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Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy
acta histochemica 113 (2011) 358–362
Contents lists available at ScienceDirect
acta histochemica journal homepage: www.elsevier.de/acthis
Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy Marta Me´ndez a,n, Magdalena Me´ndez-Lo´pez a, Laudino Lo´pez a, Marı´a A. Aller b, Jaime Arias b, Jorge L. Arias a a b
Laboratorio de Neurociencias, Departamento de Psicologı´a, Universidad de Oviedo, Plaza Feijoo s/n, 33003 Oviedo, Spain Department of Surgery, Faculty of Medicine, Complutense University of Madrid, 28040 Madrid, Spain
a r t i c l e in f o
a b s t r a c t
Article history: Received 26 November 2009 Received in revised form 15 January 2010 Accepted 18 January 2010
Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Hepatic encephalopathy Acetylcholinesterase Cirrhosis Thioacetamide Rat
Introduction Hepatic encephalopathy (HE) is a neuropsychological disorder observed in patients with acute or chronic hepatic failure. This disorder exists in three major types, depending on its origin or cause: Type A HE, associated with acute liver failure, Type B HE, associated with portal-systemic bypass and no intrinsic hepatocellular disease and Type C HE, associated with cirrhosis and portal hypertension or portal-systemic shunts (Ferenci et al., 2002). Type C HE is the most common because hepatic cirrhosis, as well as other types of acute or chronic hepatic diseases, is one of the main clinical conditions affecting the Western World. This disease requires costly treatment and has high mortality rates. More than 950,000 individuals die every year from liver diseases all over the World (WHO, 2003). Therefore, it is necessary to resort to animal experimentation in order to clarify both the behavioral and the brain dysfunction that occurs in HE (Blei et al., 1992; Chamuleau, 1996). The models proposed to study HE due to chronic cirrhosis mimic the clinical characteristics of cirrhosis and portal hypertension and require obstruction of the bile duct (Kountouras et al., 1984; Jover et al., 2005) or the administration of hepatotoxins, such
n
Corresponding author. E-mail address: [email protected] (M. Me´ndez).
0065-1281/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2010.01.009
as intoxication with carbon tetrachloride or with thioacetamide (TAA) (Dashti et al., 1989; Laleman et al., 2006). The clinical stages of HE range from subtle psychiatric and behavioral changes in the early stages, to deep coma. As HE progresses, motor function and intellectual abilities deteriorate and patients show deficits in attention and arousal (Weissenborn et al., 2005), disturbances of learning, memory and recognition (Ortiz et al., 2006) and impaired motor performance, visual perception and visuo-constructive abilities (Weissenborn et al., 2003). The factor or factors that determine the development of these alterations found in patients with HE are still unknown and, in spite of advances in recent years, there are many unanswered questions concerning HE, with regard to its etiopathogeny (Butterworth, 2003), diagnosis (Montagnese et al., 2004) or treatment (Blei and Co´rdoba, 2001). Several factors have been implicated in the etiology of HE, such as morphological cell changes, which mainly affect astrocytes (Butterworth, 2002) and alterations of neurotransmitter systems (Butterworth, 2000), hyperammonemia and other factors such as inflammation, electrolyte alterations and altered blood flow (Hazell and Butterworth, 1999; Vaquero et al., 2003). All these alterations have been regarded as possible mechanisms responsible for the neuropsychological deficits frequently seen in patients with liver disease. Also, some mechanisms of the pathophysiology of HE are associated with changes in brain cell membranes (Swapna et al., 2006a, b) and neurotransmitter uptake, particularly of glutamate (Felipo and Butterworth, 2002).
