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INTRODUCTION Cirrhosis is the most advanced stage of fibrosis and is associated with the emergence of fibrotic septa and nodules, changes in hepatic blood flow and the risk of liver failure. The hepatocyte dysfunction that results from hepatic disorder causes portal hypertension, which leads to the characteristic clinical manifestations of cirrhosis that affect the liver as well as other organ systems,,.

One of the inherent complications of cirrhosis is protein-calorie malnutrition (PCM). PCM is the loss of lean and fat mass in conjunction with metabolic alterations that compromise vital functions. The predominance of PCM in cirrhotic patients, regardless of aetiology, ranges from 20% to 60% in patients with compensated cirrhosis and can reach 100% in candidates for orthotopic liver transplantation,.

Thus far, there are no efficient, reliable or reproducible methods to assess the degree of liver involvement and to correlate it with the clinical and nutritional condition of a patient. The desired method should include parameters that identify metabolic changes and simultaneously evaluate the common characteristics of this disease, such as oedema and ascites, that cause body asymmetry. Bioelectrical impedance analysis (BIA) is a tool that utilises the phase angle (PA) to evaluate the progress of liver cirrhosis in a practical way that meets the clinical requirements by reporting on body and cell structure, even in cases of body asymmetry. The PA enables the determination of cellular structure and function, thus indirectly improving the efficacy of nutritional interventions,,,. Cirrhotic patients may present with several nutritional deficiencies, including zinc deficiency; zinc participates in homeostasis and the formation of protein structures. Because of its anti-inflammatory, anti-apoptotic and anti-oxidant properties, zinc inhibits cirrhosis-related fibrosis by controlling the function of hepatic stellate cells.

Zinc deficiency, which is commonly observed in cirrhotic patients, may promote and/or potentiate malnutrition, hypogeusia, hypoalbuminemia, a portosystemic shunt and an imbalance in hepatic zinc extraction. The human tongue is covered with gustatory papillae that have specific functions (taste receptors and signalling). The papillae allows to differentiate the sweet, bitter, sour, salty and umami flavours. Papillae can be circumvallate, fungiform, filiform or foliate, and they are all distributed on the back of the tongue. In humans, changes in taste may exacerbate PCM related to cirrhosis.

It is possible to detect changes in tongue structure that may indicate the pathophysiological process of hypogeusia. There are four types of cells (I, II, III and IV) in the gustatory papillae.

These cells, especially types II and III, are responsible for the transduction of the taste signal to the brain,. To differentiate the types of taste (bitter, sweet and umami), receptors are also present in the gustatory papillae (except for filiform papillae [FiP]) that enable the identification of changes in taste perception. The T1R family of hetero-oligomeric taste receptors comprises three specific G protein-coupled receptors, T1R1, T1R2 and T1R3, that have distinct expression patterns.

By studying these receptors, potential alterations in taste perception can be identified. To date, there have been no morphological and/or histological analyses of the tongues of cirrhotic patients, despite hypogeusia being a common complaint of these patients. Carbon tetrachloride (CCl 4) is a hepatotoxic drug that is widely used in experimental studies of hepatic cirrhosis because its effects mimic human cirrhosis,. In this study, we hypothesised that morphological and/or histological alterations occurred in the gustatory papillae of cirrhotic patients. However, performing tongue biopsies of cirrhotic and control patients was not feasible; therefore, we analysed the tongues ofcirrhotic rats, which exhibited morphological and histological structures similar to the human tongue. In this study, we aimed to correlate the changes related to PCM and cellular deficiencies with the alteration of the gustatory papillae structure in CCl 4-induced cirrhotic animals.

Bioelectrical impedance analysis To measure total body resistance (R) and total body reactance (Xc), a phase-sensitive tetrapolar impedance analyser (Biodynamics BIA 450E) was utilised with hypodermic needles as electrodes. The rats were anesthetised and placed in a prone position on a non-conductive surface to eliminate interference from the electrical induction. On the midline, source electrode 1 was placed at the anterior edge of the orbit, and source electrode 2 was placed 4 cm from the base of the tail. Detector electrode 1 was placed at the anterior opening of the ear, and detector electrode 2 was placed in the middle of the rat pelvis. The analysis was performed in two phases, the initial phase (time 1) and the final phase (time 2). Hepatic tissue lipid peroxidation measurements Frozen hepatic tissue from each rat was homogenised in ice-cold phosphate buffer (140 mM KCl and 20 mM phosphate, pH 7.4) and centrifuged at 3,000 rpm for 10 minutes.

