine in experimental animals. This blockage increased the survival time by two folds. Also, when liver injury is not 100% lethal (1.5 g/kg galactosamine), blocking NMDA receptors increases the survival rate from 23 to 62% (Cauli *et al*., 2008). On the other hand, there is experimental evidence that NMDA activation can be modulated by glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in ammonia intoxication in the cerebellum of the rats. However, it was found that this activation is not caused by increased extracellular glutamate (Hermenegildo *et al*., 2000), but due to the presence of ammonium in high concentration in the brain. This was shown by Suzuki et al in 1992, who studied using microdialysis the brain extracellular fluid (ECF) amino acid metabolism and regional level elevation of ammonia in rats with hyperammonemia, experimentally induced through

treatment scheme. In this sense, microdialysis may contribute to understanding these basic mechanisms of encephalopathy, as well as to the possible application of pharmacological treatments that could restore or improve brain function of the patients, and in this way

Hepatic encephalopathy (HE) is a neuropsychiatric disorder that often occurs as a consequence of acute or chronic liver failure where hyperammonemia plays a major role in the pathogenesis of the disease. Hyperammonemia leads to altered cerebral function and neurological alterations in patients with hepatic encephalopathy. Studies with pigs showed that there is an association between extracellular brain ammonia and intracranial pressure (ICP), given that the concentration of ammonia in the brain, when it gets to about 1,200 microM of arterial accumulated ammonia, increases ICP. This result suggests that ICP could serve as a sensitive marker for HE development (Zwirner *et al*., 2010). Also, Szerb and Redondo (1993) suggest that ammonia alters the function of astrocytes, facilitating their entry into the brain. This physiological event contributes to the development of HE. This was demonstrated with local administration of fluoroacetate in rats´ hippocampal dentate

Other studies have permitted the elucidation of the role of glutamatergic system and its receptors in HE. In 1996, Bergqvist *et al* tried to explain the participation of quinolinic acid (QUIN; a potent N-methyl-D-aspartate, NMDA-receptor agonist) and L-tryptophan (L-TRP) in HE. However, they did not find evidence suggesting their participation on observing that QUIN level and QUIN/L-TRP ratio were significantly low in rats subjected to a portacaval shunt (PCS), and that elevated L-TRP availability increased QUIN levels in a similar degree in both sham and PCS rats (Bergqvist *et al*., 1996). Whereas in rats with ischemic liver failure induced by portacaval anastomosis followed by a 24-hr hepatic artery ligation, a significant threefold increase of extracellular glycine (a positive alosteric modulator of NMDA receptor) measured by *in vivo* cerebral microdialysis was found suggesting the participation of NMDA-mediated excitatory neurotransmission in this pathological condition (Zwingmann *et al*., 2002). In other studies carried out to determine the role of NMDA receptors, it was shown that blocking these receptors by continuous administration of MK-801 or memantine (NMDA receptor antagonist) induces protection against acute liver failure (ALF) caused by galactosamine in experimental animals. This blockage increased the survival time by two folds. Also, when liver injury is not 100% lethal (1.5 g/kg galactosamine), blocking NMDA receptors increases the survival rate from 23 to 62% (Cauli *et al*., 2008). On the other hand, there is experimental evidence that NMDA activation can be modulated by glutamate-nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in ammonia intoxication in the cerebellum of the rats. However, it was found that this activation is not caused by increased extracellular glutamate (Hermenegildo *et al*., 2000), but due to the presence of ammonium in high concentration in the brain. This was shown by Suzuki et al in 1992, who studied using microdialysis the brain extracellular fluid (ECF) amino acid metabolism and regional level elevation of ammonia in rats with hyperammonemia, experimentally induced through

improve the quality of life of those suffering from this problem.

**4.1 Encephalopathy induction mechanisms**

gyrus using microdialysis (Szerb & Redondo, 1993).

**4.1.1 Hepatic encephalopathy**

**4. Microdialysis: Contributions in the study of encephalopathy**

administration of local intracerebral ammonia infusion. They found that total brain ECF amino acid level was increased by the elevation of blood ammonia and that in rats with hyperammonemia, there was an increased permeability of the blood-brain barrier. Also, they observed that only glutamate levels showed a marked elevation suggesting that high ammonia levels may increase the excitability of the brain and this situation may serve as a key to the onset of HE (Suzuki *et al*., 1992).

In addition, other experimental data showed a significant increase in extracellular hippocampal glutamate concentration in rats thioacetamide-induced liver failure (McArdle *et al*., 1996). Moreover, other studies suggest that the beneficial effect of hypothermia (35°C) in rats with hepatic devascularization that induces ALF is mediated via mechanism involving reduced blood-brain transfer of ammonia and/or reduction of extracellular brain glutamate concentrations (Rose *et al*., 2000). It was also found that extracellular glutamate increased in substantia nigra pars reticulate (SNr) of rats with chronic liver failure due to portacaval shunt (PCS), and that this provoked activation of mGluR1 in SNr which together were responsible for reduced motor activity in these rats (Cauli *et al*., 2006).

On the other hand, it was demonstrated that the involvement of glutamate-nitric oxidecyclic guanosine monophosphate (cGMP) pathway in brain contributes to cognitive impairment in HE. Rodrigo *et al* (2007) analyzed glutamate-nitric oxide-cGMP pathway function and nitric oxide synthase (NOS) activation by NMDA in an in vivo microdialysis in cerebral cortex of portacaval shunt (PCS) as well as in control and hyperammonemia rats without liver failure. They demonstrated that basal NOS activity, nitrites and cGMP are increased in cortex of rats with hyperammonemia or liver failure. These results are associated to increased inducible NOS expression. In both animal models and in neurons exposed to ammonia, impairment in NOS activation by NMDA was found while in chronic liver failure, basal NOS activity, nitric oxide and cGMP increased whereas activation of NOS induced by NMDA receptors activation decreased. Their results are evidence that hyperammonemia is responsible for both effects and these alterations could contribute to neurological alterations in HE (Rodrigo *et al*., 2007). Additionally, there is an evidence indicating that hyperammonemia increases tonic activation of NMDA receptors leading to reduced activity of nNOS and of glutamate--NO--cGMP pathway. This was demonstrated by ElMlili et al (2010), where the blockade of NMDA receptors with MK-801 increases cGMP and NO metabolites in cerebellum in *in vivo* and in slices from hyperammonemic rats. This was due to the reduction of the phosphorylation and activity of calcium-calmodulinindependent protein kinase II (CaMKII), leading to normalization of nNOS phosphorylation and activity. Also in these experiments, MK-801 increases nNOS in synaptic membranes and reduces it in cytosol (ElMlili *et al*., 2010).

There is an evidence suggesting that not only glutamatergic neurotransmission is altered. It has been proposed that alterations of excitatory and inhibitory amino acids play a role in the pathogenesis of hepatic encephalopathy in ALF. This was determined in vivo cerebral microdialysis using extracellular concentrations of amino acid samples in the frontal cortex of non-anaesthetized rats at various times during the progression of encephalopathy resulting from ALF (induced by portacaval anastomosis). The amino acids from dialysate were measured by high-performance liquid chromatography (HPLC) with fluorescence detection and the results showed that the deterioration of neurological status was accompanied by two to four-fold increases in extracellular glutamate, glutamine, and

The Use of Microdialysis in the Study of Encephalopathies 211

In addition, it was demonstrated that in rats with acute HE induced by repeated administration of thioacetamide, the impairment of modulation of striatal DA discharge and metabolism by the action of glutamate on NMDA receptors, contribute to motor disturbances in this disease (Borkowska *et al*., 1999). Cauli *et al* (2007) demonstrated that the function of the circuits that modulate motor function in rats with liver failure due to portacaval shunt (PCS) is dependent on the kind of neurotransmission. They found this result by analyzing the function of the circuits that modulate motor function in rats with liver failure due to portacaval shunt (PCS) using in vivo brain microdialysis. They inserted cannulae in the NAcc (where injected (S)-3,5-dihydroxyphenylglycine, DHPG) and microdialysis probes in NAcc, ventral pallidum (VP), substantia nigra pars reticulata (SNr), medio-dorsal thalamus (MDT), ventro-medial thalamus (VMT) or prefrontal cortex (PFCx) and analyzed the concentration of extracellular neurotransmitters in these areas. Their results indicate that in control rats, DHPG induces locomotion by activating the normal neuronal circuit: NAcc --> VP --> MDT --> PFCx and in PCS rats this circuit is not activated but DHPG injection in PCS rats activates an alternative circuit: NAcc --> SNr --> VMT --> PFCx that is not activated in control rats. These experiments suggest that dopamine increase induced by DHPG would activate the normal neuronal circuit, while an increase in

On the other side, serotonergic system is also affected in HE. This was demonstrated by Bergqvist *et al* (1996) who showed that frontal neocortical extracellular levels of 5 hydroxytryptamine (5-HT) is unaltered while its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), is increased in portacaval shunted rats in an experimental model of chronic hepatic encephalopathy (HE), prior to and after an acute coma-induction by the administration of ammonium acetate (5.2 mmol/kg, i.p.). These results suggest that an increase in brain ammonia may augment neuronal 5-HT release in HE, which in turn could

In addition, in frontal cortex of rats with thioacetamide (TAA)-induced HE, it was found that 5-HIAA and high K+-evoked 5-HT release increased in HE rats. These data support the idea that serotonergic neurotransmission is altered in this brain area of rats with this

Like other systems affected in HE, there is an experimental evidence suggesting that noradrenergic system is affected and describes that central noradrenergic mechanisms may contribute to the central nervous system manifestations of HE. Michalak *et al* (1998) showed that there is an increase of extracellular brain concentrations of noradrenalin (NA), from the extracellular compartment of frontal cortex and thalamus in rats with acute liver failure (Michalak *et al*., 1998), and this is associated to the loss of NA transporter sites resulting from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate in brain in ischemic liver failure (Michalak *et al*., 2010). These experimental data suggested that noradrenergic neurotransmission may play a role in this

On the other hand, there is an evidence that extracellular glutathione (GSH) is involved in the ammonia toxicity present in rats with HE. This was demonstrated by Hielgier *et al* (2010) where the administration of ammonium chloride (ammonia) via a microdialysis probe to the rat prefrontal cortex increased GSH. This increase was abrogated by the inhibitor of

glutamate would activate the alternative circuit (Cauli *et al*., 2007).

be a causative of severe stages of HE (Bergqvist *et al*., 1996).

pathology (Kaneko *et al*., 1998).

pathology.

glycine while concentrations of gamma - aminobutyric acid (GABA) and taurine levels remained unchanged. These results suggest that amino acid could contribute to the pathogenesis of HE induced by ALF (Michalak *et al*., 1996). However, these results are not convincing because they depend on the method of inducing ALF which can produce different changes in the level of amino acid. This is supported by the studies of Bosman *et al* (1992), who depicted that following microdialysis determinations in the cerebral cortex of rats with acute hepatic encephalopathy induced by complete liver ischemia, extracellular concentrations of the neuroactive amino acids glutamate, taurine, and glycine increased, whereas extracellular concentrations of aspartate and GABA were unaltered and glutamine decreased (Bosman *et al*., 1992). Also, in cerebral cortical microdialysates of rats with subclinical hepatic encephalopathy (SHE) induced by two intraperitoneal injections of thioacetamide, it was found that dialysate concentrations of the neuroactive amino acids taurine (Tau), glutamate (Glu) and aspartate (Asp) were 30% to 50% higher in SHE rats than in control rats (Albrecht *et al*., 2000).