M. Me´ndez et al. / acta histochemica 113 (2011) 358–362
Acetylcholinesterase (AChE) is a membrane-bound enzyme involved in cholinergic neurotransmission, which hydrolyzes acetylcholine released from presynaptic terminals obstructing acetylcholine transmission (Silman and Sussman, 2008). Recently, it has been demonstrated that AChE was severely decreased in the brain cortex of an experimental model of acute liver failure by TAA (Swapna et al., 2007), whereas in bile duct ligated rats, it was increased and no changes were observed in the portal-systemic HE (Garcı´a-Ayllo´n et al., 2008). Cholinergic neurotransmission is one of the most important means of neurotransmission in the mammalian brain. Cortical acetylcholine mediates sustained and selective attention, arousal, alertness, wakefulness and electroencephalographic desynchronization (Sarter et al., 2003). The activity of AChE is altered in neuropsychological disorders such as Alzheimer’s disease and dementia (Mesulam, 2004), which show disturbance of learning and memory processes. Considering that impaired spatial learning and active avoidance behavior have been described in rats with chronic liver failure by TAA administration (Me´ndez et al., 2008, 2009a, 2009b), we hypothesized that this model of chronic hepatic cirrhosis may show alterations in the level of AChE. For this reason, the aim of
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this study was to analyse the AChE activity of different brain limbic system regions considered to be implicated in memory processes.
Material and methods Animals A total of 16 male Wistar rats were used from the animal house of Oviedo University. All the animals had ad libitum access to food and tapwater and were maintained at constant room temperature (21 1C), with a relative humidity of 65–70% and artificial light–dark cycle of 12 h (08:00–20:00/20:00–08:00 h). The procedures and manipulation of the animals used in this study were carried out according to the Directive 86/609/EEC (The Council Directive of the European Community) concerning the protection of animals used for experimental and other scientific purposes. The national legislation, in agreement with this Directive, is defined in Royal Decree no. 1201/2005. The study was approved by the local committee for animal studies (Oviedo University).
Fig. 1. Acetylcholinesterase (AChE) histochemistry. Acetylcholinesterase (AChE) histochemistry in control (CO) and cirrhotic (TAA) rats and schematic drawings illustrating areas of analysis (indicated by the boxes) of AChE activity (A, D, G, J). AChE activity in accumbens core (ACc) and shell (ACs) (B, C), anterior thalamic nuclei (E, F) anterodorsal (TAD), anteroventral (TAV), anteromedial (TAM), hippocampal CA1, CA3 and dentate gyrus (DG) (H, I) and entorhinal cortex (EntC) (K, L). Scale bar: 500 mm.
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M. Me´ndez et al. / acta histochemica 113 (2011) 358–362
The experimental model The animals were initially split into two groups. The experimental group (TAA) (n=8) was composed of cirrhotic rats. The method used to produce cirrhosis was administration of TAA (Sigma-Aldrich, Germany) in drinking water (Xiangnong et al., 2002). The animals received continuous administration of TAA for 12 weeks and its concentration was modified weekly, depending on the animals’ weight gain or weight loss. Only TAA solution was used as drinking water for this group. The initial concentration of TAA was 0.03%. The control group (CO) (n=8) was isolated in the same way as the TAA group during 12 weeks and received normal tapwater. Experimental and control animals received commercial chow ad libitum and their body weights and fluid intake were recorded weekly.
Acetylcholinesterase (AChE) histochemistry Rats were decapitated, brains were removed intact, frozen rapidly in isopentane (Sigma-Aldrich, Germany) and stored at 40 1C. Coronal sections (30 mm) of the brain were cut in a cryostat (Leica CM1900, Leica Microsystems, Wetzlar, Germany) and mounted on gelatinized slides. The sections were stored at 80 1C until required for processing. The sections were thawed at room temperature for 1 h before histochemistry staining.
Acetylcholinesterase incubating medium (800 ml 0.05 M sodium acetate buffer at pH 5.0, 0.256 g cupric sulfate, 3.2 mg ethopropazine, 0.92 g acetylthiocholine iodide and 0.6 g glycine) was prepared immediately before use and slides were incubated for 2 h at 37 1C in the dark and then washed 8 times in distilled water for 1 min. To visualize the sites of acetylcholinesterase activity, the sections were subjected to a 1.25% ammonium sulfide solution (in distilled H2O) for 1 min. The slides were then rinsed in several distilled water incubations and fixed in buffered 10% formalin (0.1 M, pH 7.4) overnight, then dehydrated and mounted in DPX. This method was adapted from Slattery et al. (2005).
