Thioacetamide Rats Research Articles

Thioacetamide-Induced Acute Liver Injury Increases Metformin Plasma Exposure by Downregulating Renal OCT2 and MATE1 Expression and Function.

Metformin plasma exposure is increased in rats with thioacetamide (TAA)-induced liver failure. The absorption, distribution, and excretion process of metformin is mainly mediated by organic cation transporters (OCTs) and multidrug and toxin extrusion transporters (MATEs). To investigate the mechanisms of the increase in TAA-induced metformin plasma exposure, we employed intestinal perfusion and urinary excretion assays to evaluate the changes in the absorption and excretion of metformin and used Western blotting to investigate the metformin-related transport proteins' expression changes and mechanisms. The results showed that neither intestinal OCT2 expression nor metformin intestinal absorption were significantly altered by TAA-induced liver failure, while significantly decreased expression and function of renal OCT2 and MATE1 as well as impaired metformin excretion were observed in TAA rats. HK-2 cells were used as an in vitro model to explore the mechanism of liver-failure-mediated downregulation in renal OCT2 and MATE1. The results demonstrated that among numerous abnormal substances that changed in acute liver failure, elevated estrogen levels and tumor necrosis factor-α were the main factors mediating the downregulation of OCT2 and MATE1. In conclusion, this study highlights the downregulation of renal OCT2 and MATE1 in liver injury and its regulatory mechanism and reveals its roles in the increase in TAA-mediated metformin plasma exposure.

MiRNA-ome plasma analysis unveils changes in blood–brain barrier integrity associated with acute liver failure in rats

BackgroundHepatic encephalopathy (HE) symptoms associated with liver insufficiency are linked to the neurotoxic effects of ammonia and other toxic metabolites reaching the brain via the blood–brain barrier (BBB), further aggravated by the inflammatory response. Cumulative evidence documents that the non-coding single-stranded RNAs, micro RNAs (miRs) control the BBB functioning. However, miRs’ involvement in BBB breakdown in HE is still underexplored. Here, we hypothesized that in rats with acute liver failure (ALF) or rats subjected to hyperammonemia, altered circulating miRs affect BBB composing proteins.MethodsTransmission electron microscopy was employed to delineate structural alterations of the BBB in rats with ALF (thioacetamide (TAA) intraperitoneal (ip.) administration) or hyperammonemia (ammonium acetate (OA) ip. administration). The BBB permeability was determined with Evans blue dye and sodium fluorescein assay. Plasma MiRs were profiled by Next Generation Sequencing (NGS), followed by in silico analysis. Selected miRs, verified by qRT-PCR, were examined in cultured rat brain endothelial cells. Targeted protein alterations were elucidated with immunofluorescence, western blotting, and, after selected miR mimics transfection, through an in vitro resistance measurement.ResultsChanges in BBB structure and increased permeability were observed in the prefrontal cortex of TAA rats but not in the brains of OA rats. The NGS results revealed divergently changed miRNA-ome in the plasma of both rat models. The in silico analysis led to the selection of miR-122-5p and miR-183-5p with their target genes occludin and integrin β1, respectively, as potential contributors to BBB alterations. Both proteins were reduced in isolated brain vessels and cortical homogenates in TAA rats. We documented in cultured primary brain endothelial cells that ammonia alone and, in combination with TNFα increases the relative expression of NGS-selected miRs with a less pronounced effect of TNFα when added alone. The in vitro study also confirmed miR-122-5p-dependent decrease in occludin and miR-183-5p-related reduction in integrin β1 expression.ConclusionThis work identified, to our knowledge for the first time, potential functional links between alterations in miRs residing in brain endothelium and BBB dysfunction in ALF.

Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1β, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

Therapeutic efficacy of Ganoderma Lucidum on Thioacetamide-Induced Hepatic Fibrosis through Inhibition of TGF-β1 signaling in Male Rats.