Other reports demonstrated that the precursors of monoamines, as well as monoamines together with their metabolites, altered neuronal excitability and contribute to the characteristics of HE. Deshpande *et al* (2007) showed that in frontal cortex of rats with HE induced by ALF resulting from portacaval anastomosis followed by hepatic artery ligation, the extracellular brain concentrations of aromatic amino acids (AAAs) and of valine and leucine (precursors of monoamine neurotransmitters) elevated by 2 to 4 folds following hepatic devascularization, and these increases were significantly correlated to arterial ammonia concentration (Deshpande *et al*., 2007; Michalak & Butterworth, 1997). So, those alterations in monoaminergic system are present in HE. Likewise, the monoaminergic system and the dopaminergic system are affected in HE. Yano et al., (2005) demonstrated that the extracellular concentration of 3,4-dihydroxyphenylacetic acid, a metabolite of dopamine, decreased by 39% of that in sham-operated animals, although the dopamine level did not change. However, when treated with flumazenil (benzodiacepine antagonist), there was complete abolition of the decrease in the metabolite. Although, in this study the glutamate level in the injured animals decreased to 42% of that in sham-operated animals, there was no increase in the glutamate levels in animals treated with flumazenil. Also in this study, it was shown that spontaneous motor activity decreased 24 hours after surgery in animals subjected to liver ischemia and that flumazenil treatment improved spontaneous the motor activity 5 minutes after administration, but this effect was not observed after 30 minutes. For this reason, these authors proposed that the restoration of the central dopaminergic function could be a relevant factor in the improvement of HE (Yano *et al*., 2005).

Other researchers have determined that in hyperammonemic rats there are neurological and cerebral function alterations, as well as alterations on the modulation of motor and neurochemical functions. The locomotion induced by injection of mGluR agonist dihydroxyphenylglycine (DHPG) in the nucleus accumbens increased. Also in control rats, DHPG increased extracellular dopamine (400%), but glutamate remained unchanged. Whereas in hyperammonemic rats, DHPG increased extracellular glutamate (600%), and when mGluR1 receptor was blocked with 7-hydroxyiminocyclopropan[b]chromen-1acarboxylic acid ethylester (CPCCOEt), this treatment prevented DHPG effects, suggesting that this receptor mediates locomotor and neurochemical effects and showed that modulation of locomotor and neurochemical functions by mGluRs in nucleus accumbens is strongly altered in hyperammonemia (Canales *et al*., 2003).

glycine while concentrations of gamma - aminobutyric acid (GABA) and taurine levels remained unchanged. These results suggest that amino acid could contribute to the pathogenesis of HE induced by ALF (Michalak *et al*., 1996). However, these results are not convincing because they depend on the method of inducing ALF which can produce different changes in the level of amino acid. This is supported by the studies of Bosman *et al* (1992), who depicted that following microdialysis determinations in the cerebral cortex of rats with acute hepatic encephalopathy induced by complete liver ischemia, extracellular concentrations of the neuroactive amino acids glutamate, taurine, and glycine increased, whereas extracellular concentrations of aspartate and GABA were unaltered and glutamine decreased (Bosman *et al*., 1992). Also, in cerebral cortical microdialysates of rats with subclinical hepatic encephalopathy (SHE) induced by two intraperitoneal injections of thioacetamide, it was found that dialysate concentrations of the neuroactive amino acids taurine (Tau), glutamate (Glu) and aspartate (Asp) were 30% to 50% higher in SHE rats than

Other reports demonstrated that the precursors of monoamines, as well as monoamines together with their metabolites, altered neuronal excitability and contribute to the characteristics of HE. Deshpande *et al* (2007) showed that in frontal cortex of rats with HE induced by ALF resulting from portacaval anastomosis followed by hepatic artery ligation, the extracellular brain concentrations of aromatic amino acids (AAAs) and of valine and leucine (precursors of monoamine neurotransmitters) elevated by 2 to 4 folds following hepatic devascularization, and these increases were significantly correlated to arterial ammonia concentration (Deshpande *et al*., 2007; Michalak & Butterworth, 1997). So, those alterations in monoaminergic system are present in HE. Likewise, the monoaminergic system and the dopaminergic system are affected in HE. Yano et al., (2005) demonstrated that the extracellular concentration of 3,4-dihydroxyphenylacetic acid, a metabolite of dopamine, decreased by 39% of that in sham-operated animals, although the dopamine level did not change. However, when treated with flumazenil (benzodiacepine antagonist), there was complete abolition of the decrease in the metabolite. Although, in this study the glutamate level in the injured animals decreased to 42% of that in sham-operated animals, there was no increase in the glutamate levels in animals treated with flumazenil. Also in this study, it was shown that spontaneous motor activity decreased 24 hours after surgery in animals subjected to liver ischemia and that flumazenil treatment improved spontaneous the motor activity 5 minutes after administration, but this effect was not observed after 30 minutes. For this reason, these authors proposed that the restoration of the central dopaminergic function could be a

Other researchers have determined that in hyperammonemic rats there are neurological and cerebral function alterations, as well as alterations on the modulation of motor and neurochemical functions. The locomotion induced by injection of mGluR agonist dihydroxyphenylglycine (DHPG) in the nucleus accumbens increased. Also in control rats, DHPG increased extracellular dopamine (400%), but glutamate remained unchanged. Whereas in hyperammonemic rats, DHPG increased extracellular glutamate (600%), and when mGluR1 receptor was blocked with 7-hydroxyiminocyclopropan[b]chromen-1acarboxylic acid ethylester (CPCCOEt), this treatment prevented DHPG effects, suggesting that this receptor mediates locomotor and neurochemical effects and showed that modulation of locomotor and neurochemical functions by mGluRs in nucleus accumbens is

in control rats (Albrecht *et al*., 2000).

relevant factor in the improvement of HE (Yano *et al*., 2005).

strongly altered in hyperammonemia (Canales *et al*., 2003).

In addition, it was demonstrated that in rats with acute HE induced by repeated administration of thioacetamide, the impairment of modulation of striatal DA discharge and metabolism by the action of glutamate on NMDA receptors, contribute to motor disturbances in this disease (Borkowska *et al*., 1999). Cauli *et al* (2007) demonstrated that the function of the circuits that modulate motor function in rats with liver failure due to portacaval shunt (PCS) is dependent on the kind of neurotransmission. They found this result by analyzing the function of the circuits that modulate motor function in rats with liver failure due to portacaval shunt (PCS) using in vivo brain microdialysis. They inserted cannulae in the NAcc (where injected (S)-3,5-dihydroxyphenylglycine, DHPG) and microdialysis probes in NAcc, ventral pallidum (VP), substantia nigra pars reticulata (SNr), medio-dorsal thalamus (MDT), ventro-medial thalamus (VMT) or prefrontal cortex (PFCx) and analyzed the concentration of extracellular neurotransmitters in these areas. Their results indicate that in control rats, DHPG induces locomotion by activating the normal neuronal circuit: NAcc --> VP --> MDT --> PFCx and in PCS rats this circuit is not activated but DHPG injection in PCS rats activates an alternative circuit: NAcc --> SNr --> VMT --> PFCx that is not activated in control rats. These experiments suggest that dopamine increase induced by DHPG would activate the normal neuronal circuit, while an increase in glutamate would activate the alternative circuit (Cauli *et al*., 2007).

On the other side, serotonergic system is also affected in HE. This was demonstrated by Bergqvist *et al* (1996) who showed that frontal neocortical extracellular levels of 5 hydroxytryptamine (5-HT) is unaltered while its major metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), is increased in portacaval shunted rats in an experimental model of chronic hepatic encephalopathy (HE), prior to and after an acute coma-induction by the administration of ammonium acetate (5.2 mmol/kg, i.p.). These results suggest that an increase in brain ammonia may augment neuronal 5-HT release in HE, which in turn could be a causative of severe stages of HE (Bergqvist *et al*., 1996).

In addition, in frontal cortex of rats with thioacetamide (TAA)-induced HE, it was found that 5-HIAA and high K+-evoked 5-HT release increased in HE rats. These data support the idea that serotonergic neurotransmission is altered in this brain area of rats with this pathology (Kaneko *et al*., 1998).

Like other systems affected in HE, there is an experimental evidence suggesting that noradrenergic system is affected and describes that central noradrenergic mechanisms may contribute to the central nervous system manifestations of HE. Michalak *et al* (1998) showed that there is an increase of extracellular brain concentrations of noradrenalin (NA), from the extracellular compartment of frontal cortex and thalamus in rats with acute liver failure (Michalak *et al*., 1998), and this is associated to the loss of NA transporter sites resulting from depletion of central NA stores due to a reserpine-like effect of ammonia which is known to accumulate in brain in ischemic liver failure (Michalak *et al*., 2010). These experimental data suggested that noradrenergic neurotransmission may play a role in this pathology.