AChE densitometry Measurements were taken by using a computer-controlled image analysis workstation (MCID, InterFocus Imaging Ltd., Linton, England) and expressed as arbitrary units of optical density (OD). In order to establish comparisons between different staining baths, measurements were taken from AChE-stained brain standards of different thicknesses. Regression curves and coefficients between section thickness and AChE activity measured from each set of standards were calculated for each incubation bath. Optical density values measured for the brain regions selected were set at the same level using the optical
Fig. 2. Acetylcholinesterase (AChE) optical density values. Acetylcholinesterase (AChE) optical density values (+ SEM) of control (CO) and cirrhotic (TAA) group in accumbens core (ACc) and shell (ACs) (A), anterior thalamic nuclei (B) anterodorsal (TAD), anteroventral (TAV), anteromedial (TAM), hippocampal CA1, CA3 and dentate gyrus (DG) (C) and entorhinal cortex (EntC) (D). Asterisk (n) shows significant difference (po 0.05) in the AChE optical density values between control group and cirrhotic group.
M. Me´ndez et al. / acta histochemica 113 (2011) 358–362
density differences calculated from the regression plots of the brain standards. The OD of each structure was measured on the right side of bilateral structures using three consecutive sections in each animal. In each section, four, non-overlapping readings were taken using a square-shaped sampling window that was adjusted for each region size. A total of twelve measurements were taken per region. These twelve measurements were averaged to obtain one mean per region for each subject. Measurements were performed by an investigator using coded samples without knowledge of the groups. The selected brain regions that were analysed were anatomically defined according to Paxinos and Watsons atlas (2005). The stained coronal sections, from anterior to posterior, corresponded to Bregma levels 1.44 mm (accumbens core (ACc) and accumbens shell (ACs)), 1.92 mm (anterodorsal thalamic nucleus (TAD), anteroventral thalamic nucleus (TAV) and anteromedial thalamic nuleus (TAM)), 3.84 mm (hippocampal CA1 (CA1), hippocampal CA3 (CA3) and hippocampal dentate gyrus (DG)) and 5.04 mm (entorhinal cortex (EntC)) (Fig. 1). Data analysis A t-test for independent samples was used to compare AChE optical density values between the TAA and CO groups. Differences were considered statistically significant if p o0.05. The results are represented in Fig. 2.
Results AChE optical density levels of the TAA group were lower than those of the CO in all the hippocampal subfields CA1, CA3 and DG [t(14)= 4.409, p o0.001; t(14) =2.281, p o0.05 and t(14) =3.361, p =0.005, respectively]. However, TAA had higher AChE levels than those of CO in the thalamic nuclei TAD and TAV [t(14)= 3.455, p o0.005 and t(14) = 4.309, p o0.001, respectively] and in the ACc and ACs [t(14)= 3.835, po0.005 and t(14)= 5.885, p o0.001, respectively]. Also, AChE levels of the TAA group were increased in the EntC [t(14) =2.305, po0.05] (Fig. 2).