The anti-fibrosis efficacy of Ganoderma lucidum (GL) was investigated on thioacetamide (TAA)-induced liver fibrosis. Experimental fibrosis was induced by (200 mg/kg TAA, i.p.) twice weekly for 6 weeks in male Sprague-Dawley rats. GL was administered to TAA rats, either as (250/500 mg/kg, i.p) daily for further 3 weeks. Repeated administration of TAA caused liver fibrosis evidenced by significant elevation of hepatic TGF-β1 accompanied by an increase in the hydroxyproline content, oxidative stress levels and liver biomarkers. Contrarily, GL treatment significantly reduced ALT, AST, total bilirubin, MDA, NO concentrations and restored SOD activity. H & E and Masson trichrome staining confirmed that GL suppressed liver fibrosis, inflammation and attenuated the histopathological alterations. GL also elicited a statistical decrease in TGF-β1 level and hydroxyproline content with a concomitant decline in NFk-B and α-SMA immunoexpression in the TAA treated rats. Furthermore, GL downregulated TNF-α and IL-1β levels in TAA-treated rats compared to the control group. Conclusion: GL possesses a pronounced protective activity against TAA-induced fibrosis in rats; It significantly inhibits oxidative stress, inflammation and diminishes fibrosis by: inhibiting NFk-B activation, reducing TGF-β mediated by PI3K-AKT pathway thus restoring the liver function, the effect was in a dose dependent manner.

S-Adenosylmethionine Deficiency and Brain Accumulation of S-Adenosylhomocysteine in Thioacetamide-Induced Acute Liver Failure.

Background: Acute liver failure (ALF) impairs cerebral function and induces hepatic encephalopathy (HE) due to the accumulation of neurotoxic and neuroactive substances in the brain. Cerebral oxidative stress (OS), under control of the glutathione-based defense system, contributes to the HE pathogenesis. Glutathione synthesis is regulated by cysteine synthesized from homocysteine via the transsulfuration pathway present in the brain. The transsulfuration-transmethylation interdependence is controlled by a methyl group donor, S-adenosylmethionine (AdoMet) conversion to S-adenosylhomocysteine (AdoHcy), whose removal by subsequent hydrolysis to homocysteine counteract AdoHcy accumulation-induced OS and excitotoxicity. Methods: Rats received three consecutive intraperitoneal injections of thioacetamide (TAA) at 24 h intervals. We measured AdoMet and AdoHcy concentrations by HPLC-FD, glutathione (GSH/GSSG) ratio (Quantification kit). Results: AdoMet/AdoHcy ratio was reduced in the brain but not in the liver. The total glutathione level and GSH/GSSG ratio, decreased in TAA rats, were restored by AdoMet treatment. Conclusion: Data indicate that disturbance of redox homeostasis caused by AdoHcy in the TAA rat brain may represent a deleterious mechanism of brain damage in HE. The correction of the GSH/GSSG ratio following AdoMet administration indicates its therapeutic value in maintaining cellular redox potential in the cerebral cortex of ALF rats.

P: 24 Comparative Morphological Analysis of Astroglia Reactivity in the Hippocampus of Rats With Acute and Chronic Hepatic Encephalopathy

BACKGROUND: Hepatic encephalopathy (HE) is a serious complication of advanced liver failure which represents the whole neuropsychiatric abnormalities resulting from liver disease ranging from abnormal behavior to coma. Impairment of cognitive function is well described in HE patients, while mechanisms of memory impairment in HE patients is still not fully understood, however, it may involve a possible gliopathy as well as neuropathy in various brain areas, including the hippocampus known as the main brain structure associated to the memorization process. The aim of the present investigation is to assess astroglia reaction of the hippocampus, in a comparative approach between acute and chronic HE in rat. METHODS: Sprague-Dawley rats were divided into 3 groups: controls; rats were treated with saline solution (NaCl 0.9 % i.p) during 3 days, chronic HE group: rats were subjected to Bile Duct Ligation and acute HE group; rats were subjected to 3 i.p injections of thioacetamide (TAA) 300 mg/kg BW. Astroglia was assessed through an immunohistochemical analysis using anti-GFAP antibody on frontal sections of the hippocampus following. RESULTS: Our data showed in the TAA rats (3 days following 3 TAA injections) compared to controls, a significant increase of GFAP immunoreactivity within the whole hippocampal areas, while astrocytic processes length and ramification were reduced, the GFAP-immunoreactive area was increased. In contrast to BDL rats, at the cirrhotic stage (4 weeks after surgery), showing the opposite tendency with a drop of the astrocytic GFAP-immunoreactive area as well as the astrocytic length and ramification levels. CONCLUSIONS: The present finding sustains a differential astroglial reactivity within the hippocampus of acute and chronic HE rats. Astrocytic morphology changes depends to the severity of liver failure and seems to HE type-dependent, while those astroglial changes lead to a severe gliopathy which may be behind the disturbed cognitive function, especially memory seen in patients with chronic as well as acute HE.