On the other hand, there is an evidence that extracellular glutathione (GSH) is involved in the ammonia toxicity present in rats with HE. This was demonstrated by Hielgier *et al* (2010) where the administration of ammonium chloride (ammonia) via a microdialysis probe to the rat prefrontal cortex increased GSH. This increase was abrogated by the inhibitor of

The Use of Microdialysis in the Study of Encephalopathies 213

Encephalopathy associated with septic shock as well as neurological disease could be induced by the central nervous formation of reactive oxygen species (ROS) associated with inflammation process. Clement *et al* (2010) analyzed the effect of peripherally applied lipopolysaccharide (LPS, 100 mug/kg i.p.) that is used as a model for major depression and results in enhanced inflammatory processes, on the central nervous formation of ROS and interleukin-6 (IL-6) in wild-type mice and in mice lacking NADPH oxidase Nox2 subunit gp91phox using microdialysis paradigm. They found that in the wild-type mice, LPS increased ROS formation in the striatum of wild-type mice and resulted in enhanced IL-6 production. In the mice lacking NADPH oxidase Nox2 subunit gp91phox, LPS did not enhance ROS formation, whereas IL-6 increased. They conclude that gp91phox-containing NADPH oxidase complex was involved in the central nervous ROS formation after peripheral LPS stimulation and these results suggest that ROS determination could be a pharmacological target in patients with this pathological condition (Clement *et al*., 2010).

The Wernicke encephalopathy is characterized by thiamine deficiency. This pathology is a neurological disease detected in alcoholics and in patients with compromised nutrition. Todd *et al* (2001) established the role of glutamate excitotoxicity using in vivo microdialysis in the neuronal cell death due to thiamine deficiency, with the considerations of different factors (blood-brain barrier, tissue reactions to probe implantation) that may affect the probe recoveries (Todd & Butterworth, 2001). In addition, Todd and Butterworth (1998) evaluated the role of NMDA receptor-mediated glutamate excitotoxicity in the pathogenesis of neuronal loss induced by thiamine deficiency. They also determined extracellular glutamate levels using in vivo cerebral microdialysis in the ventral posterior medial thalamic nucleus. Their result suggests that NMDA receptor-mediated excitotoxicity is not responsible for early neuronal loss in this model of thiamine deficiency encephalopathy (Todd &

Other studies were directed to the study of the role of histamine in neuronal degeneration in a rat model of Wernicke's encephalopathy induced by pyrithiamine-induced thiamine deficiency (PTD). They established that the histamine enhancement of glutamate receptor activation suggests that the histamine release could be a participant in glutamate-N-methyl-D-aspartate (NMDA)-mediated excitotoxic neuronal death in this pathology (Langlais *et al*., 1994). And, that the release of histamine in rat from nerve terminals and histamine and other vasoactive substances from granulocytes could be responsible for thiamine deficiencyinduced vascular breakdown and perivascular edema in thalamus (McRee *et al*., 2000).

Espey *et al* (1998) determined in mice infected with LP-BM5 leukemia retrovirus mixture that develop a progressive immunodeficiency with associated behavioral, histological, and neurochemical alterations consistent with glutamatergic hyperactivation. They clarify the contribution of excitatory amino acids to the neurodegeneration observed in these mice by measuring extracellular glutamate levels in the striated brain area of these animals, and found that infection with an immunodeficiency-inducing retrovirus increases extracellular

**4.1.5 Encephalopathy associated with septic shock**

**4.1.6 Thiamine deficiency encephalopathy (Wernicke)**

Butterworth, 1998).

**4.1.7 Viral encephalopathy**

astrocytic energy metabolism of fluoroacetate and inhibitor of glutathione synthesis of buthionine sulfoximine. Their results suggest that in rats with hyperammonemia HA or HE ammonia specifically promote GSH synthesis may improve the availability of precursors for GSH synthesis in neurons and their resistance to ammonia toxicity present in HE (Hilgier *et al*., 2010).

#### **4.1.2 Fulminant hepatic encephalopathy**

Fulminant hepatic encephalopathy is one of the encephalopathies with high mortality. To found chemical markers that provide information leading to application of the best therapeutic treatment in these patients is a priority. The microdialysis, being a highly sensitive technique in monitoring cerebral energy metabolism and in early detection of cerebral hypoxia has been used to find these chemical markers. Beuer *et al* (2004) were the first in reporting a case of monitoring neurochemical markers using intracerebral microdialysis during cardiac resuscitation. They measured the chemical markers of energy metabolism of glucose, lactate, pyruvate, and the marker of cell membrane damage (glycerol) and found that except subcutaneous glucose, all markers showed a sudden and significant increase during resuscitation and a prolonged period afterwards with all values returning to normal after some hours (Bauer *et al*., 2004). In another study to determine the role of cerebral microdialysis as an adjunct to the management of a 49-year old woman with hepatic encephalopathy secondary to a paracetamol overdose, it was found that the application of the microdialysis technique for the detection of cerebral glucose concentration in the presence of a normal plasma glucose was useful for the continuous on line monitoring of substrate delivery and metabolism, and this may have a relevant role in the therapeutic management of patients with fulminant hepatic failure (Hutchinson *et al*., 2006).

#### **4.1.3 Posthypoxic encephalopathy**

Actually, there is only one study that focused on determining the neurochemical alterations in a seemingly posthypoxic encephalopathy.  They induced hypoxia for fortyfive minutes by reducing the fraction of inspired O2 to the maximum concentration and found that there was no association between onset of electroconvulsive activity and an increase in lactate or lactate/pyruvate (L/P) ratio which are the biochemical parameters altered in posthypoxic encephalopathy (Thoresen *et al*., 1998).

#### **4.1.4 Hypoxic-ischemic encephalopathy**

Pearing *et al* (1996) studying the effect of hypoxia in-vivo, separately analyzed the hypoxic component of hypoxic-ischemic encephalopathy. They used rats that were prepared such that their arterial oxygen pressure (paO2) was maintained at 20 mmHg. While maintaining systemic arterial pressures, the brain oxygen concentration and extracellular amino acid concentrations were monitored during 20 minutes. They did not find changes in the extracellular glutamate extracellular levels, and with additional experiments no morphologic injury was detected. Thus, they conclude that hypoxia without ischemia is well tolerated by the brain (Pearigen *et al*., 1996).

astrocytic energy metabolism of fluoroacetate and inhibitor of glutathione synthesis of buthionine sulfoximine. Their results suggest that in rats with hyperammonemia HA or HE ammonia specifically promote GSH synthesis may improve the availability of precursors for GSH synthesis in neurons and their resistance to ammonia toxicity present in HE (Hilgier *et*

Fulminant hepatic encephalopathy is one of the encephalopathies with high mortality. To found chemical markers that provide information leading to application of the best therapeutic treatment in these patients is a priority. The microdialysis, being a highly sensitive technique in monitoring cerebral energy metabolism and in early detection of cerebral hypoxia has been used to find these chemical markers. Beuer *et al* (2004) were the first in reporting a case of monitoring neurochemical markers using intracerebral microdialysis during cardiac resuscitation. They measured the chemical markers of energy metabolism of glucose, lactate, pyruvate, and the marker of cell membrane damage (glycerol) and found that except subcutaneous glucose, all markers showed a sudden and significant increase during resuscitation and a prolonged period afterwards with all values returning to normal after some hours (Bauer *et al*., 2004). In another study to determine the role of cerebral microdialysis as an adjunct to the management of a 49-year old woman with hepatic encephalopathy secondary to a paracetamol overdose, it was found that the application of the microdialysis technique for the detection of cerebral glucose concentration in the presence of a normal plasma glucose was useful for the continuous on line monitoring of substrate delivery and metabolism, and this may have a relevant role in the therapeutic

management of patients with fulminant hepatic failure (Hutchinson *et al*., 2006).

altered in posthypoxic encephalopathy (Thoresen *et al*., 1998).

Actually, there is only one study that focused on determining the neurochemical alterations in a seemingly posthypoxic encephalopathy.  They induced hypoxia for fortyfive minutes by reducing the fraction of inspired O2 to the maximum concentration and found that there was no association between onset of electroconvulsive activity and an increase in lactate or lactate/pyruvate (L/P) ratio which are the biochemical parameters

Pearing *et al* (1996) studying the effect of hypoxia in-vivo, separately analyzed the hypoxic component of hypoxic-ischemic encephalopathy. They used rats that were prepared such that their arterial oxygen pressure (paO2) was maintained at 20 mmHg. While maintaining systemic arterial pressures, the brain oxygen concentration and extracellular amino acid concentrations were monitored during 20 minutes. They did not find changes in the extracellular glutamate extracellular levels, and with additional experiments no morphologic injury was detected. Thus, they conclude that hypoxia without ischemia is well

*al*., 2010).

**4.1.2 Fulminant hepatic encephalopathy**

**4.1.3 Posthypoxic encephalopathy**

**4.1.4 Hypoxic-ischemic encephalopathy**

tolerated by the brain (Pearigen *et al*., 1996).

#### **4.1.5 Encephalopathy associated with septic shock**

Encephalopathy associated with septic shock as well as neurological disease could be induced by the central nervous formation of reactive oxygen species (ROS) associated with inflammation process. Clement *et al* (2010) analyzed the effect of peripherally applied lipopolysaccharide (LPS, 100 mug/kg i.p.) that is used as a model for major depression and results in enhanced inflammatory processes, on the central nervous formation of ROS and interleukin-6 (IL-6) in wild-type mice and in mice lacking NADPH oxidase Nox2 subunit gp91phox using microdialysis paradigm. They found that in the wild-type mice, LPS increased ROS formation in the striatum of wild-type mice and resulted in enhanced IL-6 production. In the mice lacking NADPH oxidase Nox2 subunit gp91phox, LPS did not enhance ROS formation, whereas IL-6 increased. They conclude that gp91phox-containing NADPH oxidase complex was involved in the central nervous ROS formation after peripheral LPS stimulation and these results suggest that ROS determination could be a pharmacological target in patients with this pathological condition (Clement *et al*., 2010).