Discussion Our study demonstrates alteration of AChE activity in an experimental model of HE by chronic administration of TAA. This alteration was not uniform in the entire brain. Our analysis shows that up-regulation of AChE levels was found in the EntC, TAD, TAV, ACc and ACs, whereas down-regulation was found in the hippocampus. The impaired areas are related to attention and memory function and receive extensive innervations from cholinergic neurons (Butcher and Woolf, 2004). Therefore, it is possible that the dysfunction of cholinergic neurotransmission in these limbic system regions is related to the impairment of cognition seen both in patients and in the experimental models of HE. The relationship between cholinergic system alterations and liver failure has been assessed in previous studies. Alterations in AChE kinetic properties have been described in TAA-induced acute liver failure (Swapna et al., 2007) and increased levels of AChE have been found both in cirrhotic patients and in rats with bile duct ligation (Garcı´a-Ayllo´n et al., 2008). Nonetheless, some of these studies fail to demonstrate changes in the levels of cholinergic enzymes in human or experimental portal-systemic encephalopathy or in hyperammonemic conditions (Raghavendra Rao et al., 1994; Garcı´a-Ayllo´n et al., 2008), confirming decreased cholinesterase activity in acute or subacute liver disease, but not in portal-systemic HE. Chronic liver disease could alter
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blood–brain barrier permeability, increase toxic substances in brain and produce anomalous metabolic response that may contribute to neurotransmission deregulation (Hazell and Butterworth, 1999; Butterworth, 2003). Cholinergic pathways innervate all cortical and limbic areas and are associated with the ascending reticular activating system, influencing cognition and behavior. Increased levels of AChE in the brain, which obstructs acetylcholine neurotransmission by hydrolyzing presynaptic acetylcholine, might cause a decrease of extracellular acetylcholine and affect information processing and learning acquisition. Increased AChE could lead to a pronounced decrease in the levels of the neurotransmitter acetylcholine. Therefore, these data suggests an impairment of the brain cholinergic system induced by cirrhosis, which may be associated with failure in learning and attention processes (Hasselmo and Giocomo, 2006). This study shows abnormally higher AChE levels in the entorhinal cortex. Given that cortical acetylcholine mediates detection and selection of relevant stimuli and both sustained and selective attention (Sarter et al., 2003), high AChE levels in the entorhinal cortex could cause deficits in information processing and encoding of new episodic memories. The effects of high AChE levels in the entorhinal cortex might provide support for the involvement of cholinergic transmission in the impairment in divided attention found in human subjects with HE (Amodio et al., 2005). A similar increase in AChE levels was found in the anterodorsal and anteroventral thalamic nuclei. The thalamus is a structure which influences behavior through its participation in many brain circuits involved in the processing of new and old information (Van der Werf et al., 2000). These nuclei contain neurons that code for direction (Taube, 1995; Blair et al., 1999) and theta rhythm (Vertes et al., 2001; Albo et al., 2003) and are involved in complex cognitive skills and memory processes, participating in selection of stimuli for subsequent memory storage in accordance with the connections with the hippocampal system. Hence, the impairment of cholinergic neurotransmission in these thalamic nuclei could be related to the deterioration of cognition found in this experimental model (Me´ndez et al., 2008) and is in accordance with the reduction of oxidative metabolism of these nuclei (Me´ndez et al., 2009b). Down-regulation of AChE in the accumbens core and shell nuclei suggests an impairment of reward and learning. The accumbens receives projections from several limbic regions such as the hippocampus, the cortex, the thalamic nuclei and the ventral tegmental area (Fallon and Moore, 1978; Krayniak et al., 1981; Kelley and Domesick, 1982; Ligorio et al., 2009), and its primary output is to the ventral pallidum (Zahm and Heimer, 1990). Therefore, its cholinergic interneurons are involved in motivation and reward and aversive behavior, and are considered a part of the limbic system (Mogenson et al., 1980; Hoebel et al., 2007). Contrary to the results in the previous structures, AChE levels in the cirrhotic animals were decreased in the dorsal hippocampus. This could imply an increase of acetylcholine in the hippocampal formation and might correlate with the increase in the neuronal activity assessed by cytochrome oxidase histochemistry found in the hippocampus in this experimental model (Me´ndez et al., 2009b). Cholinergic transmission enhancement could contribute to the impairment of learning found in this model (Me´ndez et al., 2008) because, contrary to the idea that increased hippocampal acetylcholine release will facilitate the acquisition of a learning task (Givens and Olton, 1994, 1995), some studies suggest that the increase in hippocampal acetylcholine by stimulation of septohippocampal cholinergic cells produces deficit in spatial learning assessed in the Morris water maze (Elvander et al., 2004). Also, the consolidation of memory
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processes should be impaired by increases in acetylcholine levels during consolidation (Bunce et al., 2004). Taking into account these results, further research is necessary to understand the role of acetylcholine neurotransmission in the cognitive deficits of HE Type C.