Cerebrovascular reactivity and cerebral perfusion of rats with acute liver failure: role of L-glutamine and asymmetric dimethylarginine in L-arginine-induced response.

Cerebral blood flow (CBF) is impaired in acute liver failure (ALF), however, the complexity of the underlying mechanisms has often led to inconclusive interpretations. Regulation of CBF depends at least partially on variations in the local brain L-arginine concentration and/or its metabolic rate. In ALF, other factors, like an increased concentration of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor and elevated level of L-glutamine, may contribute to CBF alteration. This study demonstrated strong differences in the reactivity of the middle cerebral arteries and their response to extravascular L-arginine application between vessels isolated from rats with thioacetamide (TAA)-induced ALF and control animals. Our results also showed the decrease in the cerebral perfusion in TAA rats measured by arterial spin labeling perfusion magnetic resonance. Subsequently, we aimed to investigate the importance of balance between the concentration of ADMA and L-arginine in the CBF regulation. In vivo, intraperitoneal L-arginine administration in TAA rats corrected: (i) decrease in cerebral perfusion, (ii) decrease in brain extracellular L-arginine/ADMA ratio and (iii) increase in brain L-glutamine concentration. Our study implicates that impaired vascular tone of cerebral arteries is most likely associated with exposure to high ADMA and L-glutamine levels resulting in limited availability of L-arginine and might be responsible for reduced cerebral perfusion observed in ALF.

Hepatic effect of sofosbuvir and daclatasvir in thioacetamide-induced liver injury in rats.

IntroductionThe study aimed at investigating the hepatic effect of direct acting anti-hepatitis C virus drugs (DAAs), sofosbuvir (Sof) and daclatasvir (Dac), in thioacetamide (TAA)-induced liver injury in rats.Material and methodsAnimals were allocated to 7 groups: a normal control group, a TAA group (receiving TAA 50 mg/kg, i.p. twice weekly), two TAA groups receiving either a low or a high dose of Sof (Sof-L and Sof-H; 41.1 mg/kg and 82.2 mg/kg, respectively), two TAA groups receiving either a low or a high dose of Dac (Dac-L and Dac-H; 6.2 mg/kg and 12.4 mg/kg, respectively), and a TAA group receiving both Sof-L and Dac-L.ResultsAfter 6 weeks, TAA significantly elevated the serum activities of liver enzymes, along with histopathological evidence of liver injury. These findings were associated with a significant increase in a fibrotic marker (tissue inhibitor metalloproteinase-1 – TIMP-1), proinflammatory cytokine (tumor necrosis factor alpha – TNF-α), and oxidative stress parameters (malondialdehyde [MDA] content, and superoxide dismutase [SOD] and catalase activities) in hepatic tissue. TAA rats treated with Sof-L, Dac-L, Dac-H and a combination of Sof-L plus Dac-L showed significant amelioration of TAA-induced liver injury. Their effects were accompanied by a significant reduction in TIMP-1, TNF-α and oxidative stress parameters in hepatic tissue. Interestingly, Sof-H caused no improvement in TAA-induced hepatic injury.ConclusionsThe hepatic effects of Sof and Dac in TAA-induced liver injury appeared to be mediated by anti-oxidant effects, and inhibition of TNF-α and TIMP-1.

