Effect of Blocking Neurosteroids Synthesis on Brain Electrical Activity and behaviour in Animal Models of Type A and Type C Hepatic Encephalopathy
Abdul-Qawi Nagi Saeed
a, Shaimaa Nasr Amin
2*,Suheir Ali Abd EL-Wahed
3, Mona Osman Abd El-Halim
3, Miran Atif Elkordy
4, Walaa Ibrahim Ali
5, Noha Samir Abdel Latif
31Department of Medical Pharmacology, Faculty of Medicine, Taiz University, Taiz, Yemen.
2Department of Medical Physiology, Faculty of Medicine, Cairo University, Cairo, Egypt.
3Department of Medical Pharmacology, Faculty of Medicine, Cairo University, Cairo, Egypt.
4Department of Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt.
5Department of Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt.
* Corresponding Author:
Shaimaa Nasr Amin, MD, PhD E-mail: [email protected]
ORCID: 0000-0001-9232-2389
ABSTRACT
Hepatic Encephalopathy (HE) is a brain dysfunction caused by acute or chronic liver insufficiency. Increased brain levels of Neurosteroids (NS) in HE have been implicated in the pathogenesis of HE. Hence, blockage of NS synthesis by Finasteride is postulated as a possible agent to attenuate HE, especially in those who did not respond or were intolerant to the currently used drugs. In the present study, intraperitoneal injection (IP) of rats with thioacetamide for three days induced type (A) HE, while IP injection of rats with TAA twice weekly for 12 weeks induced type (C) HE. Rats were divided into two main groups, group A (type A, HE) and group B (type C, HE), each leading group subdivided into six subgroups (6 rats in each); rats were treated with Finasteride, Lactulose, or their combination, daily for three days for type (A) HE, and two weeks for type (C) HE. Administration of TAA induced neurobehavioral changes in behavioral, clinical score, open field test (OFT), and forced swimming test (FST).
Besides, it caused EEG power spectral density (PSD) changes, derangement in liver function tests, increased serum ammonia level, and brain histological changes. Finasteride had similar effects to Lactulose in ameliorating neurobehavioral, electrophysiological, biochemical, and histopathological changes. The concomitant effect of Finasteride and Lactulose showed better outcomes for all measured parameters and tests.
Keywords
Hepatic Encephalopathy, brain, neurosteroids, EEG, spectral densities.
Introduction
Hepatic Encephalopathy (HE) is a reversible brain dysfunction caused by liver impairment (Ferenci, 2017). HE is categorized into three main types: type A, due to acute liver failure (ALF); type B, due to portosystemic shunt (PSS);
and type C, due to liver cirrhosis. Each type has different clinical presentation, treatment approaches, and prognosis;
in general, HE manifests with neuropsychiatric alterations, extended from Minimal HE (MHE) "alteration only in psychometric tests, electrophysiological and other functional brain measures," to Overt HE (OHE), showing symptomatic neuropsychiatric changes that range from a trivial impairment of intellectual and cognitive function to frank alterations in consciousness and motor function, with increased risk of cerebral edema and death (Vilstrup et al., 2014)
Many biochemical tests, including serum ammonia level, neuropsychiatric tests such as psychomotor hepatic encephalopathy score (PHES), and neurophysiological tests such as EEG, are used for diagnosis and grading the severity of HE (Press et al., 2017). However, EEG changes associated with HE needed further scientific validation.
The liver has a detoxifying function; in hepatocellular injury, the hepatic clearance of toxic materials from the gut is impaired, shifting toxins into the systemic circulation. These toxins impair brain functions, disturb brain water
homeostasis, cellular stress, dysregulated neurotransmitters and neurosteroid (NS) balance, and neuroinflammation (Tranah et al., 2015). NS, are steroids formed from cholesterol by a series of enzymatically catalyzed steps, including 5α-R by the neurons and glial cells (Butterworth, 2016)
NS potentiates GABAergic transmission contributing to the balance between excitation and inhibition of neural activity. Its brain concentrations are increased, resulting in sedation seen in HE. Furthermore, ammonia, manganese, and proinflammatory cytokines upregulateupregulate NS formation (Ahboucha &Butterworth, 2008).
Thioacetamide (TAA) induces HE due to acute or chronic liver failure; it induces behavioral, motor, and electroencephalographic manifestations similar to human HE (Farjam et al., 2012).
Several pharmacological agents have been used for the management of HE is including, Lactulose, rifaximin, metronidazole, neomycin, vancomycin, and other therapies; however, some patients are unresponsive to these agents because it minimized ammonia absorption but did not affect ammonia detoxification and sometimes associated with annoying side effects (Vilstrup et al., 2014). Drugs that interfere with the effect of GABA-steroids on the GABA-A receptor may provide a positive impact on the management of HE (Johansson et al., 2016)
Finasteride (FIN), a 5 α- reductase (5α-R) inhibitor that blocks the formation of brain NS is commonly used in benign prostatic hyperplasia (Mukaiet al., 2008). Hence, as an NS inhibitor, Finasteride may alleviate HE's manifestations by blocking one of the pathogenic steps involved in HE. This study aimed to evaluate the potential effects of Finasteride compared with Lactulose in Hepatic Encephalopathy.
Methods
The experimental steps, animal handling, sampling, and euthanasia were approved by the Ethical and Scientific Committee, Department of Pharmacology, Faculty of Medicine, Cairo University.
Experimental Animals and study design:
Seventy-two male adult albino rats (150-250 g) constituted the animal model, obtained from the Animal House, Ophthalmology Institute, Cairo University, and housed three rats/cage to avoid isolation stress. Animals were entrained on a 12-h light/dark cycle at an ambient temperature (24 ± 1°C), where food and water were given ad libitum throughout the study. Rats were accommodated for one week before initiating any experimental steps to permit habituation to the lab environment. Rats were divided into type A and type C encephalopathy main groups that further subdivided into six subgroups (six rats/subgroup), as the following:
Type A Hepatic Encephalopathy groups:
The normal control group (GA1): received a single dose of normal saline (2 ml), intraperitoneal (IP), for three consecutive days.
Finasteride control group (GA2): received only Finasteride in a dose of 50 mg\kg\day (Mladenović et al., 2012), by IP injection, once daily for three consecutive days.Finasteride (Sigma Chemical Co, St. Louis, Mo, USA) was solubilized in 20% weight/vol 2-hydroxypropyl- β-cyclodextrin and administered in a concentration of 5 mg/mL (injection volume was 0.01 mL/g animal body weight).
Thioacetamide (TAA) group (GA3):received IP injection of TAA for three consecutive days in a dose of 300 mg\kg\day (Mladenović et al., 2012). TAA (Sigma-Aldrich Chemical Co., USA)was purchased as a powder and dissolved in pure saline in 100 mg/mL concentration.
Lactulose treated group:(GA4): received Lactulose (EIPICO, Egypt) in a dose of 8 mg\kg/day (Jia & Zhang, 2005), given daily by gastric gavage, starting 2 hours before every dose of TAA for three consecutive days.
Finasteride treated group (GA5): received Finasteride in a dose of 50mg\kg\day by IP injection, once daily starting 2 hours before each TAA dose (Mladenović et al., 2012) for three consecutive days.
Finasteride and lactulose treated group (GA6): received Finasteride, in a dose of 50mg\kg\day, by IP injection, and lactulose 8 mg\kg\day, giving by gastric gavage, once daily starting 2 hours before every dose of TAA, for three consecutive days.
All rats of group A that received TAA (GA3-GA6) were given a supportive therapy which consisted of a solution containing 10% glucose solution mixed with lactate ringer (1:1), in a dose of (10 ml/kg), subcutaneously every 12 hours after the first injection of TAA to avoid hypoglycemia and electrolyte imbalance (Li et al., 2015).
Group B (Type C Hepatic Encephalopathy)
The normal control group (GB1):received standard saline IP two times per week for 12 weeks.