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Contents lists available at ScienceDirect
acta histochemica journal homepage: www.elsevier.de/acthis
Acetylcholinesterase activity in an experimental rat model of Type C hepatic encephalopathy Marta Me´ndez a,n, Magdalena Me´ndez-Lo´pez a, Laudino Lo´pez a, Marı´a A. Aller b, Jaime Arias b, Jorge L. Arias a a b
Laboratorio de Neurociencias, Departamento de Psicologı´a, Universidad de Oviedo, Plaza Feijoo s/n, 33003 Oviedo, Spain Department of Surgery, Faculty of Medicine, Complutense University of Madrid, 28040 Madrid, Spain
a r t i c l e in f o
a b s t r a c t
Article history: Received 26 November 2009 Received in revised form 15 January 2010 Accepted 18 January 2010
Patients with liver malfunction often suffer from hepatic encephalopathy, a neurological complication which can affect attention and cognition. Diverse experimental models have been used to study brain alterations that may be responsible for hepatic encephalopathy symptoms. The aim of the study was to determine whether cognitive impairment found in cirrhosis could be due to disturbance of acetylcholinesterase activity. Acetylcholinesterase activity was assessed in the brains of Wistar rats with thioacetamide-induced cirrhosis. The cirrhotic group displayed up-regulation of acetylcholinesterase levels in the entorrhinal cortex, anterodorsal and anteroventral thalamus and accumbens, whereas down-regulation was found in the CA1, CA3 and dentate gyrus of the hippocampus. Our results indicate that the experimental model of hepatic encephalopathy by chronic administration of thioacetamide presents alterations of acetylcholinesterase activity in brain limbic system regions, which play a role in attention and memory. & 2010 Elsevier GmbH. All rights reserved.
Keywords: Hepatic encephalopathy Acetylcholinesterase Cirrhosis Thioacetamide Rat
Introduction Hepatic encephalopathy (HE) is a neuropsychological disorder observed in patients with acute or chronic hepatic failure. This disorder exists in three major types, depending on its origin or cause: Type A HE, associated with acute liver failure, Type B HE, associated with portal-systemic bypass and no intrinsic hepatocellular disease and Type C HE, associated with cirrhosis and portal hypertension or portal-systemic shunts (Ferenci et al., 2002). Type C HE is the most common because hepatic cirrhosis, as well as other types of acute or chronic hepatic diseases, is one of the main clinical conditions affecting the Western World. This disease requires costly treatment and has high mortality rates. More than 950,000 individuals die every year from liver diseases all over the World (WHO, 2003). Therefore, it is necessary to resort to animal experimentation in order to clarify both the behavioral and the brain dysfunction that occurs in HE (Blei et al., 1992; Chamuleau, 1996). The models proposed to study HE due to chronic cirrhosis mimic the clinical characteristics of cirrhosis and portal hypertension and require obstruction of the bile duct (Kountouras et al., 1984; Jover et al., 2005) or the administration of hepatotoxins, such
n
Corresponding author. E-mail address: [email protected] (M. Me´ndez).
0065-1281/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2010.01.009
as intoxication with carbon tetrachloride or with thioacetamide (TAA) (Dashti et al., 1989; Laleman et al., 2006). The clinical stages of HE range from subtle psychiatric and behavioral changes in the early stages, to deep coma. As HE progresses, motor function and intellectual abilities deteriorate and patients show deficits in attention and arousal (Weissenborn et al., 2005), disturbances of learning, memory and recognition (Ortiz et al., 2006) and impaired motor performance, visual perception and visuo-constructive abilities (Weissenborn et al., 2003). The factor or factors that determine the development of these alterations found in patients with HE are still unknown and, in spite of advances in recent years, there are many unanswered questions concerning HE, with regard to its etiopathogeny (Butterworth, 2003), diagnosis (Montagnese et al., 2004) or treatment (Blei and Co´rdoba, 2001). Several factors have been implicated in the etiology of HE, such as morphological cell changes, which mainly affect astrocytes (Butterworth, 2002) and alterations of neurotransmitter systems (Butterworth, 2000), hyperammonemia and other factors such as inflammation, electrolyte alterations and altered blood flow (Hazell and Butterworth, 1999; Vaquero et al., 2003). All these alterations have been regarded as possible mechanisms responsible for the neuropsychological deficits frequently seen in patients with liver disease. Also, some mechanisms of the pathophysiology of HE are associated with changes in brain cell membranes (Swapna et al., 2006a, b) and neurotransmitter uptake, particularly of glutamate (Felipo and Butterworth, 2002).