L-Arginine Improves the Diminished Cerebral Blood Flow in Rats With Acute Liver Failure: Uncertain Role of Asymmetric Dimethylarginine

Background and Aims: Dysregulated cerebral blood flow (CBF) contributes to neurological dysfunctions accompanying hepatic encephalopathy (HE). The well-documented increase in asymmetric dimethylarginine (ADMA) in CSF of patients and brains of animals with liver failure may impair CBF by inhibition of endothelial nitric oxide synthesis (eNOS). Therefore, the correction of l-arginine (l-arg)/ADMA ratio and thereby elevation of the availability of l-arg for NO production by l-arg supplementation may improve CBF. The aim of this study was to assess whether: (i) HE in the thioacetamide (TAA) model affects CBF in the prefrontal cortex (PFC) and cerebellum (CRBL), (ii) exogenous l-arg is capable of modulating CBF and (iii) CBF changes induced by l-arg and/or TAA are correlated with extracellular cortical ADMA, l-arg, cGMP, nitrite and citrulline levels. Methods: Male Sprague Dawley rats were subjected to three intraperitoneal (i.p.) injections of TAA (300 mg/kg) at 24 h intervals. Arterial spin labeling MRI measurement was performed 24 h after the last TAA injection using a 9.4 T scanner. High resolution anatomic images were acquired with spin echo RARE sequence. For T1 relaxation time and perfusion measurement FAIR-RARE sequence was used. l-arg (200 mg/kg) was administered once by intraperitoneal cannula. The regional changes in CBF were observed before, and 30 min after l-arg injection, at first in the PFC, then in the CRBL. In the second group of anesthetized rats, bilateral microdialysis of the PFC was carried out 24 h after the last TAA dose. Six fractions were collected every 40 min. l-arg (200 mg/kg) was injected i.p. after the beginning of the 3rd fraction. Extracellular levels of l-arg, ADMA, cGMP, nitrite, citrulline were measured using positive mode electrospray LC–DMS–MS/MS, cGMP enzyme immunoassay, NO Analyzer and HPLC-FD, respectively. A repeated-measures ANOVA was applied. Results and Discussion: TAA rats exhibited a decreased basal CBF both in the PFC and CRBL (70% and 65% of the control value, respectively). l-arg increased cortical CBF both in the control (by 20%) and TAA (by 15%) rats, and cerebellar CBF in TAA rats only (by 15%). Administration of l-arg evoked durable increase of the extracellular l-arg level in the PFC of TAA rats (by 85%), while in the control group the effect was much less pronounced (by 25%) and shorter-lasting. The diminished l-arg/ADMA ratio in the TAA brain was increased after l-arg administration, whereas no effect on ADMA was observed. The increase of CBF in TAA rats was not accompanied by changes in extracellular levels of nitrite, cGMP and citrulline. It can be assumed that beneficial effect of l-arg on CBF is, as a rule, associated with eNOS activity, whereas metabolic changes noted in the extracellular fluid reflect a combination of the variable effects of acute liver failure on each of the three NOSs isoforms (Figure 1). Disclosures: This study was supported by Grant N 2013/09/B/NZ4/00536 from the National Science Centre. The equipment used for dimethylarginine determination was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by Eu. The authors have none to declare.

Treatment of HE Through Selective Alpha 2A Adrenergic Receptor Antagonism Improves Higher Brain Function in Cirrhosis