Finasteride control group (GB2):received Finasteride in a dose of 50 mg\kg\ days (Mladenović et al., 2012), through IP injection, daily for the last two weeks.
Thioacetamide (TAA) group (GB3):received IP of TAA in a dose of 200 mg\kg, twice time per week for 12 weeks (Bruck et al., 2001).
Lactulose treated group (GB4): received Lactulose in a dose of 8 mg\kg\day (Jia & Zhang, 2005) by gastric gavage daily for the last two weeks.
Finasteride treated group (GB5):received Finasteride in a dose of 50mg\kg\ day (Mladenović et al., 2012), by IP injection for the last two weeks.
Finasteride and lactulose treated group (GB6): received Finasteride in a dose of 50mg\kg\day by IP injection, and lactulose 8 mg\kg by gastric gavage, once daily for the last two weeks.
During the study period, rats' body weight was recorded weekly to monitor body weight change and determine the doses for hepatic encephalopathy and treatment induction. Cognitive and behavioral assessment performed twice: at the start of the work: after habituation to the laboratory environment; before drug administration for different groups;
and by the end of the work. After animal treatment was over, rats were fasted for 12 hours and were evaluated clinically for cognition ad behavior. Then, EEG was recorded for 30 minutes. After that, blood samples were collected from the retro-orbital venous plexus, and finally, animals euthanized were euthanized by decapitation and exsanguinated. For uniform preservation and proper histological examination of the brain, intracardiac fixation perfusion (Gage et al., 2012), the brains were extracted for histopathological staining and examination.
Outcome Measures.
Neurobehavioral Assessment.
Behavioral and psycho-mental functions were assessed in all groups during the study by the following:
-The rats' behavior clinical score:
The rats were clinically evaluated regularly for mortality rate and the clinical grade of Hepatic Encephalopathy and were scored as shown in table 1 (Farjam et al., 2012).
Table. 1: The Rats' Behavior Clinical Score (Farjam et al., 2012) Clinical score Definition
0 Normal behavior
1 Mild lethargy
2 Decreased motor activity, poor gesture control, diminished pain perception.
3 Severe ataxia, no spontaneous righting reflex 4 No righting reflex, no reaction to pain stimuli
5 Death
-Open Field (OF)Behavioral Test:
Apparatus and Procedure:the OF apparatus in this study was constructed from common household wood and measured 72 x 72 cm with 36 cm walls, the floor of the box was divided by red marker into sixteen squares (18 x 18 cm), the bottom of the OF arena should be mildly rough to enable animals to move without sliding, the open field arena was equipped with a webcam (20-Megapixel, Gigaware, UK), the camera was connected to a computer system in a separate room where all activities were monitored and recorded (Brown et al., 1999)
The box has placed in the corner of a quiet room and lit by a lamp, avoiding unintentional illumination and light reflections, which influences the behavior and possibly the tracking procedure. Each rat was placed into one of the four corners of the open field and allowed to explore the apparatus for 10 minutes and monitored for: latency time (time in seconds elapsed until the rat start to move), ambulation frequency (number of squares crossed by rats with all four paws), rearing frequency (time number the rat stood on their hind legs in the field), grooming frequency (time number the rat licking or scratching itself while stationary). The OF arena was cleaned with 70 % ethyl alcohol and permitted to dry between subjects' tests to avoid internal olfactory cues, which may change the following animal's exploratory pattern (Tatem et al., 2014).
-Forced Swimming Behavioral Test
The Forced swimming behavioral test (FST) is used to study the depressive-like behavior as reflected by the periods of immotility if the animals are subjected to the forced swimming in a cylindrical container of water from which it cannot escape (Porsolt et al.,1978).
Apparatus and Procedure:
The transparent cylindrical plastic containers measuring 50 cm in height and a diameter of 20 cm, was filled with water at room temperature (23 ± 1 °C), and a webcam (20 Megapixel, Gigaware, UK) was fixed in front of the containers and the camera connected to a computer system in a separate room where all activities were monitored and recorded. The test was performed in 2 sessions, 24 hours apart, the first session is the pre-test stage (15 min), and the second session is the test stage (5 min) (Kawai et al., 2012). Each rat was placed in the water-filled cylinder container for 5 min; then, the rat was removed from the container and dried by a pad, and placed in the transient drying cage with the heat lamp above it and the heat pad under it. The water was changed after every session to avoid any influence on the next rats' behavior. Rats monitored for the following during FST: climbing(upward-directed movements of the forepaws along the side of the swim chamber), swimming (the movement throughout the swimming chamber that also includes crossing into another quadrant), immobility (no additional activity is observed other than that required to keep the rat's head above the water). (Yankelevitch-Yahav et al., 2015).
Electroencephalography (EEG) Recording.
After 24 hours of the last dose, rats were subjected to EEG recording for 30 minutes under urethane (1gm/kg). Rats were implanted with three small stainless steel needle electrodes placed over the right parietal cortex, the left parietal cortex, and a third reference electrode over the cerebellum. The EEG electrodes were connected to EEG amplifier through recording cables which were connected to the Power Lab system (AD Instruments Power Lab 7.3.7); the EEG signals were digitized at 400 Hz (AD Instruments) with 16-bit analog-to-digital conversion (A/D) and stored in a PC through the lab Chart program software (Mladenovic et al., 2013).
The EEG signals were processed and converted to power spectra by the fast Fourier transform system using 0.1 - 45 Hz bandpass filter and Hamming window type, the frame size used was 1024 points (50% overlap), the resolution was 0.195 Hz, and the sampling rate was 200 samples/sec, the integrated energy signals were expressed as µV2/Hz, and the absolute and relative power spectral densities were calculated, and the obtained averaged power spectra were segmented into four frequency: delta (0.1–4 Hz); theta (4.1–8 Hz); alpha (8.1–14 Hz) and beta (14.1–30 Hz), the power of each frequency band was relative (percentage) to thetotal power (0.1–30 Hz) of the bands for comparison purpose (Mladenovic et al., 2013).
Biochemical Assessment
-Determination of serum ammonia activity:Serum ammonia level was determined calorimetrically, using a test reagent kit (Bio diagnostic, Egypt) according to (Konitzer & Voigt, 1963).
-Determination of aspartate transaminase (AST) and alanine aminotransferase (ALT) activity:The serum AST and ALT activity were assessed calorimetrically, using test reagent kits (Bio diagnostic, Egypt ( according to manufacturer's instructions.
-Determination of bilirubin level: Serum bilirubin level was determined by the colorimetric method according to (Scherlock 1951) method, using Randox laboratories kit (UK).
-Determination of albumin level:Serum albumin measured according to the method described by Doumas et al.
(1971), kits used was purchased from Bio diagnostic Egypt.
Brain histopathology:
Rats were euthanized by decapitation and exsanguinated. For uniform preservation and proper histological examination of the brain, intracardiac fixation perfusion (Gage et al., 2012), then the brain was were placed in 10%
formaldehyde for 2 hours then removed and placed in a new formaldehyde solution for 24 hours before being dehydrated using ethanol, then cleaned in xylene and embedded in paraffin. Coronal section cuts were made with a microtome (Leica RM 2025, Germany) at 5 µm thicknesses at the level of the prefrontal cortex, mounted on glass slides, and stained with the hematoxylin and eosin (Sidoryk-Wegrzynowiczet al., 2011).
Statistical analysis
All results were expressed as mean ± standard deviation (SD) for all studied groups. Comparison of quantitative data between studied groups was made using a one-way analysis of variance (ANOVA) test followed by a post hoc Tukey-Kramer multiple comparison test for comparing more than two groups. Results were significant if p-values <
0.05. All statistical calculations were done using Statistical Package for the Social Science (SPSS), v.22 for Microsoft Windows.