M. Me´ndez et al. / acta histochemica 113 (2011) 358–362
Acetylcholinesterase (AChE) is a membrane-bound enzyme involved in cholinergic neurotransmission, which hydrolyzes acetylcholine released from presynaptic terminals obstructing acetylcholine transmission (Silman and Sussman, 2008). Recently, it has been demonstrated that AChE was severely decreased in the brain cortex of an experimental model of acute liver failure by TAA (Swapna et al., 2007), whereas in bile duct ligated rats, it was increased and no changes were observed in the portal-systemic HE (Garcı´a-Ayllo´n et al., 2008). Cholinergic neurotransmission is one of the most important means of neurotransmission in the mammalian brain. Cortical acetylcholine mediates sustained and selective attention, arousal, alertness, wakefulness and electroencephalographic desynchronization (Sarter et al., 2003). The activity of AChE is altered in neuropsychological disorders such as Alzheimer’s disease and dementia (Mesulam, 2004), which show disturbance of learning and memory processes. Considering that impaired spatial learning and active avoidance behavior have been described in rats with chronic liver failure by TAA administration (Me´ndez et al., 2008, 2009a, 2009b), we hypothesized that this model of chronic hepatic cirrhosis may show alterations in the level of AChE. For this reason, the aim of
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this study was to analyse the AChE activity of different brain limbic system regions considered to be implicated in memory processes.
Material and methods Animals A total of 16 male Wistar rats were used from the animal house of Oviedo University. All the animals had ad libitum access to food and tapwater and were maintained at constant room temperature (21 1C), with a relative humidity of 65–70% and artificial light–dark cycle of 12 h (08:00–20:00/20:00–08:00 h). The procedures and manipulation of the animals used in this study were carried out according to the Directive 86/609/EEC (The Council Directive of the European Community) concerning the protection of animals used for experimental and other scientific purposes. The national legislation, in agreement with this Directive, is defined in Royal Decree no. 1201/2005. The study was approved by the local committee for animal studies (Oviedo University).
Fig. 1. Acetylcholinesterase (AChE) histochemistry. Acetylcholinesterase (AChE) histochemistry in control (CO) and cirrhotic (TAA) rats and schematic drawings illustrating areas of analysis (indicated by the boxes) of AChE activity (A, D, G, J). AChE activity in accumbens core (ACc) and shell (ACs) (B, C), anterior thalamic nuclei (E, F) anterodorsal (TAD), anteroventral (TAV), anteromedial (TAM), hippocampal CA1, CA3 and dentate gyrus (DG) (H, I) and entorhinal cortex (EntC) (K, L). Scale bar: 500 mm.
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The experimental model The animals were initially split into two groups. The experimental group (TAA) (n=8) was composed of cirrhotic rats. The method used to produce cirrhosis was administration of TAA (Sigma-Aldrich, Germany) in drinking water (Xiangnong et al., 2002). The animals received continuous administration of TAA for 12 weeks and its concentration was modified weekly, depending on the animals’ weight gain or weight loss. Only TAA solution was used as drinking water for this group. The initial concentration of TAA was 0.03%. The control group (CO) (n=8) was isolated in the same way as the TAA group during 12 weeks and received normal tapwater. Experimental and control animals received commercial chow ad libitum and their body weights and fluid intake were recorded weekly.
Acetylcholinesterase (AChE) histochemistry Rats were decapitated, brains were removed intact, frozen rapidly in isopentane (Sigma-Aldrich, Germany) and stored at 40 1C. Coronal sections (30 mm) of the brain were cut in a cryostat (Leica CM1900, Leica Microsystems, Wetzlar, Germany) and mounted on gelatinized slides. The sections were stored at 80 1C until required for processing. The sections were thawed at room temperature for 1 h before histochemistry staining.