Background and aims: Hepatic encephalopathy (HE) has been shown to affect daily functioning, quality of life, driving and overall mortality. However, HE is a multifactorial disease not clinically easy to assess. Many mechanisms have been proposed, one of which is altered adrenergic signaling. One animal model available for studying HE resulting from cirrhosis is chronic administration of the hepatotoxin thioacetamide (TAA) which mimics the clinical characteristics of cirrhosis including portal hypertension and HE. The aim of this study was to assess the effects of the alpha 2a adrenergic receptor (Adra2a) antagonist, BRL44408, on memory processes already altered in the TAA-induced model of cirrhosis. Methods: Rats were divided into groups: Control rats without TAA administration (CN, n = 10) and rats administered TAA intraperitoneally (IP) for 11 weeks (TAA, n = 10). On the 7th week we tested the animals (n = 10) in a reference memory task using the Barnes maze. On the 11th week, TAA animals were divided into two groups: rats treated IP with 10 mg/kg BRL44408 (BRL, n = 4) and rats treated with IP saline (SAL, n = 6). Treatments were given acutely -twice in 24 hrs with the second dose given 1hr before rats were challenged in the same task. Latencies (time to reach the hole within the Barnes maze) time spent in quadrants and locomotor activity were all studied. Results: TAA animals were not able to reach the behavioural criterion, in contrast to the CN group. No changes in locomotor activity were found between the groups. However, TAA animals treated with BRL44408 improved their latencies compared to TAA plus SAL groups. The underlying liver disease was not overtly affected by such short treatment dose as shown by a lack of significant change in liver biochemistry or haemodynamics. Conclusions: Our data suggests that selective Adra2a adrenergic receptor antagonism may be beneficial as an additional therapy for improving brain function in hepatic encephalopathy patients. Disclosures: Rajiv Jalan is a consultant to Ocera Therapeutics Inc.; has received research grants from Sequana Medical AG, Ocera Therapeutics Inc., and Yaqrit, Ltd.; has accepted speaker fees from Norgine; is involved with inventions for Ornithine Phenylacetate licensed by University College London (UCL) to Ocera Therapeutics; UCL spinout Yaqrit, Ltd., which will in-license 5 of the following inventions for which Rajiv Jalan is the main inventor (YAQ001; DIALIVE; DASIMAR: biomarker for liver failure; Neutrophil function test; TLR4 as a target for liver failure). The authors have none to declare.

Automated evaluation of liver fibrosis in thioacetamide, carbon tetrachloride, and bile duct ligation rodent models using second-harmonic generation/two-photon excited fluorescence microscopy

Animal models provide a useful platform for developing and testing new drugs to treat liver fibrosis. Accordingly, we developed a novel automated system to evaluate liver fibrosis in rodent models. This system uses second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy to assess a total of four mouse and rat models, using chemical treatment with either thioacetamide (TAA) or carbon tetrachloride (CCl4), and a surgical method, bile duct ligation (BDL). The results obtained by the new technique were compared with that using Ishak fibrosis scores and two currently used quantitative methods for determining liver fibrosis: the collagen proportionate area (CPA) and measurement of hydroxyproline (HYP) content. We show that 11 shared morphological parameters faithfully recapitulate Ishak fibrosis scores in the models, with high area under the receiver operating characteristic (ROC) curve (AUC) performance. The AUC values of 11 shared parameters were greater than that of the CPA (TAA: 0.758–0.922 vs 0.752–0.908; BDL: 0.874–0.989 vs 0.678–0.966) in the TAA mice and BDL rat models and similar to that of the CPA in the TAA rat and CCl4 mouse models. Similarly, based on the trends in these parameters at different time points, 9, 10, 7, and 2 model-specific parameters were selected for the TAA rats, TAA mice, CCl4 mice, and BDL rats, respectively. These parameters identified differences among the time points in the four models, with high AUC accuracy, and the corresponding AUC values of these parameters were greater compared with those of the CPA in the TAA rat and mouse models (rats: 0.769–0.894 vs 0.64–0.799; mice: 0.87–0.93 vs 0.739–0.836) and similar to those of the CPA in the CCl4 mouse and BDL rat models. Similarly, the AUC values of 11 shared parameters and model-specific parameters were greater than those of HYP in the TAA rats, TAA mice, and CCl4 mouse models and were similar to those of HYP in the BDL rat models. The automated evaluation system, combined with 11 shared parameters and model-specific parameters, could specifically, accurately, and quantitatively stage liver fibrosis in animal models.