Results
Result of Neurobehavioral Assessment
Table 2: The Effect of Finasteride and Lactulose on Behavioral Clinical Score of Rats with Hepatic Encephalopathy Induced by Thioacetamide (TAA).
Rats' behavior clinical score
Acute HE (GA) Chronic HE (GB)
Normal Control Group (G1) 0 0
Finasteride Control Group (G2) 0 0
TAA Group (G3) 3±0.25* 1.66±0.21#
Lactulose Treated Group (G4) 2.5±0.20* 1.5±0.22
Finasteride Treated Group (G5) 2.5±0.22* 1.33±0.21
Finasteride and Lactulose Treated Group (G6) 2±0.25*- # # 1.16±0.16
Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test.
* Significantly different from the normal group at p*<0.01.
# Significantly different from control (TAA) group at p#<0.01 and p## <0.05
Effect of Finasteride and Lactulose on Behavioural Clinical Score of Rats with Hepatic Encephalopathy Induced by Thioacetamide (TAA) As shown in table 2:
Type A, Hepatic Encephalopathy: Administration of TAA to rats in the control group (GA 3), at 300 mg/day, for three days, produced significant changes in rats'behaviours that represented as a mean score of (3 ± 0.25, P ˂ 0.001) compared to the control group (GA 1). Rats in the Finasteride control group (GA 2) exhibited an insignificant change in clinical behaviours for the control group (GA 1); both had behavioural scores of 0. Rats treated by lactulose (GA 4) or Finasteride (GA 5) produced slight and similar improvement in behavioural scores compare to TAA control group (GA 3), but with insignificant result (2.5 ± 0.25 vs 3 ± 0.25, P = 0.48 & 2.5 ± 0.22 vs 3 ± 0.25, P = 0.48, respectively). However, rats treated with Lactulose and Finasteride (GA 6) produced significant improvement in clinical behavioural changes in contrast to the TAA control group (GA 3), with a score of (1.16 ± 0.16 vs. 3 ± 0.25, p
˂ 0.05).
Type C, Hepatic Encephalopathy groups:Meanwhile, administration of TAA at 100 mg/kg/days (twice weekly) for 12 weeks produced a model of liver cirrhosis and hepatic Encephalopathy, which is characterized by abnormal behavioural changes that are observed clinically and represented with significant abnormal higher behaviour score relative to the negative control group (GB 1) (1.66 ± 0.21 vs. 0, P ˂ 0.001).Rats in Finasteride control groups (GB 2) revealed non-significant changes in clinical behaviours relative to the control group (GB 1) and produced no changes in the clinical score (0 ± 0) for both groups. Administration of Lactulose (GB 4) produced a little bit of improvement in rat's behaviour score compared to TAA controlled group (GB 3), but with an insignificant result (1.5 ± 0.22 vs.
1.66 ± 0.21, P = 0.89). Similarly, rats treated by Finasteride alone (GB 5) or with lactulose (GB 6), showed non- significant improvement in clinical behaviour score relative to TAA control group (GB 3) (1.33 ± 0.21 vs 1.66 ± 0.21, P = 69 & 1.16 ± 0.16 vs 1.66 ± 00.21, P = 0.30, respectively)
Table 3: Effect of Finasteride and Lactulose on Open Field Behaviors of Rats with Hepatic Encephalopathy Induced by Thioacetamide (TAA).
Open field behaviors Latency time
(in a sec)
Frequency of ambulation
Frequency of rearing
Frequency of grooming Type A Hepatic Encephalopathy
Normal Control Group (GA 1) 1.16 ± 0.21 120.16±3.6 38±1.98 5.16±0.47
Finasteride Control Group (GA 2) 1.33± 0.21 115.16±3.32 35.5±1.94 5.5±0.42
TAA Group (GA 3) 5 ± 0.36* 31.83±3.3* 11.5±0.76* 9.66±0.33*
Lactulose Treated Group (GA 4) 3.33± 0.33# 60.33±2.37# 23.83±1.07# 6.16±0.3#
Finasteride Treated Group (GA 5) 3.66± 0.33# 50.16±1.4# 21.66±1.17# 6.33±0.42#
Finasteride &Lactulose Treated Group (GA 6) 2.16± 0.3# 75.66±2.53# ◊ ∞ 30±0.96 # ◊ ∞∞ 5.16±0.3#
Type C Hepatic Encephalopathy
Normal Control Group (GB 1) 1.08 ± 0.2 110±3.45 27.83±1.4 6.16±0.47
Finasteride Control Group (GB 2) 1± 0.12 116.33±3.7 30.33±1.2 6.83±0.74
TAA Group (GB 3) 5.16 ± 0.47* 22.83±1.97* 9.66±0.55* 10.16±0.47*
Lactulose Treated Group (GB 4) 3.25± 0.17# 50.5±3.43# 19.5±0.42# 7.33±0.55#
Finasteride Treated Group (GB 5) 3.33± 0.24# 42.16±2.12# 17.5±0.76# 7.5±0.42#
Finasteride&Lactulose Treated Group(GBA6) 2.25± 0.17# 68.16±3.48#∞◊ 24.66±1.22 # ∞∞◊ 6±0.36#
Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test.
* Significantly different from the normal group at p*<0.01.and p**<0.05
# Significantly different from control (TAA) group at p#<0.01 and p## <0.05
∞ Significantly different from the Lactulose treated group (GA4) at p∞<0.01 and p∞∞ <0.05
◊ Significantly different from Finasteride treated group (GA 5) at p<0.01
Effect of Finasteride and Lactulose on Open Field Behavioural Test of Rats with Hepatic Encephalopathy Induced by Thioacetamide (TAA) as shown in table 3:Type A, Hepatic Encephalopathy Groups:Rats receiving TAA (GA 3) exhibited significant prolongation of latency time and increasing in grooming frequency in contrast to the control group (GA 1) (P ˂ 0.01). Meanwhile, the ambulation and rearing frequencies were significantly reduced relative to the control group (P ˂ 0.01).Rats in Finasteride control group (GA 2) exhibited insignificant change in latency time (P = 0.89), frequency of ambulation (P = 0.79), frequency of grooming (P = 0.89) and frequency of rearing (P = 0.77) in contrast to control group (GA 1).
In contrast to the TAA control group, lactulose administration significantly reduced the latency time, grooming frequency (P ˂ 0.01) and significantly increased the ambulation & rearing frequencies (P ˂ 0.01). Similarly, the administration of Finasteride produced a significant reduction in latency time and grooming frequency (P ˂ 0.01) and significantly improved the ambulation and rearing frequencies compared to the TAA control group (P ˂ 0.01). Rats that received Lactulose and Finasteride (GA 6) resulted in a significant reduction in the latency time and grooming frequency (P ˂ 0.01) and significantly increased ambulation and rearing frequencies (P ˂0.01) in respect to the TAA control group. However, rats treated with Finasteride revealed insignificant changes in latency time, ambulation frequency, rearing frequency and grooming frequency in comparison with rats treated with lactulose (GA 4) (P = 0.89, P = 0.15, P = 0.87, P = 0.89, respectively), rats receiving lactulose and Finasteride (GA 6), showed significant improvement in rearing and ambulation frequencies compared to lactulose group (P < 0.05) and Finasteride group (P
< 0.01).
Type C, Hepatic Encephalopathy groups:In contrast to the control group (GB 1), rats receiving TAA (GB 3) for 12 weeks induced behavioural changes in OFT that resulted in a significant prolongation of latency time and increased in frequency of grooming (P ˂ 0.01). It also significantly reduced rearing and ambulation frequencies (P ˂ 0.01, P ˂ 0.01), respectively.Rats in Finasteride control group (GB 2) exhibited insignificant change in latency time (P = 0.89), frequency of ambulation (P = 0.67), frequency of rearing (P = 0.49) and frequency of grooming (P = 0.89) in contrast to control group (GB 1). In contrast to the TAA control group, administration of Lactulose (8 mg/kg) significantly reduced the latency time and grooming frequency (P ˂ 0.01) respectively and increased the ambulation and rearing frequencies (P ˂ 0.01).