#### **4.1.6 Thiamine deficiency encephalopathy (Wernicke)**

The Wernicke encephalopathy is characterized by thiamine deficiency. This pathology is a neurological disease detected in alcoholics and in patients with compromised nutrition. Todd *et al* (2001) established the role of glutamate excitotoxicity using in vivo microdialysis in the neuronal cell death due to thiamine deficiency, with the considerations of different factors (blood-brain barrier, tissue reactions to probe implantation) that may affect the probe recoveries (Todd & Butterworth, 2001). In addition, Todd and Butterworth (1998) evaluated the role of NMDA receptor-mediated glutamate excitotoxicity in the pathogenesis of neuronal loss induced by thiamine deficiency. They also determined extracellular glutamate levels using in vivo cerebral microdialysis in the ventral posterior medial thalamic nucleus. Their result suggests that NMDA receptor-mediated excitotoxicity is not responsible for early neuronal loss in this model of thiamine deficiency encephalopathy (Todd & Butterworth, 1998).

Other studies were directed to the study of the role of histamine in neuronal degeneration in a rat model of Wernicke's encephalopathy induced by pyrithiamine-induced thiamine deficiency (PTD). They established that the histamine enhancement of glutamate receptor activation suggests that the histamine release could be a participant in glutamate-N-methyl-D-aspartate (NMDA)-mediated excitotoxic neuronal death in this pathology (Langlais *et al*., 1994). And, that the release of histamine in rat from nerve terminals and histamine and other vasoactive substances from granulocytes could be responsible for thiamine deficiencyinduced vascular breakdown and perivascular edema in thalamus (McRee *et al*., 2000).

#### **4.1.7 Viral encephalopathy**

Espey *et al* (1998) determined in mice infected with LP-BM5 leukemia retrovirus mixture that develop a progressive immunodeficiency with associated behavioral, histological, and neurochemical alterations consistent with glutamatergic hyperactivation. They clarify the contribution of excitatory amino acids to the neurodegeneration observed in these mice by measuring extracellular glutamate levels in the striated brain area of these animals, and found that infection with an immunodeficiency-inducing retrovirus increases extracellular

The Use of Microdialysis in the Study of Encephalopathies 215

When VEN was administered at 5 mg/kg as a single subcutaneous challenge to portacaval shunted rats, it resulted in elevated levels of VEN in serum, brain parenchyma, and brain dialysate in about 300 minutes after the injection. Therefore, this result suggests that when the dose of VEN administered to experimental HE was reduced 50%, important pharmacokinetic alterations are presented in these animals (Wikell *et al*., 2001), in the same

Citalopram (CIT) is an antidepressant drug of the selective serotonin reuptake inhibitor used in patients with HE that display neuropsychiatric symptoms like affective disturbances. The simultaneous pharmacokinetic and pharmacodynamic outcome of the commonly used serotonin-selective thymoleptic drugs in liver-impaired subjects with HE is

Berqvist *et al* (1997) studied the effects of neocortical administration of CIT (1.0 microM), and CIT (5 mg/kg subcutaneously) on brain 5-hydroxytryptamine release in portacaval shunted rats with an experimental chronic hepatic encephalopathy. They found that neocortical administration of CIT increased the brain 5-HT output in the same way as in portacaval shunted sham-operated rats. These data do not explain the increased 5-HT turnover and unchanged release in PCS rats by an accelerated brain 5-HT reuptake. For this reason, the administration of CIT (5 mg/kg subcutaneously) resulted in a pronounced decrease of brain 5-HT release in PCS rats than in sham-operated controls. This may be due to a higher susceptibility to indirect 5-HT1A autoreceptor activation in experimental portalsystemic encephalopathy. Their experiments with potassium chloride (60 mM) challenge in the presence of locally CIT (1 microM) induced an increase of 5-HT response in PCS rats than in sham-operated rats, confirmed an abnormal increase of 5-HT available for depolarization-induced release in PCS rats. These results suggested that central nervous system 5-HT-active drugs could perhaps pose a potential hazard in patients with liver

In addition, Apelqvist *et al* (2000) investigated the effects of chronic treatment with CIT (10 mg/Kg day) in the frontal neocortex of rats with and without portacaval shunts in 5-HT, 5- HIAA, noradrenaline (NA), and dopamine (DA) output. The rats with PCS increased by 2-3 folds the levels of CIT than rats undergoing a sham treatment with CIT in all compartments. This treatment induced in neocortical output differences between PCS rats and control rats within 5-HT and DA systems but not in NA system. Their data suggest pharmacokinetic and pharmacodynamic changes in an equal-dose chronic treatment with CIT in PCS rats, changes that were not observed in sham rats. Interestingly, these authors indicate that although there are pharmacokinetic and pharmacodynamic alterations with CIT-treatment

Lubeluzole is a neuroprotectant which is effective in the treatment of experimental stroke in rats, mainly by inhibition of glutamate-activated NO pathway and also by counteracting osmotic stress (mechanism associated to the release of the active amino acid taurine). Zielinska *et al* (2001) showed that lubeluzole administered intraperitoneally decreases by 25% the high (50 mM) K+-evoked accumulation of Taurine (Tau) in striatal microdialysates

in PCS rats, the beneficial behavioral response remains (Apelqvist *et al*., 2000).

way that the studies done with dose of 10 mg/kg (Wikell *et al*., 1998, 2002).

**<IP_ADDRESS> Citalopram studies**

not totally understood today.

**<IP_ADDRESS> Lubeluzole study**

failure with or without HE (Bergqvist *et al*., 1997).

free glutamate levels in this brain region. Based on this, they suggest that these changes contribute to neurodegenerative and cognitive deficits observed in this experimental model of viral encephalopathy (Espey *et al*., 1998).

#### **4.1.8 Portal-systemic encephalopathy**

Portal-systemic encephalopathy (PSE) is characterized by psychiatric symptoms progressing through stupor and coma. Actually, there is experimental evidence suggesting that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. Rao *et al* (1995) studying this changes, did found changes in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, and serine in cerebral frontal cortex of portacaval-shunted rats that were administered ammonium acetate (3.85 mmol/kg, i.p.) to induce PSE. (Rao *et al*. 1995).

In addition, PSE is associated with an increased brain tissue of serotonin (5-HT). The involvement of serotoninergic neurotransmission was demonstrated by Bergqvist *et al* (1996, 1997) who found that potassium chloride (KCI) challenged induced increase of 5-HT release in neocortical region of the rats with a portacaval shunt (PCS) (Bergqvist *et al*., 1996, 1997).

#### **4.2 Microdialysis applied in the study of encephalopathy therapeutic treatments**

#### **4.2.1 Hepatic encephalopathy**

#### **<IP_ADDRESS> L-ornithine and L-ornithine-L-aspartate study**

Vogels *et al* (1997) established that the L-ornithine (ORN) and L-ornithine-L-aspartate (OA) therapeutic treatment had a beneficial effects on the symptomatologies of rats with hyperammonemia-induced encephalopathy by portacaval shunted for the fact that ORN and OA treatments decrease ammonia concentrations in blood by 34% and 39%, and in brain by 42% and 22%. Also they found that these substances increased urea production by 39% and 86%, with a significant smaller increase in brain glutamine and lactate concentrations than in controls. However, the effect of ornithine should be taken with care due to the fact that this substance induces high brain extracellular levels of glutamate and aspartate (excitatory amino acids), suggesting a possible overstimulation of NMDA receptors (Vogels *et al*., 1997).

#### **<IP_ADDRESS> Venlafaxine studies**

Patients with chronic hepatic encephalopathy (HE) may present affective symptoms and antidepressant drug treatment due to HE displays monoaminergic perturbations together with affective symptoms. Venlafaxine (VEN) is an antidepressant, and serotoninnorepinephrine reuptake inhibitor that is used in the treatment of patients with HE. The liver impairment present in HE patients may induce pharmacokinetic alterations of antidepressant drug, which in turn can modify monoaminergic function. Because of this, Wikell *et al* studied this possible alterations, and determined that in rats with chronic portacaval shunted (PCS; experimental hepatic encephalopathy), VEN (10 mg/kg) administered in a unique doses (subcutaneous) and daily during 14 days (continous delivery by osmotic minipumps) exhibits both pharmacokinetic and pharmacodynamic alterations in these rats (Wikell *et al*., 1998, 2002).

When VEN was administered at 5 mg/kg as a single subcutaneous challenge to portacaval shunted rats, it resulted in elevated levels of VEN in serum, brain parenchyma, and brain dialysate in about 300 minutes after the injection. Therefore, this result suggests that when the dose of VEN administered to experimental HE was reduced 50%, important pharmacokinetic alterations are presented in these animals (Wikell *et al*., 2001), in the same way that the studies done with dose of 10 mg/kg (Wikell *et al*., 1998, 2002).

#### **<IP_ADDRESS> Citalopram studies**

214 Miscellanea on Encephalopathies – A Second Look

free glutamate levels in this brain region. Based on this, they suggest that these changes contribute to neurodegenerative and cognitive deficits observed in this experimental model

Portal-systemic encephalopathy (PSE) is characterized by psychiatric symptoms progressing through stupor and coma. Actually, there is experimental evidence suggesting that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. Rao *et al* (1995) studying this changes, did found changes in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, and serine in cerebral frontal cortex of portacaval-shunted rats that were administered ammonium acetate (3.85 mmol/kg, i.p.) to

In addition, PSE is associated with an increased brain tissue of serotonin (5-HT). The involvement of serotoninergic neurotransmission was demonstrated by Bergqvist *et al* (1996, 1997) who found that potassium chloride (KCI) challenged induced increase of 5-HT release in neocortical region of the rats with a portacaval shunt (PCS) (Bergqvist *et al*., 1996, 1997).

Vogels *et al* (1997) established that the L-ornithine (ORN) and L-ornithine-L-aspartate (OA) therapeutic treatment had a beneficial effects on the symptomatologies of rats with hyperammonemia-induced encephalopathy by portacaval shunted for the fact that ORN and OA treatments decrease ammonia concentrations in blood by 34% and 39%, and in brain by 42% and 22%. Also they found that these substances increased urea production by 39% and 86%, with a significant smaller increase in brain glutamine and lactate concentrations than in controls. However, the effect of ornithine should be taken with care due to the fact that this substance induces high brain extracellular levels of glutamate and aspartate (excitatory amino acids), suggesting a possible overstimulation of NMDA

Patients with chronic hepatic encephalopathy (HE) may present affective symptoms and antidepressant drug treatment due to HE displays monoaminergic perturbations together with affective symptoms. Venlafaxine (VEN) is an antidepressant, and serotoninnorepinephrine reuptake inhibitor that is used in the treatment of patients with HE. The liver impairment present in HE patients may induce pharmacokinetic alterations of antidepressant drug, which in turn can modify monoaminergic function. Because of this, Wikell *et al* studied this possible alterations, and determined that in rats with chronic portacaval shunted (PCS; experimental hepatic encephalopathy), VEN (10 mg/kg) administered in a unique doses (subcutaneous) and daily during 14 days (continous delivery by osmotic minipumps) exhibits both pharmacokinetic and pharmacodynamic

**4.2 Microdialysis applied in the study of encephalopathy therapeutic treatments**

of viral encephalopathy (Espey *et al*., 1998).