Intracerebral Administration of S-Adenosylhomocysteine or S-Adenosylmethionine Attenuates the Increases in the Cortical Extracellular Levels of Dimethylarginines Without Affecting cGMP Level in Rats with Acute Liver Failure

Alterations in brain nitric oxide (NO)/cGMP synthesis contribute to the pathogenesis of hepatic encephalopathy (HE). An increased asymmetrically dimethylated derivative of l-arginine (ADMA), an endogenous inhibitor of NO synthases, was observed in plasma of HE patients and animal models. It is not clear whether changes in brain ADMA reflect its increased local synthesis therefore affecting NO/cGMP pathway, or are a consequence of its increased peripheral blood content. We measured extracellular concentration of ADMA and symmetrically dimethylated isoform (SDMA) in the prefrontal cortex of control and thioacetamide (TAA)-induced HE rats. A contribution of locally synthesized dimethylarginines (DMAs) in their extracellular level in the brain was studied after direct infusion of the inhibitor of DMAs synthesizing enzymes (PRMTs), S-adenosylhomocysteine (AdoHcy, 2 mM), or the methyl donor, S-adenosylmethionine (AdoMet, 2 mM), via a microdialysis probe. Next, we analyzed whether locally synthesized ADMA attains physiological significance by determination of extracellular cGMP. The expression of PRMT-1 was also examined. Concentration of ADMA and SDMA, detected by positive mode electrospray LC–DMS–MS/MS, was greatly enhanced in TAA rats and was decreased (by 30 %) after AdoHcy and AdoMet infusion. TAA-induced increase (by 40 %) in cGMP was unaffected after AdoHcy administration. The expression of PRMT-1 in TAA rat brain was unaltered. The results suggest that (i) the TAA-induced increase in extracellular DMAs may result from their effective synthesis in the brain, and (ii) the excess of extracellular ADMA does not translate into changes in the extracellular cGMP concentration and implicate a minor role in brain NO/cGMP pathway control.

Decreased excitability and voltage-gated sodium currents in aortic baroreceptor neurons contribute to the impairment of arterial baroreflex in cirrhotic rats.

Cardiovascular autonomic dysfunction, which is manifested by an impairment of the arterial baroreflex, is prevalent irrespective of etiology and contributes to the increased morbidity and mortality in cirrhotic patients. However, the cellular mechanisms that underlie the cirrhosis-impaired arterial baroreflex remain unknown. In the present study, we examined whether the cirrhosis-impaired arterial baroreflex is attributable to the dysfunction of aortic baroreceptor (AB) neurons. Biliary and nonbiliary cirrhotic rats were generated via common bile duct ligation (CBDL) and intraperitoneal injections of thioacetamide (TAA), respectively. Histological and molecular biological examinations confirmed the development of fibrosis in the livers of both cirrhotic rat models. The heart rate changes during phenylephrine-induced baroreceptor activation indicated that baroreflex sensitivity was blunted in the CBDL and TAA rats. Under the current-clamp mode of the patch-clamp technique, cell excitability was recorded in DiI-labeled AB neurons. The number of action potential discharges in the A- and C-type AB neurons was significantly decreased because of the increased rheobase and threshold potential in the CBDL and TAA rats compared with sham-operated rats. Real-time PCR and Western blotting indicated that the NaV1.7, NaV1.8, and NaV1.9 transcripts and proteins were significantly downregulated in the nodose ganglion neurons from the CBDL and TAA rats compared with the sham-operated rats. Consistent with these molecular data, the tetrodotoxin-sensitive NaV currents and the tetrodotoxin-resistant NaV currents were significantly decreased in A- and C-type AB neurons, respectively, from the CBDL and TAA rats compared with the sham-operated rats. Taken together, these findings implicate a key cellular mechanism in the cirrhosis-impaired arterial baroreflex.

Carnosine Reduces Oxidative Stress and Reverses Attenuation of Righting and Postural Reflexes in Rats with Thioacetamide-Induced Liver Failure.