Oxidative stress was determined by measuring the concentration of aldehydic products (malondialdehyde [MDA]) using thiobarbituric acid reactive substances (TBARS). The absorbance (535 nm) of the supernatant was measured by spectrophotometry, and the values were expressed as nmol/mg protein. Scanning Electron Microscopy The dorsal region of the tongue was collected and immersed in a fixative solution containing 4% glutaraldehyde. The samples were dehydrated in a graded acetone series: 30%, 50%, 70%, 95% and 100%. A CPD 030 Critical Point Dryer (Leica Microsystems, Buffalo Grove, IL) was utilised to remove moisture from the samples and to reach the critical point. The metallisation was performed with a gold/palladium alloy. The samples were covered with an approximately 15-nm layer using a Metalizer Med 020 (Leica Microsystems).

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Images were captured with a Philips XL20 Scanning Electron Microscope at 40-800X magnification. The taste buds were counted by pathologists who were blinded to the groups. The buds in each field were counted, and the mean was calculated for each sample. Image-Pro Plus (Media Cybernetics, Bethesda, MD, USA) was utilised to determine the diameter of the gustatory papillae and the taste buds. PGP 9.5, T1R2 and T1R3 The expression of PGP 9.5, T1R2 and T1R3 in the tongue was determined by immunohistochemistry.

Antigen recovery was performed using citrate butter at 100ºC, and endogenous peroxidase activity was blocked by incubating the slides at room temperature with absolute methanol containing 3% hydrogen peroxide. The slides were preincubated with 10% rabbit serum at room temperature to block potential undesirable reactions with the secondary antibody. The slides were incubated with polyclonal rabbit antibodies against PGP 9.5, T1R2 or T1R3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4ºC followed by an incubation with the secondary antibody for 1 hour at room temperature. After 60 minutes at room temperature, the slides were treated with EnVision reagents and washed 3 times with hydrogen peroxide (in PBS). The nuclei were counterstained with hematoxylin.

The primary antibody was diluted in PBS containing bovine serum albumin; this buffer without a primary antibody was utilised as a negative control. The results were evaluated without prior knowledge of the groups using a microscope equipped with a digital camera to capture the images using Image-Pro Plus (Media Cybernetics). RESULTS After 16 weeks, we obtained histological confirmation of cirrhosis in all the treated animals; there was no cirrhosis in the control animals. One animal was lost due to death before 16 weeks. The animals were randomized according to the phase angle. In addition to the histological evidence, the cirrhotic animals displayed significant changes in liver function (AST, ALT and ALP), indicating the presence of liver damage. The zinc levels were lower in the CCl 4 group than in the control group ().

Variables Control (n = 7) CCl4 (n = 6) P Mean ± SD Mean ± SD AST (U/L) 171 ± 32.2 1016 ± 305. Control (n = 7)Mean ± SD CCl 4 (n = 6)Mean ± SD P between groups Body Initial 239.4 ± 35.7 211.8 ± 52.8 0.287 Weight Final 296.1 ± 39.8 243.9 ± 17.0** 0.013 (g) ∆ 56.6 32.1 0.193 (final - initial) (45.1 - 68.1)* (-9.1 - 73.3) Phase Initial 7.64 ± 0.75 9.30 ± 0.95 0.005 Angle Final 7.60 ± 1.51 7.73 ± 1.14 0.863 (º) ∆ 0.04 1.57 0.120 (final - initial) (-1.67 - 1.76) (0.22 - 2.92)* * P≤0.05. The results are expressed as the mean ± SD. ** Weight of animals with ascites. The PA was evaluated at the same timepoints (times 1 and 2). There was a statistically significant difference in the CCl 4 group between times 1 and 2, as evidenced by the 2-degree decrease from the initial value to the final value.

FIGURE 2 Histological analysis of the rat tongues by H/E staining. (A) FiP in the control group exhibited normal structure; the arrow indicates a FiP (100X magnification). (B) FiP in the cirrhosis group had an abnormal structure, and there were fewer papillae; the arrow indicates a FiP (100X magnification). SEM analysis of the morphological structure of the tongue revealed that tapering of the gustatory papillae occurred, particularly in the base diameter and the apical region, in the CCl 4 group ( and ). Compared with the control animals, the taste buds in the cirrhotic animals displayed morphological changes ().