Furthermore, administration of Finasteride (50mg/kg) produced significant reduction of latency time and grooming frequency (P ˂ 0.05 & P ˂ 0.01) and significantly improvement the ambulation and rearing frequencies compared to TAA control group (P ˂ 0.01 & P ˂ 0.01 respectively).Administration of both lactulose and Finasteride (GB 6) resulted in significant reduction in the latency time and grooming frequencies (P ˂ 0.01), and significant increasing in the ambulation and rearing frequencies (P ˂ 0.01) in respect to TAA control group. However, rats treated with Finasteride (GB 5) revealed insignificant differences in latency time (P = 0.89), frequency of ambulation (P = 0.42), frequency of rearing (P = 0.69) and frequency of grooming (P = 0.89) relative to rats treated with lactulose (GB 4).
Rats received combination of lactulose and Finasteride (GB 6), exhibited significant improvement in rearing (P <
0.05 & P < 0.01) and ambulation frequencies (P < 0.01 & P <, 0.01) compared to lactulose group (GB 4) and Finasteride group (GB 5) respectively.
Table. 4: Effect of Finasteride and Lactulose on forced swimming behavior test in Rats with Hepatic Encephalopathy Induced by Thioacetamide (TAA).
Test (time in seconds) Groups
Immobility Swimming Climbing
Type A Hepatic Encephalopathy
Normal Control Group (GA 1) 168.33±4.04
56.1±1.34%
69.5±3.06 23.16±1.02%
63.83±4.04 21.27±1.34 Finasteride Control Group (GA 2) 162.5±4.02
54.16±1.34%
76.16±2.58 25.38±0.86%
61.33±1.76 20.44±0.58%
TAA Group (GA 3) 206.33±3.73*
68.77±1.24%
48.83±2.07*
16.27±0.69%
44.83±2.08*
14.94±0.69%
Lactulose Treated Group (GA 4) 177.5±2.78#
59.16±0.92%
63±1.73#
20.99±0.57%
59.5±1.08#
19.83±0.36%
Finasteride Treated Group (GA 5) 183±2.62#
60.99±0.87%
58.83±1.6##
19.6±0.53%
58.16±1.22#
19.38±0.4%
Finasteride&Lactulose Tested Group(GA6) 170.16±2.25#
56.72±0.75%
66.66±1.4#
22.21±0.46%
63.16±2.31#
21.05±0.77%
Type C Hepatic Encephalopathy
Normal Control Group (GB 1) 169.83±3.42
56.55±1.14%
65±1.36 21.66±0.45%
66.16±3.13 21.71±1.04 Finasteride Control Group (GB 2) 166.83±3.01
55.6±1.00%
70.5±1.99 23.66±0.62%
64.16±2.35 21.38±0.78%
TAA Group (GB 3) 207.33±4.31*
69.1±1.43%
47.16±2.28*
15.77±0.74%
45.33±2.29*
15.1±0.76%
Lactulose Treated Group (GB 4) 176.66±3.33 58.88±1.11%
62.5±1.43#
20.83±0.47%
60.83±1.95#
20.27±0.65%
Finasteride Treated Group (GB 5) 180.33±1.87#
60.10±0.62%
59.5±0.42#
19.83±0.14%
60.16±1.68#
20.05±0.56%
Finasteride&Lactulose Treated Group (GB6) 168±2.5#
55.99±0.83%
67.66±1.14# ∞ 22.55±0.38%
64.33±1.49#
21.44±0.49%
Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test.
* Significantly different from the normal group at p*<0.01.and p**<0.05 # Significantly different from control (TAA) group at p#<0.01 and p## <0.05
Effect of Finasteride and Lactulose on Forced Swimming Behavioural Test in Rats with Hepatic Encephalopathy Induced by Thioacetamide (TAA) as shown in table 4:
Type A, Hepatic Encephalopathy groups: Rats in Finasteride control group (GA 2) exhibited insignificant change in immobility (P = 0.78), swimming (P = 0.27) or climbing time (P = 0.89) in respect to control group (GA 1).In contrast to the control group, TAA administration increased the immobility time (P ˂ 0.001) and decreased the swimming and climbing time (P ˂ 0.001 & P ˂ 0.001, respectively). Administration of Lactulose (P ˂ 0.001) increased the swimming time and climbing time by 4.72% (P ˂ 0.001) and 4.89% (P ˂0.001) compared to TAA- treated rats.
Administration of Finasteride after each dose of TAA produced a significant reduction of immobility time by 7.78%
(P ˂ 0.001) and an increase in the swimming and climbing time by 3.33% (P ˂ 0.05) and 4.44% (P ˂ 0.001) respectively. Rats that received Lactulose and Finasteride (GA 6) showed a significant reduction in immobility and increased swimming and climbing time than the TAA control group (P ˂ 0.001). Rats treated with Finasteride (GA 5) showed non-significant differences in FST including immobility (P = 0.82), swimming (P = 0.72) and climbing time (P = 0.89) in contrast to rats treated with lactulose. Furthermore, rats treated with Finasteride and lactulose showed insignificant effect on immobility, swimming and climbing time in contrast to rats treated with lactulose (P = 0.61, P = 0.81, P = 0.85, respectively), and in contrast to rats treated with Finasteride (P = 0.09, P = 0.13, P = 0.62, respectively)
Type C, Hepatic Encephalopathy groups:Rats in the Finasteride control group (GB 2) showed an insignificant difference in immobility, swimming, and climbing behaviours relative to the normal group (GB 1). Concerning the control group, administration of TAA for 12 weeks significantly increased the immobility time (P ˂ 0.001) and reduced the swimming and climbing times (P ˂ 0.001).
In contrast to the disease group (GB 3), administration of Lactulose daily for the last two weeks to rats with liver cirrhosis and HE induced by TAA for 12 weeks, significantly stabilizing the depressed behavioural alterations through reduction of immobility time by 10.22% (P ˂ 0.001) and increasing swimming and climbing times by 5.06%
(P ˂ 0.001) and 5.17% (P ˂ 0.001) respectively. Rats treated with Finasteride daily, for the last two weeks of the study, had a significant reduction of immobility time by 9% (P ˂ 0.001), raising the swimming time by 4.06% (P ˂ 0.001) and increasing in the climbing time by 4.95% (P ˂ 0.001).
Rats treated with Finasteride revealed insignificant difference in immobility time (P = 0.89), swimming time (P = 0.72), and climbing time (P = 0.89) in contrast to rats treated with lactulose. Meanwhile, rats receiving both Lactulose and Finasteride (GA 6) resulted in a significant reduction in immobility time by 13.11% (P ˂ 0.001); an increase in swimming time by 6.78% (P ˂ 0.001) and an increase in climbing time by 6.34% (P ˂ 0.001) in contrast to TAA control group. Rats treated with Lactulose and Finasteride combined resulted in a significant increase in swimming time (P < 0.01) compared to the Finasteride group.
Result of EEG Assessment
EEG Changes in Type A hepatic Encephalopathy (GA) (Table 5 & Figure 1):In comparison with the control group (GA1), rats received TAA showed the EEG spectral changes that characterized by a significant reduction in absolute (P < 0.001) and relative (P < 0.01) PSD of the alpha band; significant reduction in absolute (P < 0.001) and relative PSD of theta band; and a significant reduction in absolute PSD of delta band ( P < 0.001), and a significant increase in relative PSD of delta band (P < 0.001). No significant changes in absolute and relative PSD of the beta band.
Rats in Finasteride control groups (GA 2) showed a significant increase in EEG absolute PSD of all bands with a significant reduction in relative PSD of the delta band (P < 0.001) relative to the control group.