**4.1.8 Portal-systemic encephalopathy**

induce PSE. (Rao *et al*. 1995).

**4.2.1 Hepatic encephalopathy**

receptors (Vogels *et al*., 1997). **<IP_ADDRESS> Venlafaxine studies**

**<IP_ADDRESS> L-ornithine and L-ornithine-L-aspartate study**

alterations in these rats (Wikell *et al*., 1998, 2002).

Citalopram (CIT) is an antidepressant drug of the selective serotonin reuptake inhibitor used in patients with HE that display neuropsychiatric symptoms like affective disturbances. The simultaneous pharmacokinetic and pharmacodynamic outcome of the commonly used serotonin-selective thymoleptic drugs in liver-impaired subjects with HE is not totally understood today.

Berqvist *et al* (1997) studied the effects of neocortical administration of CIT (1.0 microM), and CIT (5 mg/kg subcutaneously) on brain 5-hydroxytryptamine release in portacaval shunted rats with an experimental chronic hepatic encephalopathy. They found that neocortical administration of CIT increased the brain 5-HT output in the same way as in portacaval shunted sham-operated rats. These data do not explain the increased 5-HT turnover and unchanged release in PCS rats by an accelerated brain 5-HT reuptake. For this reason, the administration of CIT (5 mg/kg subcutaneously) resulted in a pronounced decrease of brain 5-HT release in PCS rats than in sham-operated controls. This may be due to a higher susceptibility to indirect 5-HT1A autoreceptor activation in experimental portalsystemic encephalopathy. Their experiments with potassium chloride (60 mM) challenge in the presence of locally CIT (1 microM) induced an increase of 5-HT response in PCS rats than in sham-operated rats, confirmed an abnormal increase of 5-HT available for depolarization-induced release in PCS rats. These results suggested that central nervous system 5-HT-active drugs could perhaps pose a potential hazard in patients with liver failure with or without HE (Bergqvist *et al*., 1997).

In addition, Apelqvist *et al* (2000) investigated the effects of chronic treatment with CIT (10 mg/Kg day) in the frontal neocortex of rats with and without portacaval shunts in 5-HT, 5- HIAA, noradrenaline (NA), and dopamine (DA) output. The rats with PCS increased by 2-3 folds the levels of CIT than rats undergoing a sham treatment with CIT in all compartments. This treatment induced in neocortical output differences between PCS rats and control rats within 5-HT and DA systems but not in NA system. Their data suggest pharmacokinetic and pharmacodynamic changes in an equal-dose chronic treatment with CIT in PCS rats, changes that were not observed in sham rats. Interestingly, these authors indicate that although there are pharmacokinetic and pharmacodynamic alterations with CIT-treatment in PCS rats, the beneficial behavioral response remains (Apelqvist *et al*., 2000).

#### **<IP_ADDRESS> Lubeluzole study**

Lubeluzole is a neuroprotectant which is effective in the treatment of experimental stroke in rats, mainly by inhibition of glutamate-activated NO pathway and also by counteracting osmotic stress (mechanism associated to the release of the active amino acid taurine). Zielinska *et al* (2001) showed that lubeluzole administered intraperitoneally decreases by 25% the high (50 mM) K+-evoked accumulation of Taurine (Tau) in striatal microdialysates

The Use of Microdialysis in the Study of Encephalopathies 217

which may accumulate during cerebral ischemia. Corbett *et al* (1998) hypothesized that postischemic DCA administration affects lactate and acid clearance in different extents in immature versus mature brain. Their results indicate that postischemic DCA administration helps to resolve cerebral acidosis to a greater degree in immature than in more mature brain, and this suggests that DCA could have cerebroprotective properties for neonatal hypoxic-

Indometacin or indomethacin is a non-steroidal anti-inflammatory drug commonly used to reduce fever, pain, stiffness, and swelling. It works by inhibiting the production of prostaglandins, molecules known to cause these symptoms. To try understanding the role of this drug in the possible protective effect on hypoxic ischemic encephalopathy, Ogasawara *et al* (1999) examined the effects of indomethacin on extracellular dopamine (DA) in the striatum of immature rats submitted to anoxia using in vivo microdialysis and HPLC to quantify DA. They found that during anoxia the DA level reached 1185+/-400% of the basal level and the peak levels of DA were only 307+/-63%, 153+/-35% in indomethacin groups. They conclude that the suppression that induces indomethacin is possibly one of the mechanisms that helps to avoid the dopamine alterations on hypoxic ischemic

Microdialysis is a powerful tool widely used in neurosciences. The use in Neurology has been on the increase on the discovery of its importance in understanding the possible mechanisms underlying neuropathies, and its application in the study of new therapeutic

Although, the use in in-vivo experimental studies and in animal models dates back to years, now there is an increase in the tendency to use this technique to study the neuropathology in humans, and this has helped to make the most appropriate decisions for the treatment of patients. It is noteworthy that studies in humans are very limited, since they involve legal and ethical permissions, but however, data from the few studies in human have shown

On the other hand, microdialysis is a technique that allows the measurement of molecules which may be involved in pathophysiological changes of various diseases as encephalopathy, finding molecular targets for early diagnosis and timely treatment of patients with this disease that result in the greater likelihood of remission and minimizing the potential consequences that such conditions could leave, improving the prognosis of

We thank professor Aristides III Sampieri Hernández for his technical assistance, and also to Dr. Abel Santamaría del Ángel, from the laboratory of excitatory amino acids, National Institute of Neurology and Neurosurgery, Mexico, who kindly provided us with a photography from his microdyalisis equipment to illustrate our work. Finally we thank to

Dr Cyrill Ndidi Nwoye a native English speaker for translating this manuscript.

favorable results that have contributed to the implementation of specific treatments.

ischemic encephalopathy (Corbett *et al*., 1998).

encephalopathy (Ogasawara *et al*., 1999).

**5. Conclusions**

substances.

these patients.

**6. Acknowledgements**

**<IP_ADDRESS> Indomethacin study**

of healthy rats and by 34% in rats with thioacetamide-induced hepatic failure, suggesting increased extracellular of Tau in ongoing hepatic encephalopathy. These data indicate that lubeluzole could be effective in ameliorating ionic or osmotic stress in rats with hepatic failure (Zielińska *et al*., 2001).

#### **<IP_ADDRESS> Sildenafil study**

Patients with liver disease with overt or minimal hepatic encephalopathy have impaired intellectual capacity and the underlying molecular mechanism remains unknown. Interestingly, rats with portacaval anastomosis or with hyperammonemia without liver failure also show impaired learning ability and impaired function of the glutamate-nitric oxide-cyclic guanine monophosphate (glutamate-NO-cGMP) pathway in brain. Erceg *et al* (2005) hypothesized that pharmacological manipulation of this pathway (glutamate-NOcGMP) could restore learning ability. They showed that in vivo brain microdialysis, chronic oral administration of sildenafil (an inhibitor of the phosphodiesterase that degrades cGMP), normalizes the function of the glutamate-NO-cGMP pathway and extracellular cGMP in the brain of rats with portacaval anastomosis or with hyperammonemia. They determined that impairment of learning ability in rats with chronic liver failure or with hyperammonemia are the result of impairment of glutamate-NO-cGMP pathway and that the chronic treatment with sildenafil normalizes the function of the pathway and restores learning ability in rats with portacaval shunts or with hyperammonemia (Erceg *et al*., 2005).

#### **<IP_ADDRESS> Ibuprofen study**

Patients with hepatic encephalopathy show altered motor function, psychomotor slowing, and hypokinesia, which are reproduced in rats with portacaval shunts (PCS, an experimental model of HE). The neurochemical alterations induced by hypokinesia in PCS rats are attributed to the increase of extracellular glutamate in substantia nigra pars reticulate (SNr), but the mechanisms by which liver failure leads to increased extracellular glutamate in SNr remain unclear. However, it was seen that inflammation acts synergistically with hyperammonemia to induce neurological alterations in hepatic encephalopathy and by this way, the inflammation alterations can contribute to motor alterations in HE. For this reason Cauli *et al* (2009) assessed if the treatment with an antiinflammatory, ibuprofen, is able to normalize extracellular glutamate in SNr and/or improve hypokinesia in PCS rats. They found that ibuprofen at 15 or 30 (but not at 5 mg/kg/day), completely eliminates hypokinesia and restore normal motor activity. This supports the supposition that inflammation is the principal contributor to the induction of hypokinesia in HE and this data suggests that therapeutic treatment of inflammation in the motor deficits in patients with this pathology could be beneficial (Cauli *et al*., 2009).

#### **4.2.2 Hypoxic-ischemic encephalopathy**

#### **<IP_ADDRESS> Dichloroacetate study**

Dichloroacetic acid, often abbreviated as DCA, the salts of which has been studied as potential drugs due to their inhibition of pyruvate dehydrogenase kinase enzyme. The common use of DCA is in the treatment of cancer, and actually several studies using adult animal models suggest that DCA may have neuroprotective properties by virtue of its ability to increase rates of metabolism and, therefore, clearance of brain lactic acidosis, which may accumulate during cerebral ischemia. Corbett *et al* (1998) hypothesized that postischemic DCA administration affects lactate and acid clearance in different extents in immature versus mature brain. Their results indicate that postischemic DCA administration helps to resolve cerebral acidosis to a greater degree in immature than in more mature brain, and this suggests that DCA could have cerebroprotective properties for neonatal hypoxicischemic encephalopathy (Corbett *et al*., 1998).