Cerebral oxidative stress (OS) contributes to the pathogenesis of hepatic encephalopathy (HE). Existing evidence suggests that systemic administration of l-histidine (His) attenuates OS in brain of HE animal models, but the underlying mechanism is complex and not sufficiently understood. Here we tested the hypothesis that dipeptide carnosine (β-alanyl-l-histidine, Car) may be neuroprotective in thioacetamide (TAA)-induced liver failure in rats and that, being His metabolite, may mediate the well documented anti-OS activity of His. Amino acids [His or Car (100 mg/kg)] were administrated 2 h before TAA (i.p., 300 mg/kg 3× in 24 h intervals) injection into Sprague–Dawley rats. The animals were thus tested for: (i) brain prefrontal cortex and blood contents of Car and His, (ii) amount of reactive oxygen species (ROS), total antioxidant capacity (TAC), GSSG/GSH ratio and thioredoxin reductase (TRx) activity, and (iii) behavioral changes (several models were used, i.e. tests for reflexes, open field, grip test, Rotarod). Brain level of Car was reduced in TAA rats, and His administration significantly elevated Car levels in control and TAA rats. Car partly attenuated TAA-induced ROS production and reduced GSH/GSSG ratio, whereas the increase of TRx activity in TAA brain was not significantly modulated by Car. Further, Car improved TAA-affected behavioral functions in rats, as was shown by the tests of righting and postural reflexes. Collectively, the results support the hypothesis that (i) Car may be added to the list of neuroprotective compounds of therapeutic potential on HE and that (ii) Car mediates at least a portion of the OS-attenuating activity of His in the setting of TAA-induced liver failure.

Brain extracellular levels of dimethylarginines (ADMA, SDMA) and cGMP and their modulation by exogenous S-adenosylmethionine in rats with acute liver failure

Acute liver failure (ALF) instantly evokes symptoms of hepatic encephalopathy (HE), mainly attributed to hyperammonemia. Ammonia neurotoxicity is related to disturbances in the nitric oxide (NO)/cGMP pathway. The methylated derivative of L-arginine (MDALs) - asymmetric dimethylarginine (ADMA) but not symmetrically methylated arginine (SDMA) is an endogenous inhibitor of nitric oxide synthase. Elevated blood and brain ADMA was reported in HE patients and experimental animals. The question arose whether ALF evokes changes in the incorporation of the methyl donor S-adenosylmethionine (SAM) to MDALs, and how they affect cGMP accumulation. To this end, we investigated the effect of intracortical perfusion of SAM on the brain extracellular levels of ADMA, SDMA and cGMP in rats with ALF in the thioacetamide (TAA) model. Sprague Dawley rats (250–270 g) received three i.p. injections of TAA (300 mg/kg) at 24 h intervals. Bilateral microdialysis of the prefrontal cortex was carried out 24 h after the last TAA administration. SAM at 2 mM concentration in artificial cerebrospinal fluid was infused for 40 min and then the medium was changed back. The extracellular levels of ADMA and SDMA were analyzed using positive mode electrospray LC–DMS–MS/MS and cGMP was determined with cGMP enzyme immunoassay. ALF resulted in the increased extracellular levels of ADMA (by ~800%) and SDMA (by ~250%). Moreover, in ALF rats infusion of SAM decreased the ADMA and SDMA (~30%) as compared to the basal value. It seems reasonable that an excessive amount of substrate inhibited the enzymes synthesizing MDALs. On the other hand, the cGMP level did not differ between control and TAA rats. SAM increased by ~90% cGMP only in the control group, what indicates affected NO/cGMP pathway in TAA model. The study demonstrates, that ALF modulates methylation of arginine to MDALs in a manner affecting cGMP accumulation.

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Citrulline uptake in rat cerebral cortex slices: Modulation by Thioacetamide -Induced hepatic failure