The EEG changes in rats treated with Lactulose revealed a significant reduction in absolute PSD of delta band (P <
0.01) and insignificant higher absolute PSD of beta (P = 0.34), alpha (P = 0.89), and theta (P = 0.89) bands in contrast to TAA control group. However, the relative PSD of beta (P < 0.01), alpha (P < 0.05), and theta (P < 0.01) were significantly higher than the TAA control group, while the relative PSD of the delta band was significantly lower than that TAA control group (P < 0.001).
Table 5: Effects of Finasteride on absolute and relative mean power spectral densities (PSD) (µV2/Hz) of beta, alpha, theta, and delta waves in rats with type A hepatic encephalopathy induced by TAA and anesthetized by urethane.
Absolute Mean Power Spectral Densities (PSD) (µv2/Hz) Group
Wave
Normal Control Group (GA 1)
Finasteride Control Group (GA 2)
TAA Group (GA 3)
Lactulose Treated Group (GA 4)
Finasteride Treated Group (GA 5)
Finasteride and Lactulose Tested Group (GA 6)
Beta Wave 27.95±2.09 135.37±14.83* 13.01±1.57 34.77±2.05 158.95±9.52*#∞ 204.7±3.46*#∞◊
Alpha Wave 56.99±12.76 220.21±16.27* 11.65±0.53** 19.9±.66 154.95±5.14*#∞ 155.15±2.22*#∞
Theta Wave 92.57±11.61 359.85±26.51* 8.95±1.39* 24.47±1.42** 252.32±10.73*#∞ 285.2±3.75*#∞
Delta Wave 270.5±20.01 835.25±24.58* 136.5±11.31* 105.35±6.31* 672.7±32.1*#∞ 636.2±12.5*#∞
Relative Mean Power Spectral Densities (PSD) (µv2/Hz)
Beta Wave 6.31±0.53 8.65±0.61 7.58±0.32 18.9±1.07*# 12.84±0.46*#∞ 15.97±0.14*#∞∞◊◊
Alpha Wave 12.2±1.66 14.12±0.38 6.95±0.44*# 10.85±0.66## 12.6±0.66# 12.11±0.18#
Theta Wave 20.44±0.49 23.1±0.56* 5.24±0.48* 13.26±0.51*# 20.41±0.21#∞ 22.25±0.15#∞
Delta Wave 60.75±2.17 53.99±1.22* 80.39±0.44* 56.96±1.17# 54.39±0.28**# 49.64±0.29*#∞
Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test. (µV2/Hz = amplitude2/frequency),
* Significantly different from the normal group at p*<0.01.and p**<0.05
# Significantly different from control (TAA) group at p#<0.01 and p## <0.05
∞ Significantly different from the Lactulose Treated control group (GA 4) at p∞<0.01 and p∞∞ <0.05
◊ Significantly different from Finasteride treated group (GA 5) at p<0.01
Figure 1: The average power spectra transformed from recorded EEG on rats with acute hepatic Encephalopathy induced by TAA, using FFT analysis software. Showing power bands in different groups. 1; normal control, 2; Finasteride control
group (50 mg/kg), 3; thioacetamide control group (300mg/kg for 3 days, 4; lactulose (8mg/kg) treated group, 5; Finasteride treated group, and 6; Finasteride and lactulose treated group. TAA produced low voltage EEG with an increase in PSD of delta
band, which Finasteride partially reversed.
In contrast to the TAA control group, the EEG changes in rats treated with Finasteride showed a significant increase in absolute PSD of the beta band (P < 0.001), the alpha band (P < 0.001), and theta band (P < 0.001) and a significant reduction in PSD of delta band (P < 0.001) as well as it revealed significant rising in relative PSD of the beta band (P
< 0.001), the alpha band (P < 0.01) and theta band (P < 0.001); and a significant reduction in the delta band (P ˂ 0.05). Similarly, the EEG changes in rats treated with Finasteride were associated with a significant increase in absolute PSD of all bands and relative PSD of theta band (P < 0.01) compared to the Lactulose treated group. On the other hand, the rats treated with Lactulose showed a more significant rise in the beta band's relative PSD than those treated with Finasteride (P < 0.01). Moreover, rats received both Lactulose and Finasteride (GA 6) resulted in significant improvement in EEG changes in the form of an increase in absolute (P < 0.001) and relative (P < 0.05) PSD of the beta-band compared to the lactulose group (GA 4) and Finasteride groups (GA 5) respectively. It was also associated with a significant reduction in absolute and relative EEG PSD of delta band (P < 0.001) compared to lactulose treated group (GA 4) with no significant changes from Finasteride treated group.
Table 6: Effects of Finasteride on absolute and relative mean power spectral densities (PSD) (µV2/Hz) of beta, alpha, theta, and delta waves in rats with type C hepatic Encephalopathy induced by TAA and anesthetized by urethane.
Absolute Mean Power Spectral Densities (PSD) (µv2/Hz) Group
Wave
Normal Control Group (GB 1)
Finasteride Control Group (GB 2)
TAA Group (GB 3)
Lactulose Treated Group (GB 4)
Finasteride Treated Group (GB 5)
Finasteride
&Lactulose Treated Group (GB 6)
Beta Wave 56.22±5.6 118.95±3.87* 19.71±2.52* 52.89±4.1# 110.83±2.99*#∞ 176.7±4.2*#∞◊
Alpha Wave 68.27±5.52 196.42±5.59* 24.41±1.97* 30.98±2.36* 110.73±3.8*#∞ 104.16±4.19*#∞
Theta Wave 96.3±4.94 283.6±4.27* 20.4±2.1* 49.26±4.39*# 186.35±4.64*#∞ 183.74±2.9*#∞
Delta Wave 351.7±23.5 3
661.87±12.57* 183.21±8.95* 168.85±4.69* 493.54±1.73*#∞ 471.63±4.77*# ∞ Relative Mean Power Spectral Densities (PSD) (µv2/Hz)
Beta Wave 9.75±0.37 9.33±0.26 7.92±0.79 17.55±0.96*# 12.3±0.35#∞ 18.87±0.42*#◊
Alpha Wave 11.91±0.48 15.42±0.37* 9.82±0.48 10.25±0.72 12.27±0.33## 11.12±0.42 Theta Wave 16.88±0.43 22.26±0.2* 8.17±0.51* 16.26±1.14# 20.67±0.41*#∞ 19.62±0.23**#∞
Delta Wave 61.43±0.52 51.96±0.4* 74.05±0.96* 55.97±0.69*# 54.78±0.35*# 50.37±0.23*#∞◊
Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test. (µV2/Hz = amplitude2/frequency)
* Significantly different from the normal group at p*<0.01.and p**<0.05.
# Significantly different from control (TAA) group at p#<0.01 and p## <0.05.
∞ Significantly different from the Lactulose treated group (GA 4) at p∞<0.01 and p∞∞ <0.05.
◊ Significantly different from Finasteride treated group (GA 5) at p◊<0.01 and p◊◊ <0.05.
Figure 2: The average power spectra transformed from recorded EEG on rats with chronic hepatic Encephalopathy induced by TAA, using FFT analysis software. Showing low voltage power bands that corresponding to the model of Hepatic Encephalopathy (B), relative to the normal control group (A), Finasteride increased the spectral powers as shown in figure (C) .
Result of EEG Changes in Type C, hepatic Encephalopathy (GB) (table 6& Figure 2): The EEG spectral analysisinrats received TAA revealed a significant reduction in absolute PSD of the beta band (P = 0.89) without changes in relative beta band SPD; significant reduction in absolute (P < 0.001) without changes in relative PSD of the alpha band; significant reduction in absolute (P < 0.001) and relative (P < 0.001) PSD of theta band; and a significant reduction in absolute PSD of delta band (P < 0.001) with the significant rising of relative PSD band (P <
0.001) in contrast to the normal control group (GB 1).