#### **<IP_ADDRESS> Indomethacin study**

216 Miscellanea on Encephalopathies – A Second Look

of healthy rats and by 34% in rats with thioacetamide-induced hepatic failure, suggesting increased extracellular of Tau in ongoing hepatic encephalopathy. These data indicate that lubeluzole could be effective in ameliorating ionic or osmotic stress in rats with hepatic

Patients with liver disease with overt or minimal hepatic encephalopathy have impaired intellectual capacity and the underlying molecular mechanism remains unknown. Interestingly, rats with portacaval anastomosis or with hyperammonemia without liver failure also show impaired learning ability and impaired function of the glutamate-nitric oxide-cyclic guanine monophosphate (glutamate-NO-cGMP) pathway in brain. Erceg *et al* (2005) hypothesized that pharmacological manipulation of this pathway (glutamate-NOcGMP) could restore learning ability. They showed that in vivo brain microdialysis, chronic oral administration of sildenafil (an inhibitor of the phosphodiesterase that degrades cGMP), normalizes the function of the glutamate-NO-cGMP pathway and extracellular cGMP in the brain of rats with portacaval anastomosis or with hyperammonemia. They determined that impairment of learning ability in rats with chronic liver failure or with hyperammonemia are the result of impairment of glutamate-NO-cGMP pathway and that the chronic treatment with sildenafil normalizes the function of the pathway and restores learning ability in rats with portacaval shunts or with hyperammonemia (Erceg *et al*., 2005).

Patients with hepatic encephalopathy show altered motor function, psychomotor slowing, and hypokinesia, which are reproduced in rats with portacaval shunts (PCS, an experimental model of HE). The neurochemical alterations induced by hypokinesia in PCS rats are attributed to the increase of extracellular glutamate in substantia nigra pars reticulate (SNr), but the mechanisms by which liver failure leads to increased extracellular glutamate in SNr remain unclear. However, it was seen that inflammation acts synergistically with hyperammonemia to induce neurological alterations in hepatic encephalopathy and by this way, the inflammation alterations can contribute to motor alterations in HE. For this reason Cauli *et al* (2009) assessed if the treatment with an antiinflammatory, ibuprofen, is able to normalize extracellular glutamate in SNr and/or improve hypokinesia in PCS rats. They found that ibuprofen at 15 or 30 (but not at 5 mg/kg/day), completely eliminates hypokinesia and restore normal motor activity. This supports the supposition that inflammation is the principal contributor to the induction of hypokinesia in HE and this data suggests that therapeutic treatment of inflammation in the

motor deficits in patients with this pathology could be beneficial (Cauli *et al*., 2009).

Dichloroacetic acid, often abbreviated as DCA, the salts of which has been studied as potential drugs due to their inhibition of pyruvate dehydrogenase kinase enzyme. The common use of DCA is in the treatment of cancer, and actually several studies using adult animal models suggest that DCA may have neuroprotective properties by virtue of its ability to increase rates of metabolism and, therefore, clearance of brain lactic acidosis,

failure (Zielińska *et al*., 2001).

**<IP_ADDRESS> Sildenafil study**

**<IP_ADDRESS> Ibuprofen study**

**4.2.2 Hypoxic-ischemic encephalopathy**

**<IP_ADDRESS> Dichloroacetate study**

Indometacin or indomethacin is a non-steroidal anti-inflammatory drug commonly used to reduce fever, pain, stiffness, and swelling. It works by inhibiting the production of prostaglandins, molecules known to cause these symptoms. To try understanding the role of this drug in the possible protective effect on hypoxic ischemic encephalopathy, Ogasawara *et al* (1999) examined the effects of indomethacin on extracellular dopamine (DA) in the striatum of immature rats submitted to anoxia using in vivo microdialysis and HPLC to quantify DA. They found that during anoxia the DA level reached 1185+/-400% of the basal level and the peak levels of DA were only 307+/-63%, 153+/-35% in indomethacin groups. They conclude that the suppression that induces indomethacin is possibly one of the mechanisms that helps to avoid the dopamine alterations on hypoxic ischemic encephalopathy (Ogasawara *et al*., 1999).

#### **5. Conclusions**

Microdialysis is a powerful tool widely used in neurosciences. The use in Neurology has been on the increase on the discovery of its importance in understanding the possible mechanisms underlying neuropathies, and its application in the study of new therapeutic substances.

Although, the use in in-vivo experimental studies and in animal models dates back to years, now there is an increase in the tendency to use this technique to study the neuropathology in humans, and this has helped to make the most appropriate decisions for the treatment of patients. It is noteworthy that studies in humans are very limited, since they involve legal and ethical permissions, but however, data from the few studies in human have shown favorable results that have contributed to the implementation of specific treatments.

On the other hand, microdialysis is a technique that allows the measurement of molecules which may be involved in pathophysiological changes of various diseases as encephalopathy, finding molecular targets for early diagnosis and timely treatment of patients with this disease that result in the greater likelihood of remission and minimizing the potential consequences that such conditions could leave, improving the prognosis of these patients.

#### **6. Acknowledgements**

We thank professor Aristides III Sampieri Hernández for his technical assistance, and also to Dr. Abel Santamaría del Ángel, from the laboratory of excitatory amino acids, National Institute of Neurology and Neurosurgery, Mexico, who kindly provided us with a photography from his microdyalisis equipment to illustrate our work. Finally we thank to Dr Cyrill Ndidi Nwoye a native English speaker for translating this manuscript.

The Use of Microdialysis in the Study of Encephalopathies 219

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**10**

*USA*

**Portal-Systemic Encephalopathy in Emergency**

Bleeding esophageal varices (BEV) is a common and highly lethal complication of cirrhosis of the liver (Graham & Smith, 1981; Smith & Graham, 1982; Burroughs et al., 1989; Bornman et al., 1994; Khan et al., 2006; Orloff et al., 1977; Mikkelsen, 1974; Terblanche et al., 1989; & D'Amico et al., 1995) The mortality rate associated with BEV is highest during the period surrounding the episode of acute bleeding. If the varices remain untreated after recovery from a bout of acute bleeding, we ( Orloff et al., 1977) and others observed a 95% incidence of recurrent bleeding, and death within 2 to 5 years in 90% to 100% of the patients. Recurrent bleeding has been reported to develop most often within the first few days after the acute bleeding episode (Graham & Smith, 1981; Smith & Graham, 1982).  Our studies have been distinguished by three features that, together, make them different from other reported investigations. All patients admitted to our institution with cirrhosis and BEV ("all comers"), regardless of their condition, were included without selection; the specific emergency treatment undergoing evaluation was administered within 24 hours and usually within 8 hours of initial contact; and our studies were prospective, meaning that a welldefined protocol was consistently used and data were collected on-line. Evaluation, prevention, and treatment of portal-systemic encephalopathy (PSE) were an important part

In 1877, Nikolai V. Eck (1849-1908), a 29-year-old military surgeon working in the Military Medical Academy in St. Petersburg, published a brief article describing his experiments and

patients with portal hypertension and esophagogastric varices.

**1. Introduction**

of each of our studies.

**2. The Eck fistula**

**Treatment of Cirrhosis and Bleeding**

**Esophageal Varices**

*University of California, San Diego, Medical Center*

Marshall J. Orloff

### **Portal-Systemic Encephalopathy in Emergency Treatment of Cirrhosis and Bleeding Esophageal Varices**

Marshall J. Orloff *University of California, San Diego, Medical Center USA*

#### **1. Introduction**

224 Miscellanea on Encephalopathies – A Second Look

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Lubeluzole attenuates K(+)-evoked extracellular accumulation of taurine in the striatum of healthy rats and rats with hepatic failure. *Brain Res*. Vol.904, No.1, (June

(2002). Reduced expression of astrocytic glycine transporter (Glyt-1) in acute liver

Extracellular brain ammonia levels in association with arterial ammonia, intracranial pressure and the use of albumin dialysis devices in pigs with acute Bleeding esophageal varices (BEV) is a common and highly lethal complication of cirrhosis of the liver (Graham & Smith, 1981; Smith & Graham, 1982; Burroughs et al., 1989; Bornman et al., 1994; Khan et al., 2006; Orloff et al., 1977; Mikkelsen, 1974; Terblanche et al., 1989; & D'Amico et al., 1995) The mortality rate associated with BEV is highest during the period surrounding the episode of acute bleeding. If the varices remain untreated after recovery from a bout of acute bleeding, we ( Orloff et al., 1977) and others observed a 95% incidence of recurrent bleeding, and death within 2 to 5 years in 90% to 100% of the patients. Recurrent bleeding has been reported to develop most often within the first few days after the acute bleeding episode (Graham & Smith, 1981; Smith & Graham, 1982). Thus, it is clear that emergency treatment of acute bleeding is of paramount importance in the care of patients with portal hypertension and esophagogastric varices.

 Our studies have been distinguished by three features that, together, make them different from other reported investigations. All patients admitted to our institution with cirrhosis and BEV ("all comers"), regardless of their condition, were included without selection; the specific emergency treatment undergoing evaluation was administered within 24 hours and usually within 8 hours of initial contact; and our studies were prospective, meaning that a welldefined protocol was consistently used and data were collected on-line. Evaluation, prevention, and treatment of portal-systemic encephalopathy (PSE) were an important part of each of our studies.

#### **2. The Eck fistula**

In 1877, Nikolai V. Eck (1849-1908), a 29-year-old military surgeon working in the Military Medical Academy in St. Petersburg, published a brief article describing his experiments and

Portal-Systemic Encephalopathy

in Emergency Treatment of Cirrhosis and Bleeding Esophageal Varices 227

extraction by the liver (Cordoba & Blei, 1997). In chronic liver failure, hepatic urea synthesis declines and this, in addition to portal-systemic shunting, results in increased blood ammonia concentrations. Furthermore, cirrhotic patients are hypersensitive to ammoniagenic conditions such as an oral protein load or gastrointestinal hemorrhage. An illustration of this hypersensitivity is provided by a report of studies in the 1950s in which attempts were made to treat ascites in cirrhotic patients with ion-exchange resins that absorbed sodium but released ammonium ions (Gabuzda et al., 1952). This treatment led to a significant reduction in ascitic volume but precipitated severe PSE in many of the patients treated. If present in high concentrations, ammonia has the potential to adversely affect central nervous system (CNS) function by several mechanisms, which include a direct effect of the ammonium ion on inhibitory and excitatory neurotransmission as well as inhibition of the tricarboxylic acid cycle enzyme ketoglutarate dehydrogenase, with potential impairment of brain energy metabolism (Szerb & Butterworth, 1992; Lai & Cooper, 1986) However, brain energy metabolism does not appear to be impaired in chronic liver failure until very late stages associated with isoelectric electroencephalography (EEG) traces (Hindfelt et al., 1977). On the other hand, increases of cerebrospinal fluid lactate have been described both in cirrhotic patients with PSE (Yao et al., 1987) and in experimental animals with chronic liver failure and ammonia-precipitated encephalopathy (Therrien et al., 1991) , findings that are

consistent with an inhibitory effect of ammonia on cerebral glucose oxidation.