Acetylcholinesterase incubating medium (800 ml 0.05 M sodium acetate buffer at pH 5.0, 0.256 g cupric sulfate, 3.2 mg ethopropazine, 0.92 g acetylthiocholine iodide and 0.6 g glycine) was prepared immediately before use and slides were incubated for 2 h at 37 1C in the dark and then washed 8 times in distilled water for 1 min. To visualize the sites of acetylcholinesterase activity, the sections were subjected to a 1.25% ammonium sulfide solution (in distilled H2O) for 1 min. The slides were then rinsed in several distilled water incubations and fixed in buffered 10% formalin (0.1 M, pH 7.4) overnight, then dehydrated and mounted in DPX. This method was adapted from Slattery et al. (2005).
AChE densitometry Measurements were taken by using a computer-controlled image analysis workstation (MCID, InterFocus Imaging Ltd., Linton, England) and expressed as arbitrary units of optical density (OD). In order to establish comparisons between different staining baths, measurements were taken from AChE-stained brain standards of different thicknesses. Regression curves and coefficients between section thickness and AChE activity measured from each set of standards were calculated for each incubation bath. Optical density values measured for the brain regions selected were set at the same level using the optical
Fig. 2. Acetylcholinesterase (AChE) optical density values. Acetylcholinesterase (AChE) optical density values (+ SEM) of control (CO) and cirrhotic (TAA) group in accumbens core (ACc) and shell (ACs) (A), anterior thalamic nuclei (B) anterodorsal (TAD), anteroventral (TAV), anteromedial (TAM), hippocampal CA1, CA3 and dentate gyrus (DG) (C) and entorhinal cortex (EntC) (D). Asterisk (n) shows significant difference (po 0.05) in the AChE optical density values between control group and cirrhotic group.
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density differences calculated from the regression plots of the brain standards. The OD of each structure was measured on the right side of bilateral structures using three consecutive sections in each animal. In each section, four, non-overlapping readings were taken using a square-shaped sampling window that was adjusted for each region size. A total of twelve measurements were taken per region. These twelve measurements were averaged to obtain one mean per region for each subject. Measurements were performed by an investigator using coded samples without knowledge of the groups. The selected brain regions that were analysed were anatomically defined according to Paxinos and Watsons atlas (2005). The stained coronal sections, from anterior to posterior, corresponded to Bregma levels 1.44 mm (accumbens core (ACc) and accumbens shell (ACs)), 1.92 mm (anterodorsal thalamic nucleus (TAD), anteroventral thalamic nucleus (TAV) and anteromedial thalamic nuleus (TAM)), 3.84 mm (hippocampal CA1 (CA1), hippocampal CA3 (CA3) and hippocampal dentate gyrus (DG)) and 5.04 mm (entorhinal cortex (EntC)) (Fig. 1). Data analysis A t-test for independent samples was used to compare AChE optical density values between the TAA and CO groups. Differences were considered statistically significant if p o0.05. The results are represented in Fig. 2.
Results AChE optical density levels of the TAA group were lower than those of the CO in all the hippocampal subfields CA1, CA3 and DG [t(14)= 4.409, p o0.001; t(14) =2.281, p o0.05 and t(14) =3.361, p =0.005, respectively]. However, TAA had higher AChE levels than those of CO in the thalamic nuclei TAD and TAV [t(14)= 3.455, p o0.005 and t(14) = 4.309, p o0.001, respectively] and in the ACc and ACs [t(14)= 3.835, po0.005 and t(14)= 5.885, p o0.001, respectively]. Also, AChE levels of the TAA group were increased in the EntC [t(14) =2.305, po0.05] (Fig. 2).