Rats in Finasteride control groups (GB 2) showed a significant increase in EEG absolute PSD of all bands and a significant increase in relative PSD of the alpha band (P < 0.001) and theta band (P < 0.001) while associating with a significant reduction in the relative delta band (P < 0.001) relative to the normal control group. Administration of Lactulose significantly increased the absolute PSD of the beta band (P < 0.001) and theta band (P < 0.001) without significant changes in absolute PSD of alpha and delta bands. However, it is associated with a significant increase in relative PSD of the beta band (P < 0.001); theta band (P < 0.001), and reduced in the absolute delta band (P < 0.001) without changes in absolute PSD of the alpha band, in contrast to TAA control group (GB 3).
Moreover, the EEG changes in rats treated with Finasteride showed a significant increase in absolute PSD of the beta band (P < 0.001), the alpha band (P < 0.001), and theta band (P < 0.001) with a significant reduction in absolute PSD of delta band (P < 0.001). Additionally, it revealed significant rising in relative PSD of beta (P < 0.01), alpha (P <
0.05) and theta (P < 0.01) bands and significant reduction in delta band (P ˂ 0.001), in contrast to TAA control group. Furthermore, rats treated with Finasteride showed a significant increase in absolute PSD of all bands and relative PSD of theta band (P < 0.001) relative to the Lactulose treated group (GB 4). Meanwhile, rats treated with Lactulose showed a more significant rising in relative PSD of beta-band than rats treated with Finasteride (P < 0.01).
Rats treated with Lactulose and Finasteride (GB 6) showed a significant increase in absolute (P < 0.01) and relative (P < 0.01) PSD of the beta band and a significant reduction in the relative delta band (P< 0.01) in contrast to Finasteride treated group (GB 5). It is also significantly associated with higher absolute EEG PSD of all bands and a lower relative delta band (P < 0.01) compared to the Lactulose treated group (GB 4).
Result of Biochemical Assessment {AST ALT, Total Bilirubin, Albumin, and Blood Ammonia Level} (table 7):
Type A Hepatic Encephalopathy Groups:Rats received TAA for three consecutive days, produced a model of acute hepatic injury, characterized by significant elevation ALT (P ˂ 0.01), AST (P ˂ 0.01), and total bilirubin ( P ˂ 0.01), as well as it produced a significant reduction in serum albumin level (P ˂ 0.01), whereas rats in Finasteride control group (GA 2) showed an insignificant difference in ALT (P = 0.89 ), AST (P = 0.65), total bilirubin (P = 0.8), and serum albumin (P = 0.89) relative to control group (GA1).
Rats treated with lactulose showed insignificant reduction in ALT (P = 0.77), AST (P = 0.89), and insignificant increased in serum albumin level (P = 0.89) and showed significant reduction in total bilirubin (P < 0,01) in contrast to TAA control group. Similarly, rats treated with Finasteride revealed insignificant reduction in ALT (P = 0.77), AST (P = 0.89), and insignificant increased in serum albumin level (P = 0.89) and showed significant reduction in total bilirubin (P < 0,01) in contrast to TAA control group. Relative to TAA control group, rats treated by Finasteride and lactulose resulted in significant reduction in total bilirubin (P < 0.01) and showed insignificant reduction in ALT (P = 0.89), AST (P = 0.89) and insignificant increased in serum albumin (P = 0.89).
Blood ammonia level was significantly elevated in rats receiving TAA relative to the control group (P ˂ 0.01). Rats in Finasteride control groups showed no significant ammonia level from rats in the control group (P = 0.89).
Meanwhile, rats treated with Lactulose showed a significant reduction in serum ammonia level compared to the TAA control group (P ˂ 0.01) and resulted in a more significant reduction in serum ammonia level than the group treated by Finasteride (P ˂ 0.05). The rats treated with Finasteride resulted in a significant reduction in ammonia level (P <
0.01) compared to the TAA control group. Moreover, rats received both Lactulose and Finasteride (GA 6) resulted in a significant reduction in ammonia level in contrast to the TAA control group (P ˂ 0.001) and produced more reduction in ammonia level than rats treated with Finasteride (P < 0.05) or Lactulose (P = 0.37) alone.
Type C Hepatic Encephalopathy Groups:Administration of TAA twice weekly for 12 weeks associated with significant elevation in ALT (P ˂ 0.01), AST (P ˂ 0.01), and total bilirubin (P ˂ 0.01). Additionally, it produced significant reduction in serum albumin level (P ˂ 0.01), whereas rats in Finasteride control group (GB 2) showed insignificant differences in ALT (P = 0.89), AST (P = 0.89), total bilirubin (P = 0.89), and serum albumin (P = 0.89) relative to control group (GB 1).
Rats treated with lactulose showed significant reduction in total bilirubin (P < 0.05) and insignificant reduction in ALT (P = 0.08), AST (P = 0.69), and revealed insignificant increase in serum albumin level (P = 0.89) in contrast to TAA control group. Furthermore, rats treated with Finasteride revealed a significant reduction in total bilirubin (P <
0.01), an insignificant reduction in ALT, AST, and an insignificant increase in serum albumin level compared to the TAA control group.
Relative to the TAA control group, rats treated with Finasteride and Lactulose resulted in a significant reduction in total bilirubin (P < 0.01) and showed an insignificant reduction in ALT, AST, and an insignificantly increased in serum albumin. In contrast to the control group, blood ammonia levels were significantly elevated in rats receiving TAA (P ˂ 0.01). Meanwhile, rats treated with Lactulose 8 mg/kg/day showed a significant reduction in serum ammonia level than the TAA control group (P ˂ 0.01). Also, they produced a more significant reduction in serum ammonia level than the group treated by Finasteride (P ˂ 0.01).
Moreover, rats treated with Finasteride (GB 5) were associated with an insignificant reduction in serum ammonia level than the TAA control group (P = 0.66). Rats receiving both Lactulose and Finasteride (GB 6) resulted in a significant reduction in ammonia level in contrast to the TAA control group (P ˂ 0.01) and produced more reduction in ammonia level than rats treated by Finasteride (P ˂ 0.01) or Lactulose (P ˂ 0.01).
Table 7: Effect of Finasteride and Lactulose on Blood Ammonia and Liver Function Tests (ALT, AST, Bilirubin, and Albumin) in Rats with Type A and type C Hepatic Encephalopathy Induced by Thioacetamide (TAA).
Type A Hepatic Encephalopathy Control
Group (GA 1)
Finasteride Control
Group (GA 2)
TAA Group (GA 3)
Lactulose Treated Group
(GA 4)
Finasteride Treated
Group (GA 5)
Finasteride
&Lactulose Treated Group
(GA 6) ALT (U/L) 24.66±1.56 26.33±1.62 65.83±1.83* 57.38±1.77* 61.5±2.47* 55.63±2.44*
AST (U/L) 46.83±3.38 53.66±2.80 109.16±3.23* 103.33±3.51* 99.33±2.87* 100.83±3.59*
Bilirubin (mg/dl) 0.33±0.02 0.38±0.02 0.91±0.01* 0.64±0.04*# 0.75±0.02*# 0.64±0.02*#
Albumin (g/dl) 3.75±0.09 3.78±0.06 2.24±0.15* 2.28±0.11* 2.26±0.15* 2.29±0.12*
Ammonia (µmol/l) 68.16±2.98 73.5±2.06 187.83±3.90* 143.5±3.85#∞∞ 162.83±3.23# 133±5.53# ◊◊
Type C Hepatic Encephalopathy Control
Group (GB 1)
Finasteride Control Group (GB 2)
TAA Group (GB 3)
Lactulose Treated Group (GB 4)
Finasteride Treated
Group (GB 5)
Finasteride
&Lactulose Treated Group (GB 6) ALT (U/L) 22.5±1.25 24±1.82 76.5±2.45* 68.16±2.57* 72±2.44* 68.5±1.64*
AST (U/L) 51.33±4.63 53.83±2.61 117.5±5.92* 107.33±5.35* 115.66±6.50* 109.66±4.71*
Bilirubin (mg/dl) 0.30±0.01 0.33±0.02 0.97±0.05* 0.81±0.02*## 0.77±0.02*# 0.78±0.03*#
Albumin (g/dl) 3.95±0.07 3.9±0.05 2.24±0.15* 2.25±0.08* 2.26±0.25* 2.28±0.22*
Ammonia(µmol/L) 66.33±3.07 65.66±3.68 173.16±6.10* 128.33±4.12# 164.66±2.64 114.66±4.12# ∞ ◊
Statistical analysis was carried out by one-way ANOVA followed by Tukey-Kramer multiple comparison test.