& Norenberg, 1996; Knecht et al., 1997) a major excitatory neurotransmitter.

Other effects of ammonia on cerebral function include a stimulatory effect on L-arginine uptake by brain preparations resulting in increased production of nitric oxide (Raghavendra Rao et al., 1995) and inhibition of the capacity of astrocytes to accumulate glutamate (Bender

In 1963, we performed a study that subsequently influenced the use of PCS in the treatment of portal hypertension and variceal hemorrhage. We examined the influence of the stomal size of the portacaval anastomosis, and in turn of the blood flow rate through the anastomosis, on peripheral blood ammonia levels (Orloff et al., 1963). Induced ammoniemia was studied in two groups of dogs, one of which had large end-to-side PCS measuring at autopsy 2.0-4.2 cm in greatest diameter, and the other of which had small end-to-side PCS measuring 0.5-1.8 cm in greatest diameter. A third group of intact dogs served as controls. Ammoniemia was induced by three standardized methods that consisted of the administration by gavage of ammonium citrate (0.5 g/kg), gavage of fresh whole blood (30 mL/kg) plus urease (4 U/mL), and gavage of fresh whole blood (30 mL/kg) alone. All three techniques of ammonium loading resulted in significantly higher mean peak blood ammonia levels in the dogs with large shunts than in those with small shunts. Furthermore, in the animals with large shunts, markedly elevated blood ammonia concentrations persisted for longer periods of time. Measurements of the pressure gradients across the shunts at operation revealed a mean gradient of 31.0 mm saline in the small-shunt group as compared to only 3.0 mm in the large-shunt group. A consistent inverse relationship was demonstrated between the height of the blood ammonia level and the magnitude of the pressure gradient across the portacaval anastomosis. Application of Gorlins' hydraulic formula to these studies provided support for the presumption that the magnitude of ammoniemia was directly related to the blood flow rate through the shunt. We concluded that the stomal size of end-to-side PCS, and in turn the rate of blood flow through the shunts, have a definite influence on the concentration of ammonia in the peripheral blood of

technique for creating a portacaval shunt (Eck fistula) in eight dogs (Eck, 1877). This was not only the beginning of the surgical treatment of portal hypertension but also the first vascular anastomosis. In his article, Eck stated: "I am conducting these experiments with the purpose of clarifying some physiologic problems as well as to determine whether it would be possible to treat some cases of mechanical ascites by means of forming such a fistula. I operated on 8 dogs, one recovered completely and lived in the laboratory for 2.5 months. Because of lack of attention, he ran away. I had to postpone further experiments because I was called to join the active army."

Eck's work would probably have remained unknown outside Russia had it not been for Ivan Pavlov (1849-1936), who used the procedure extensively in laboratory studies of liver physiology and named the operation the Eck fistula, giving full credit to Eck for its invention (Hahn et al., 1893; Pavlov, 1893). The landmark description by Pavlov et al. of portal-systemic encephalopathy (PSE) following feeding meat to dogs with an Eck fistula, which they called "meat intoxication," was part of the work that resulted in Pavlov's receiving the Nobel Prize in 1904 for his contributions on the physiology of digestion. Pavlov spent his entire career at the Military Medical Academy in St. Petersburg, the same institution where Eck developed the portacaval shunt. Since its original description, the Eck fistula has been used extensively for a wide range of studies in animals and humans and clinically for the treatment of the life-threatening complications of portal hypertension.

#### **3. Portal-Systemic Encephalopathy (PSE)**

Hepatic encephalopathy is a neuropsychiatric disorder that results from impaired liver function. It occurs in two distinct forms: (1) in acute or fulminant hepatic failure it takes the form of a neurological disorder that progresses from altered mental status to coma, generally within hours or days, and is associated with increased intracranial pressure caused by massive brain edema; (2) portal-systemic encephalopathy (PSE), which commonly occurs in cirrhosis, in which there are spontaneous or created portal-systemic shunts that permit toxins of intestinal origin to bypass the liver into the systemic circulation. Neurologically, PSE develops slowly, starting with sleep abnormalities, shortened attention span, and muscular incoordination, progressing through lethargy, ataxia, stupor, and coma. PSE is often precipitated by event such as gastrointestinal bleeding, uremia, hypokalemia, or ingestion of excessive amounts of protein. Neuropathologically, PSE is characterized by astrocytic rather than neuronal changes, and brain edema and intracranial hypertension are not found.

Since the description in 1893 of "meat intoxication" in the Eck fistula dog by the Pavlov group, animals with a portacaval shunt (PCS) (Eck fistula) have been used extensively for research studies of the pathogenesis and treatment of PSE (Hahn et al., 1893). The PCS rat has been consistently reported to have neurobehavioral abnormalities when subjected to sophisticated testing (Bengtsson et al., 1980; Tricklebank et al., 1978). One of the best animal models of chronic recurrent PSE is the dog with a congenital or surgically created PCS (Maddison et al., 1991). Animal and human studies have demonstrated that chronic liver failure and spontaneous or created PCS result in the accumulation of neurotoxic substances in the brain. Two such substances are ammonia and manganese.

Ammonia derived from colonic bacteria as well as from the deamination of glutamine in the small bowel is absorbed by passive diffusion and normally undergoes a high first-pass

technique for creating a portacaval shunt (Eck fistula) in eight dogs (Eck, 1877). This was not only the beginning of the surgical treatment of portal hypertension but also the first vascular anastomosis. In his article, Eck stated: "I am conducting these experiments with the purpose of clarifying some physiologic problems as well as to determine whether it would be possible to treat some cases of mechanical ascites by means of forming such a fistula. I operated on 8 dogs, one recovered completely and lived in the laboratory for 2.5 months. Because of lack of attention, he ran away. I had to postpone further experiments because I

Eck's work would probably have remained unknown outside Russia had it not been for Ivan Pavlov (1849-1936), who used the procedure extensively in laboratory studies of liver physiology and named the operation the Eck fistula, giving full credit to Eck for its invention (Hahn et al., 1893; Pavlov, 1893). The landmark description by Pavlov et al. of portal-systemic encephalopathy (PSE) following feeding meat to dogs with an Eck fistula, which they called "meat intoxication," was part of the work that resulted in Pavlov's receiving the Nobel Prize in 1904 for his contributions on the physiology of digestion. Pavlov spent his entire career at the Military Medical Academy in St. Petersburg, the same institution where Eck developed the portacaval shunt. Since its original description, the Eck fistula has been used extensively for a wide range of studies in animals and humans and clinically for the treatment of the life-threatening complications of portal hypertension.

Hepatic encephalopathy is a neuropsychiatric disorder that results from impaired liver function. It occurs in two distinct forms: (1) in acute or fulminant hepatic failure it takes the form of a neurological disorder that progresses from altered mental status to coma, generally within hours or days, and is associated with increased intracranial pressure caused by massive brain edema; (2) portal-systemic encephalopathy (PSE), which commonly occurs in cirrhosis, in which there are spontaneous or created portal-systemic shunts that permit toxins of intestinal origin to bypass the liver into the systemic circulation. Neurologically, PSE develops slowly, starting with sleep abnormalities, shortened attention span, and muscular incoordination, progressing through lethargy, ataxia, stupor, and coma. PSE is often precipitated by event such as gastrointestinal bleeding, uremia, hypokalemia, or ingestion of excessive amounts of protein. Neuropathologically, PSE is characterized by astrocytic rather

than neuronal changes, and brain edema and intracranial hypertension are not found.

in the brain. Two such substances are ammonia and manganese.

Since the description in 1893 of "meat intoxication" in the Eck fistula dog by the Pavlov group, animals with a portacaval shunt (PCS) (Eck fistula) have been used extensively for research studies of the pathogenesis and treatment of PSE (Hahn et al., 1893). The PCS rat has been consistently reported to have neurobehavioral abnormalities when subjected to sophisticated testing (Bengtsson et al., 1980; Tricklebank et al., 1978). One of the best animal models of chronic recurrent PSE is the dog with a congenital or surgically created PCS (Maddison et al., 1991). Animal and human studies have demonstrated that chronic liver failure and spontaneous or created PCS result in the accumulation of neurotoxic substances

Ammonia derived from colonic bacteria as well as from the deamination of glutamine in the small bowel is absorbed by passive diffusion and normally undergoes a high first-pass

was called to join the active army."

**3. Portal-Systemic Encephalopathy (PSE)**

extraction by the liver (Cordoba & Blei, 1997). In chronic liver failure, hepatic urea synthesis declines and this, in addition to portal-systemic shunting, results in increased blood ammonia concentrations. Furthermore, cirrhotic patients are hypersensitive to ammoniagenic conditions such as an oral protein load or gastrointestinal hemorrhage. An illustration of this hypersensitivity is provided by a report of studies in the 1950s in which attempts were made to treat ascites in cirrhotic patients with ion-exchange resins that absorbed sodium but released ammonium ions (Gabuzda et al., 1952). This treatment led to a significant reduction in ascitic volume but precipitated severe PSE in many of the patients treated. If present in high concentrations, ammonia has the potential to adversely affect central nervous system (CNS) function by several mechanisms, which include a direct effect of the ammonium ion on inhibitory and excitatory neurotransmission as well as inhibition of the tricarboxylic acid cycle enzyme ketoglutarate dehydrogenase, with potential impairment of brain energy metabolism (Szerb & Butterworth, 1992; Lai & Cooper, 1986) However, brain energy metabolism does not appear to be impaired in chronic liver failure until very late stages associated with isoelectric electroencephalography (EEG) traces (Hindfelt et al., 1977). On the other hand, increases of cerebrospinal fluid lactate have been described both in cirrhotic patients with PSE (Yao et al., 1987) and in experimental animals with chronic liver failure and ammonia-precipitated encephalopathy (Therrien et al., 1991) , findings that are consistent with an inhibitory effect of ammonia on cerebral glucose oxidation.