Discussion Our study demonstrates alteration of AChE activity in an experimental model of HE by chronic administration of TAA. This alteration was not uniform in the entire brain. Our analysis shows that up-regulation of AChE levels was found in the EntC, TAD, TAV, ACc and ACs, whereas down-regulation was found in the hippocampus. The impaired areas are related to attention and memory function and receive extensive innervations from cholinergic neurons (Butcher and Woolf, 2004). Therefore, it is possible that the dysfunction of cholinergic neurotransmission in these limbic system regions is related to the impairment of cognition seen both in patients and in the experimental models of HE. The relationship between cholinergic system alterations and liver failure has been assessed in previous studies. Alterations in AChE kinetic properties have been described in TAA-induced acute liver failure (Swapna et al., 2007) and increased levels of AChE have been found both in cirrhotic patients and in rats with bile duct ligation (Garcı´a-Ayllo´n et al., 2008). Nonetheless, some of these studies fail to demonstrate changes in the levels of cholinergic enzymes in human or experimental portal-systemic encephalopathy or in hyperammonemic conditions (Raghavendra Rao et al., 1994; Garcı´a-Ayllo´n et al., 2008), confirming decreased cholinesterase activity in acute or subacute liver disease, but not in portal-systemic HE. Chronic liver disease could alter
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blood–brain barrier permeability, increase toxic substances in brain and produce anomalous metabolic response that may contribute to neurotransmission deregulation (Hazell and Butterworth, 1999; Butterworth, 2003). Cholinergic pathways innervate all cortical and limbic areas and are associated with the ascending reticular activating system, influencing cognition and behavior. Increased levels of AChE in the brain, which obstructs acetylcholine neurotransmission by hydrolyzing presynaptic acetylcholine, might cause a decrease of extracellular acetylcholine and affect information processing and learning acquisition. Increased AChE could lead to a pronounced decrease in the levels of the neurotransmitter acetylcholine. Therefore, these data suggests an impairment of the brain cholinergic system induced by cirrhosis, which may be associated with failure in learning and attention processes (Hasselmo and Giocomo, 2006). This study shows abnormally higher AChE levels in the entorhinal cortex. Given that cortical acetylcholine mediates detection and selection of relevant stimuli and both sustained and selective attention (Sarter et al., 2003), high AChE levels in the entorhinal cortex could cause deficits in information processing and encoding of new episodic memories. The effects of high AChE levels in the entorhinal cortex might provide support for the involvement of cholinergic transmission in the impairment in divided attention found in human subjects with HE (Amodio et al., 2005). A similar increase in AChE levels was found in the anterodorsal and anteroventral thalamic nuclei. The thalamus is a structure which influences behavior through its participation in many brain circuits involved in the processing of new and old information (Van der Werf et al., 2000). These nuclei contain neurons that code for direction (Taube, 1995; Blair et al., 1999) and theta rhythm (Vertes et al., 2001; Albo et al., 2003) and are involved in complex cognitive skills and memory processes, participating in selection of stimuli for subsequent memory storage in accordance with the connections with the hippocampal system. Hence, the impairment of cholinergic neurotransmission in these thalamic nuclei could be related to the deterioration of cognition found in this experimental model (Me´ndez et al., 2008) and is in accordance with the reduction of oxidative metabolism of these nuclei (Me´ndez et al., 2009b). Down-regulation of AChE in the accumbens core and shell nuclei suggests an impairment of reward and learning. The accumbens receives projections from several limbic regions such as the hippocampus, the cortex, the thalamic nuclei and the ventral tegmental area (Fallon and Moore, 1978; Krayniak et al., 1981; Kelley and Domesick, 1982; Ligorio et al., 2009), and its primary output is to the ventral pallidum (Zahm and Heimer, 1990). Therefore, its cholinergic interneurons are involved in motivation and reward and aversive behavior, and are considered a part of the limbic system (Mogenson et al., 1980; Hoebel et al., 2007). Contrary to the results in the previous structures, AChE levels in the cirrhotic animals were decreased in the dorsal hippocampus. This could imply an increase of acetylcholine in the hippocampal formation and might correlate with the increase in the neuronal activity assessed by cytochrome oxidase histochemistry found in the hippocampus in this experimental model (Me´ndez et al., 2009b). Cholinergic transmission enhancement could contribute to the impairment of learning found in this model (Me´ndez et al., 2008) because, contrary to the idea that increased hippocampal acetylcholine release will facilitate the acquisition of a learning task (Givens and Olton, 1994, 1995), some studies suggest that the increase in hippocampal acetylcholine by stimulation of septohippocampal cholinergic cells produces deficit in spatial learning assessed in the Morris water maze (Elvander et al., 2004). Also, the consolidation of memory
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processes should be impaired by increases in acetylcholine levels during consolidation (Bunce et al., 2004). Taking into account these results, further research is necessary to understand the role of acetylcholine neurotransmission in the cognitive deficits of HE Type C.
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