* Significantly different from the normal group at p*<0.01.and p**<0.05
# Significantly different from control (TAA) group at p#<0.01 and p## <0.05
∞ Significantly different from the Lactulose treated group (GA 4) at p∞<0.01 and p∞∞ <0.05
◊ Significantly different from Finasteride treated group (GA 5) at p<0.01
Result of Histopathological Assessment (Figures 3-5):
Figure 3: Photomicrograph of a section from a rat brain tissue at the prefrontal cortex in normal control and Finasteride (50mg/kg/day) control groups showing normal brain tissues (H&E, 200X).
Type A, Hepatic Encephalopathy: microscopic examination of brain sections of the prefrontal cortex after staining with H&E, in acute TAA induced HE, showed astrocyte swelling, perineural edema, and vascular congestion.
Administered Lactulose or Finasteride associated with partial amelioration of astrocyte swelling and perineural edema, and the best result was obtained from rats treated with a combination of Lactulose and Finasteride.
Figure 4: Photomicrograph of a section from a rat brain tissue at the prefrontal cortex in groups of type A hepatic encephalopathy (H&E, 400X).
Type C, Hepatic Encephalopathy: Chronic administration of TAA produced a moderate increase in many astrocytes in the cortex with characteristic Alzheimer type II changes (enlarged astrocytes with pale, large nuclei, and prominent nucleoli). Rats treated with Finasteride or Lactulose are associated with increased cortical astrocyte and moderate amelioration of Alzheimer's type II changes. However, most brain histopathological changes associated with TAA were ameliorated by administering a combination of Lactulose and Finasteride.
Figure 5: Photomicrograph of a section from a rat brain tissue at the prefrontal cortex in type C hepatic Encephalopathy (H&E, 400X).
Discussions
The present study demonstrated that administration of TAA in a dose of (300 mg/kg/day) for three consecutive days or in a dose of (200 mg/kg/ twice weekly) for 12 weeks significantly induced neurobehavioral changes represented by a significant increase in the behavioural, clinical score; prolongation of latency period and grooming frequency and decreased in ambulation and rearing frequencies in OFT; and a significant increase in the immobility time and reduced in the swimming and climbing time in FST.
These results are in accordance with Farajm et al. (2012), who found that TAA induced neurobehavioral changes in rats with HE due to ALF, which presented by increasing in coma stage as measured by clinical behaviour score.
Moreover, Leke et al. (2012) found that rats with HE due to liver cirrhosis exhibited reduced locomotor and exploratory activities when examined by OFT.
In agreement with Abd-Elsalam et al. (2014), who found that HE induced by TAA led to depressive-like symptoms that manifested clinically by increasing in passive behaviour responses such as latency period and grooming frequency in OFT and prolonged in immobility time in FST and reduced active behaviour responses such as ambulation and rearing frequencies in OFT and swimming and climbing times in the FST.
In contrast, Mendez et al. (2008) did not find differences in locomotor behaviour in TAA-induced liver failure in rats. Furthermore, Kawai et al. (2012) demonstrated that chronic TAA-induced cirrhosis in rats is associated with aggressive and irritated mood behaviours manifested by decreased immobility and increased swimming time in FST.
Amodio et al. (2004) suggested that the psychomotor slowness in HE could be related to dysfunction in neuronal network circuits between basal ganglia, thalamus, and prefrontal cortex play a role in the modulation of motor activity. These could be related to various neurochemical changes in HE and resulted in a spectrum of locomotor and mood behavioural changes that extend from depressed mood and motor hypoactivity to motor hyperactivity and maniac behavioural changes. On the other hand, in an early stage of HE, including clinical behaviour scores 1 and 2, the examined animal activity needs close observation and complemented with further evaluation by electrophysiological studies, including EEG, to detect subclinical neurological changes.
Administration of Lactulose (8 mg/kg/day, p.o.), in both type A and type C HE, ameliorated the rats' neurobehavioral changes induced by TAA, which represented by improvement in the behavioural, clinical score; reduced the latency time and grooming frequency and increased the ambulation and rearing frequencies in OFT; and decreased in the immobility time and increased in the swimming and climbing times in the FST.
In harmony with Abd-Elsalam et al. (2014), Lactulose was associated with improved neurobehavioral changes in OFT and FST in rats with type A HE induced by TAA. Furthermore, Kawai et al. (2012) reported that Lactulose improved some neurobehavioral manifestations of HE, such as memory and mood disturbances, by modifying ammonia metabolism in the intestine.
This study revealed that administration of Finasteride (50mg/kg) to rats with type A and C HE resulted in improvement in neurobehavioral changes, manifested by a reduction in the behavioural, clinical score; reduction in latency time and grooming frequency, and improvement in ambulation and rearing frequencies in OFT; and reduction of immobility time and increased in the swimming and climbing time in FST. These data are according to Mladenovi'c et al. (2014), who reported that Finasteride improved motor dysfunctions and total behavioural scores and halted HE's progression to a more severe HE form (stage IV).
Cauli et al. (2009) suggested that alterations of cognitive behaviours and the sleep-wake cycle in rats with HE resulted fromincreased synthesis of positive NS modulators such as ALLO and THDOC and reduced synthesis of negative NS modulators such as DHEAS. Furthermore, Mukai et al. (2008) demonstrated that Finasteride depletes the neuroactive steroids and reduced stress on brain NS in rats.
Since NS interacts allosterically with GABAA receptors via specific NS recognition binding sites located within the transmembrane domains of the α- and β- subunits, distinct from that of the GABA, benzodiazepine, and barbiturate sites. Furthermore, act to increase the GABA-ergic effect by increasing the frequency and duration of chloride channel openings, and at higher concentrations, NS directly activate the GABAA receptor–channel complex in the absence of GABA and suppress the excitatory neurotransmission (Hosie et al., 2006; Akk et al., 2007). Thus, Finasteride's inhibition of brain 5-αR activity led to a reduction in NS synthesis and subsequently ameliorated its contributory effects on GABAergic tone and other various neuronal activity.
In the present study, Finasteride in combination with Lactulose was more effective in improving neurobehavioral changes of type A and type C HE induced by TAA than Finasteride or Lactulose given separately; this synergistic action could be related to the reduction in both brain NS and its GABAergic modulation, and blood and brain hyperammonia and its subsequent deleterious effect.
In the present study, electrophysiological changes were evaluated by analysis of EEG spectral power density. EEG revealed that acute administration of TAA induced EEG spectral changes characterized by a significant rise in relative delta band with a significant reduction in absolute and relative PSD of alpha and theta bands, with no significant absolute and relative PSD changes beta band. Similarly, chronic administration of TAA induced EEG spectral changes characterized by a significant reduction in absolute PSD of all spectral bands, with the significant rising of relative PSD of delta band and did not show significant changes in alpha and beta relative bands.
The above results agree with Mladenovic et al. (2016), who confirmed thatTAA produced progressive EEG spectral changes depending on the severity of HE. In mild HE, EEG was associated with increased absolute alpha and beta
bands, increased relative alpha band, and decreased relative beta band. EEG in moderate to severe HE, associated with an increase in PSD of delta band, which is usually accompanied with clinical observed neurobehavioral changes; and in HE with deep coma, EEG is usually associated with an increase in absolute and relative PSD of delta band with or without triphasic waves appearance which can progress to flat EEG due to neuronal hyperpolarization.