Other effects of ammonia on cerebral function include a stimulatory effect on L-arginine uptake by brain preparations resulting in increased production of nitric oxide (Raghavendra Rao et al., 1995) and inhibition of the capacity of astrocytes to accumulate glutamate (Bender & Norenberg, 1996; Knecht et al., 1997) a major excitatory neurotransmitter.

In 1963, we performed a study that subsequently influenced the use of PCS in the treatment of portal hypertension and variceal hemorrhage. We examined the influence of the stomal size of the portacaval anastomosis, and in turn of the blood flow rate through the anastomosis, on peripheral blood ammonia levels (Orloff et al., 1963). Induced ammoniemia was studied in two groups of dogs, one of which had large end-to-side PCS measuring at autopsy 2.0-4.2 cm in greatest diameter, and the other of which had small end-to-side PCS measuring 0.5-1.8 cm in greatest diameter. A third group of intact dogs served as controls. Ammoniemia was induced by three standardized methods that consisted of the administration by gavage of ammonium citrate (0.5 g/kg), gavage of fresh whole blood (30 mL/kg) plus urease (4 U/mL), and gavage of fresh whole blood (30 mL/kg) alone. All three techniques of ammonium loading resulted in significantly higher mean peak blood ammonia levels in the dogs with large shunts than in those with small shunts. Furthermore, in the animals with large shunts, markedly elevated blood ammonia concentrations persisted for longer periods of time. Measurements of the pressure gradients across the shunts at operation revealed a mean gradient of 31.0 mm saline in the small-shunt group as compared to only 3.0 mm in the large-shunt group. A consistent inverse relationship was demonstrated between the height of the blood ammonia level and the magnitude of the pressure gradient across the portacaval anastomosis. Application of Gorlins' hydraulic formula to these studies provided support for the presumption that the magnitude of ammoniemia was directly related to the blood flow rate through the shunt. We concluded that the stomal size of end-to-side PCS, and in turn the rate of blood flow through the shunts, have a definite influence on the concentration of ammonia in the peripheral blood of

Portal-Systemic Encephalopathy

(Raghavendra Rao et al., 1998).

in Emergency Treatment of Cirrhosis and Bleeding Esophageal Varices 229

There is evidence to suggest that the increased expression of PTBRs in brain in chronic liver failure is the consequence of exposure to ammonia and/or manganese. The precise mechanism whereby increased expression or activation of PRBRs results in altered brain excitability characteristic of PSE has not been established. PCS in the rat results in increased gene expression of the constitutive (neuronal) isoform of nitric oxide synthase (nNOS) in brain (Raghavendra Rao et al., 1997a). Increased nNOS mRNA is accompanied by increased nNOS protein (Raghavendra Rao et al., 1997a) and by increased nNOS enzyme activities. Recent evidence suggests that, in addition to an induction in nNOS gene expression, increased nNOS activities may also result from a stimulatory effect of ammonia on Larginine uptake by neuronal preparations shown both *in vitro* and *in vivo* (Raghavendra Rao et al., 1997b). Increased production of NO as a consequence of increased nNOS activities could contribute to the alterations of cerebral perfusion observed in chronic liver disease

The appearance of extrapyramidal symptoms, particularly rigidity, in cirrhotic patients with end-stage liver disease has prompted, by analogy with the well-established dopamine deficit in Parkinson's disease, evaluations of the dopamine system in relation to PSE. Studies in autopsied brain tissue from cirrhotic patients (Bergeron et al., 1989) and from rats with PCS (Bergeron et al., 1995) reveal several-fold increases in concentration of the dopamine metabolite homovanillic acid, a finding that could result from increased activities of monoamine oxidase reported in the same material (Mousseau et al., 1997). In another study, densities of the postsynaptic dopamine D2 receptor were significantly reduced in pallidum/putamen from cirrhotic patients (Mousseau et al., 1993) a finding that could have resulted from manganese

Strategies aimed at the prevention and treatment of PSE in chronic liver failure are of two major types, namely, ammonia-lowering strategies and approaches aimed directly at the CNS (Cordoba & Blei, 1997; Ferenci et al., 1996). Since PSE is frequently precipitated by ammoniagenic situations such as an oral protein load or a gastrointestinal hemorrhage, various treatment modalities are aimed at the gut. Such strategies include reduction of the absorption of nitrogenous substances arising from bacterial action in the colon. Colonic cleansing reduces the luminal ammonia content and lowers blood ammonia content in cirrhotic patients (Wolpert et al., 1970). Nonabsorbable disaccharides are routinely used to decrease ammonia production in the gut. The action of the most popular substance in this class, lactulose, involves increased fecal nitrogen excretion by facilitation of the incorporation of ammonia into bacteria as well as a cathartic effect (Cordoba & Blei, 1997). Lactulose administered orally reaches the cecum, where it is metabolized by enteric bacteria, causing a fall in pH (Brown et al., 1974). The dose is adjusted to produce two or three soft

Antibiotics such as neomycin are also useful for lowering blood ammonia, mainly by an effect on ammonia production by intestinal bacteria. However, neomycin therapy may be associated with some toxic side effects (Cordoba & Blei, 1997). More recently, rifaximin, a nonabsorbable derivative of the antibiotic rifamycin, has been shown to be effective in lowering blood ammonia and has proven to be of superior efficacy compared with lactulose (Bismuth et al., 2011; Munoz, 2008). Most of an orally administered dose is eliminated unchanged in the feces. Thus, it lacks significant toxicity and side effects because of minimal

deposition in the brains of these patients (Pomier Layrargues et al., 1995).

bowel movements daily (Cordoba & Blei, 1997).

gastrointestinal absorption.

the dog. These results suggested the possibility of a relationship between shunt size and postshunt PSE. The results of this study served as the basis for the subsequent use of reduced-caliber PCS in the treatment of variceal bleeding (Sarfeh & Rypins, 1994; Zervox et al., 1998).

In addition to ammonia, chronic liver failure and portal-systemic shunting result in increased blood and brain concentrations of manganese (López-Novoa, 1988; Fitzhugh & Nelson, 1948; Dashti et al., 1989; & Nuber et al., 1980). Manganese is neurotoxic, affecting both neuronal and astrocytic integrity. In the case of astrocytes, exposure to manganese results in altered expression of several key astrocytic proteins (Hazell et al., 1999a; Hazell et al., 1999b) and Alzheimer type II changes (Weissenborn et al., 1995).

Other toxins in addition to ammonia and manganese are known to increase in the systemic circulation in chronic liver failure. Such toxins include mercaptans, phenols, and short-chain fatty acids (Zieve et al., 1974). While there is no convincing evidence that these toxins alone cause cerebral dysfunction in chronic liver failure, they could combine with ammonia or manganese to act synergistically (Zieve, 1989).

Recently, attention has been focused on changes in brain neurotransmitter systems as the likely mediators of the neuropsychiatric manifestations of PSE in chronic liver failure. Recent studies using molecular biological approaches continue to confirm that, when liver fails, brain responds with significant alterations in gene expression. In many cases, these alterations involve genes that code for neurotransmitter-related proteins, many of which are essential for CNS function. Many of the symptoms of early PSE in chronic liver failure, such as altered personality, depression, and inverted sleep patterns, are symptoms that have classically been associated with alterations in biogenic amine function. FNA extracts of brain tissue obtained at autopsy from cirrhotic patients who died in hepatic coma have been found to show increased expression of the neuronal isoform of the monoaminemetabolizing enzyme MAO-A (Mousseau et al., 1997). This increase in MAO-A gene expression was found to be associated with increased activities of the enzyme and increased densities of catalytic sites on the enzyme protein (Mousseau et al., 1997). Moreover, studies of the same brain extracts revealed increased concentrations of homovanillic and – hydroxyindoleacetic acids, the final metabolites of dopamine and serotonin, respectively (Bergeron et al., 1989). Increased concentrations of 5-hydroxyindoleacetic acid were also reported in cerebrospinal fluid from patients (Young & Lai, 1980) and experimental animals (Bergeron et al., 1995) with chronic liver failure. On the basis of these findings, it has been suggested that altered monoaminergic function may be responsible for the early neuropsychiatric symptoms of PSE in chronic liver disease (Bergeron et al., 1995; Bergeron et al., 1990).

The *"peripheral-type" benzodiazepine receptor (PTBR)* is a heterooligomeric protein complex located (like MAO-A) on the outer mitochondrial membrane of the astrocyte. Increased PTBR gene expression has been reported in brain extracts from rats with PCS (Desjardins et al., 1997). This increased gene expression resulted in increased receptor sites in the brains and peripheral tissues of these animals as revealed by quantitative receptor autoradiography and the high selective PTBR ligant [3H] PK 11195 (Giguère et al., 1992; Raghavendra Rao et al., 1994). Increased [3H] PK 11195 binding sites were also reported in autopsied brain tissue from cirrhotic patients who died in hepatic coma (Lavoie et al., 1990).

the dog. These results suggested the possibility of a relationship between shunt size and postshunt PSE. The results of this study served as the basis for the subsequent use of reduced-caliber PCS in the treatment of variceal bleeding (Sarfeh & Rypins, 1994; Zervox et

In addition to ammonia, chronic liver failure and portal-systemic shunting result in increased blood and brain concentrations of manganese (López-Novoa, 1988; Fitzhugh & Nelson, 1948; Dashti et al., 1989; & Nuber et al., 1980). Manganese is neurotoxic, affecting both neuronal and astrocytic integrity. In the case of astrocytes, exposure to manganese results in altered expression of several key astrocytic proteins (Hazell et al., 1999a; Hazell et

Other toxins in addition to ammonia and manganese are known to increase in the systemic circulation in chronic liver failure. Such toxins include mercaptans, phenols, and short-chain fatty acids (Zieve et al., 1974). While there is no convincing evidence that these toxins alone cause cerebral dysfunction in chronic liver failure, they could combine with ammonia or