L-citrulline (Cit) is a co-product of NO synthesis and a direct L-arginine (Arg) precursor for de novo NO synthesis. Acute liver failure (ALF) is associated with increased nitric oxide (NO) and cyclic GMP (cGMP) synthesis in the brain, indirectly implicating a role for active transport of Cit. In the present study we characterized [3H]Cit uptake to the cortical brain slices obtained from control rats and rats with thioacetamide (TAA)-induced ALF (“TAA slices”). In both control and TAA slices the uptake was partially Na+-dependent and markedly inhibited by substrates of systems L and N, including L-glutamine (Gln), which accumulates in excess in brain during ALF. Cit uptake was not affected by Arg, the y+/y+L transport system substrate, nor by amino acids taken up by systems A, xc −or XAG. The Vmax of the uptake in TAA slices was ~60 % higher than in control slices. Chromatographic (HPLC) analysis revealed a ~30 % increase of Cit concentration in the cerebral cortical homogenates of TAA rats. The activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), the two enzymes of Cit-NO cycle catalyzing synthesis of Arg, showed an increase in TAA rats, consistent with increased ASS and ASL protein expression, by ~30 and ~20 %, respectively. The increased Cit-NO cycle activity was paralleled by increased expression of mRNA coding for inducible nitric oxide synthase (iNOS). Taken together, the results suggest a role for Cit in the activation of cerebral NO synthesis during ALF.

Regression of Fibrosis and Reversal of Cirrhosis in Rats by Galectin Inhibitors in Thioacetamide-Induced Liver Disease

Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA) and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan) or GM-CT-01 (galactomannan). In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip) or GM-CT-01 (180 mg/kg ip) given once weekly during weeks 8–11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis.

Behavioral and biochemical characterization of rats treated chronically with thioacetamide: proposal of an animal model for hepatic encephalopathy associated with cirrhosis

Hepatic encephalopathy (HE) is a syndrome observed in patients with liver dysfunction such as hepatitis and cirrhosis, and is characterized by cognitive impairment, personality changes, and a depressed level of consciousness. The detailed mechanism underlying the pathogenesis of HE remains unclear. In the present study, our aim was to establish an animal model for HE with cirrhosis. Therefore, we carried out behavioral and biochemical analysis of cirrhotic rats after treatment with thioacetamide (TAA) for 20 weeks. The rats subjected to chronic TAA treatment (TAA rats) showed reduction of cognitive scores in the novel object recognition test (NOR), and a decrease in immobility and an increase in swimming in the forced swim test (FST). In biochemical analyses, the TAA rats exhibited elevated blood levels of ammonia, and increased metabolic activities of serotonergic and noradrenergic neurons in the brain, while the levels of Glu and GABA were not affected. Post-oral treatment of lactulose, a clinically utilized drug for HE, effectively reduced the elevated blood ammonia levels, and restored the reduced cognitive scores and the decreased immobility, without any effects on neurotransmitter contents in the brain, compared with the control. These results indicated lactulose-restorable memory disturbance and irritated mood in the TAA rats. In other words, rats treated chronically with TAA are a potential model for cirrhosis-HE, and the combination of NOR and FST in TAA rats may be useful as a simple assay system for the screening and development of anti-HE agents.

Pravastatin for thioacetamide-induced hepatic failure and encephalopathy

Nitric oxide (NO) inhibition aggravates hepatic damage and encephalopathy and increases mortality in rats with thioacetamide (TAA)-induced acute liver failure. Statins enhance NO production but whether they influence the above parameters are unknown. Male Sprague-Dawley rats were used. In the first series, TAA (350 mg/kg per day, i.p. for 3 days) was administered to induce acute liver failure. Control rats received saline. Rats received distilled water or pravastatin (20 mg/kg per day, p.o.) from 2 days before to 3 days after TAA or saline injection. In the second series, liver cirrhosis was induced by common bile duct ligation (BDL). Sham-operated rats served as controls. Rats received distilled water or pravastatin for 5 or 14 days until the 42nd day after operation. On the last day of treatment, survival, motor activities, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, ammonia and brain histology were evaluated. Thioacetamide and BDL rats showed higher ALT, AST, bilirubin and ammonia levels and lower motor activity counts compared with their corresponding control groups. In TAA rats, pravastatin elicited higher total and ambulatory motor activity counts and lower AST and total bilirubin levels. Survival was improved, whereas brain H&E staining was not significantly different in TAA rats with or without pravastatin treatment. In BDL groups, rats with or without pravastatin treatment were not different in motor activity counts and liver biochemistry. Pravastatin ameliorates hepatic encephalopathy and liver biochemistry and improves survival in rats with acute liver failure, but not in those with cirrhosis.