Increased in slow frequencies and decreased in fast frequencies of EEG signals in HE induced by TAA could be explained by diffuse post-synaptic cortical neurons hyperpolarization state, which can progress into flat EEG in the severe form of HE. TAA induced direct and indirect changes in cortical neurons and astrocytes through induction of hyperammonia. Hyperammonia mediated cortical neurons hyperpolarization via direct enhancement of GABA-A inward chloride channels opening; reduction uptake of GABA by astrocytes near the synaptic cleft, and increase expression of the peripheral type benzodiazepine receptor (PTBR) on astrocytes with subsequent activation of PTBR increased production of Neurosteroids which directly and indirectly stimulate GABA-A receptors (Gunn et al., 2011).
In the present study, Lactulose was associated with some degree of improvement in EEG changes, including an increase in absolute and relative PSD of beta, alpha, and theta bands and a decrease in PSD of delta band in type A HE. Similarly, Lactulose increased in absolute and relative PSD of beta and theta bands and reduced absolute PSD of delta band in type C HE.
This finding is confirmed by Singh et al. (2016), who demonstrated that cirrhotic patients with MHE and abnormal EEG power spectral analysis, including high theta relative power > 35%, were improved significantly after lactulose therapy.
In the present study, EEG of rats treated by Finasteride only showed a significant increase in absolute PSD of all bands, a significant increase in relative PSD of alpha and theta bands, and a reduced relative delta band. Meanwhile, administration of Finasteride to rats with type A and type C HE led to some degree of improvement in EEG changes, including increased absolute and relative PSD of beta, alpha, and theta bands and reduced in absolute and relative PSD of delta band.
These are in harmony with Mladenovic et al. (2016), who reported that rats with HE induced by TAA,associated with EEG changes of mild HE, characterized by an increase in alpha and beta bands and a decrease in the relative power of delta band whenpre-treated with Finasteride.
The present study showed that rats treated with a combination of Lactulose and Finasteride associated with a significant increase in absolute and relative PSD of the beta band and reduction in absolute and relative PSD of delta band in both type A and type C HE, in contrast to rats treated by Lactulose or Finasteride alone.
Regarding biochemical changesin the present study, both acute and chronic administration of TAA induced hepatic injury, as evidenced by elevation in serum levels of ALT, AST, and bilirubin and serum albumin level reduction.
These results agree with Li et al. (2017) and Ghobadi Pour et al. (2017).
TAA is a thionosulfur-containing compound. It metabolizes in the liver via cytochrome P450 dependent mixed- function oxidase (CYP2E1) and monooxygenase systems into various toxic metabolites, including acetamide and thioacetamide-S-oxide (TASO), sulfene, and thioacetamide-S-dioxide. These reactive compounds bind to hepatic cells such as cell membrane, mitochondria, and DNA, resulting in protein oxidation, peroxidation of lipid, increased cell membrane permeability, impairment of mitochondrial oxidative phosphorylation, and increased intracellular Ca+2 concentration, and excessive release of free oxygen radicals. These cellular changes lead to hepatocytes apoptosis and necrosis and excessive release of hepatic intracellular stored enzymes like AST and ALT into plasma, which are used as sensitive tests of hepatocyte injury (Chen et al., 2008; Li et al., 2015)
Furthermore, Ahmed et al. (2014) confirmed that TAA's excessive liver damage impairs hepatic synthetic, metabolic, and excretory functions, resulting in elevation of bilirubin, abnormal coagulopathy, reduced serum albumin level, and increase in ammonia level both in serum and brain.Besides, TAA administration induced hepatic and brain inflammation through an increase in tissue ROS level and overconsumption of antioxidants such as tissue glutathione (Ganesan et al., 2017)
In the present study, TAA administration is associated with marked elevation in serum ammonia level in both acute and chronic HE models, according to Mladenovic et al. (2012);Kawai et al. (2012).
Because of liver damage induced by TAA, systemic accumulation of ammonia is increased due to reduced functioning hepatocyte mass; decreased ammonia metabolism into urea and glutamine; and intrahepatic extrahepatic portal-systemic shunting in cirrhotic rats (Morgan, 2011; Tranah et al., 2015).
Excess brain ammonia upregulates peripheral-type benzodiazepine receptors (PTBRs) in astrocytic mitochondria, thereby enhancing the synthesis of NS as a potent ligand of neuronal GABA receptors (Cagnin et al., 2005)
In the present study, although Lactulose was associated with a marked reduction in ammonia level in both HE, A, and C, it was associated with a slight improvement in hepatic function tests manifested by a decrease in ALT, AST, and bilirubin and an increase in albumin level. These results agree with Ghobadi Pour et al. (2017), who confirmed that lactulose administration is associated with improved liver function profile, as manifested by a reduction in ALT, AST, and serum bilirubin and elevation in serum albumin.
Moreover, Alfawaz & Aljumah (2012) found that Lactulose reduced ammonia level, improved clinical features of HE, and prevented progression of HE into a more severe form. Lactulose reduces the intestinal productionand absorption of ammonia due to its cathartic effect and acidification of gut lumen; it also inhibits intestinal glutaminase activity and interferes with the uptake and conversion of glutamine into ammonia (Leise et al., 2014).
In addition to the prominent role of Lactulose in the reduction of ammonia level and its consequences on brain functions, it produced hepatic and brain-protective effects, as manifested by a reduction in liver enzymes, and improvement in hepatic and brain histological changes induced by TAA in rats, via activation of endogenous antioxidants and anti-inflammatory effects, through up-regulation of Nuclear factor-like 2 (Nrf2) and down- regulation of serum TNF-α and S100-β (Saeed et al., 2017)
This study showed that Finasteride treated groups associated with slight but insignificant amelioration of biochemical indices of liver damage, manifested by decreased ALT, AST, and bilirubin and increased albumin level in both types A and C, HE. These results are in accordance with Mladenovic et al. (2014), who found that Finasteride treated rats associated with less intensive liver tissue damage than the TAA group.
Furthermore, Mladenović et al. (2015) showed that the 5α-R inhibitory activity of Finasteride caused changes in antioxidative enzymes' activities in different brain regions, resulting in cortical alleviation lipid peroxidation, which occurred in acute HE induced by TAA. Moreover, Finasteride modulates brain oxidative stress via enhancement of catalase activity and inhibition of AchE, helping in ROS consumption.
Duborija-Kovacevic et al. (2008) have demonstrated that chronic Finasteride administration in rats with formalin- induced foot paw edema led to significant amelioration of paw edema due to the anti-inflammatory properties of Finasteride, which could be mediated by inhibition of prostaglandin synthesis.
Sauver et al. (2006) suggested that Finasteride ameliorated the BPH features by anti-inflammatory activity in addition to competitive inhibition of 5α-R. Thus, the hepatoprotective effects of Finasteride might be due to its potential antioxidative and anti-inflammatory properties.
Moreover, this study showed that Finasteride treated groups were associated with a significant reduction in ammonia level in both types A and C, HE. Hyperammonaemia, proinflammatory cytokines (IL-1β, TNF-α) in type A HE and manganese in type C HE, mediate up-regulation of TSPO on the astrocytic mitochondrial membrane by increasing the binding site densities of the peripheral like benzodiazepine ligand (Cagnin et al., 2005)
Activation of this protein facilitates the intramitochondrial flux of cholesterol. It thereby increases the availability of cholesterol to the cytochrome P450 side-chain cleavage enzyme, an enzyme located in the inner mitochondrial membrane that converts cholesterol into pregnenolone. It is a crucial intermediate for NS biosynthesis, including allopregnanolone, tetrahydro deoxycorticosterone (THDOC), and androstanediol (Badoni et al., 2013).