Control Mean ± SD CCl 4 Mean ± SD P Papilla angle µm 102.47 ± 9.707 121.25 ± 9.802 0.0001 Apex width µm 16.13 ± 2.644 11.67 ± 1.794 0.0001 Base width µm 43.93 ± 3.138 28.37 ± 4.231 0.0001 Papilla height µm 151.33 ± 9.233 76.34 ± 4.961 0.0001 Taste bud area µm 3.69 ± 0.439 2.57 ± 0.514 0.0001 Taste bud diameter µm 73.72 ± 12.63 55.05 ± 7.659 0.0008 In the animals in the cirrhosis group, some tongues had only one taste bud per field, whereas others contained no taste buds. In the control group, the presence of taste buds per field ranged between 2 and 6. The immunohistochemical analysis of the innervation of the gustatory papillae using a PGP 9.5 antibody revealed that the control and CCl 4 animals had fungiform papillae (FuP) and FiP in the dorsal region of the tongue. An immunohistochemical analysis of the T1R receptor family (T1R2 and T1R3) in the gustatory papillae of rats was performed to determine the potential for the sweet taste. T1R2 receptor immunoreactivity was observed in both groups. There was positive immunoreactivity for T1R3 receptor in the control animals but not in the cirrhotic animals. DISCUSSION The classic CCl 4-induced cirrhosis model is widely accepted in the literature because it has similar characteristics to those of human cirrhosis.

AST and ALT are enzymes that are sensitive to hepatocellular injury. The release of large amounts of these enzymes into the bloodstream is associated with centrilobular necrosis, degeneration and decreased liver function. In our study, we observed a significant increase in the liver enzyme levels in cirrhotic animals, confirming what has been previously reported. Zinc is hepatoprotective and required for ALP activity. Zinc is critical to a large number of structural proteins, enzymatic processes, and transcription factors. Zinc deficiency can result in a spectrum of clinical manifestations, such as poor of appetite, loss of body hair, altered taste and smell, testicular atrophy, cerebral and immune dysfunction, and diminished drug elimination capacity.

These are common symptoms in patients with chronic liver diseases, especially liver cirrhosis. Reinforcing the characteristics of this metal, low zinc levels have been observed in cirrhotic animals, and this was confirmed in the present study. The CCl 4-induced cirrhosis model involves various signalling pathways that result in liver injury; this was confirmed in this study by the TBARS values that indicated a relationship between CCl 4-induced changes in the liver parenchyma and the formation of reactive oxygen species (ROS) by the hepatic microsomal system, potentially through the formation of trichloromethyl (CCl 3•) and trichloromethyl peroxide (CCl 3OO•) radicals. This oxidative process causes cellular damage and may increase ion permeability and membrane and structural disintegration, which can be measured by bioelectrical impedance analysis (the PA). The reduced PA in the cirrhotic animals was accompanied by an increase in liver enzyme levels and in lipid peroxidation. The body structure of the cirrhotic animals changed as they lost weight. The cirrhotic animals exhibited body asymmetry resulting from the development of ascites, a symptom that is characteristic of cirrhosis.

This change in body composition difficult the determination of the actual nutritional status of the analysed organism when conventional methods, such as anthropometric measurements, are utilised that are based primarily on the amount of body fat. The PA has been used as a prognostic factor that assesses cell functionality, and it is associated with other indices that are indicative of the health status. Currently, there have been no studies using this method to evaluate animals.

Cells in the nerve plexus in taste buds are divided based on function into type I, II, III and IV cells. Only type II and III cells are responsive to stimuli and are called 'light' cells. Type II cells receive a stimulus and transmit the signal to type III cells, which form a synapse. In 1982, Nagy et al. Demonstrated that there are nerve fibres in the fungiform and circumvallate papillae of rats and that it is possible to record them. We confirmed these findings by identifying nerve fibres in the FuP in both animal groups. Reported that PGP 9.5 immunoreactivity in the gustatory papillae indicated the presence of type III cells.

Five types of taste have been described; in this study, because of the disease characteristics, we chose to analyse the sweet taste because it is linked to a higher percentage of daily caloric intake. The sweet taste is present in all types of gustatory papillae, and its response to stimuli is similar in different mammals. The sweet taste can be detected via the combination of two receptors (T1R2 and T1R3) that determine the function of the heteromeric T1R receptor. T1R receptors are G protein-coupled receptors (GPCRs); class C GPCRs have a long N-terminus, similar to metabotropic glutamate receptors, GABA B receptors and calcium signal receptors.

Reported in 1997 that the majority of T1R1 receptors are expressed in FuP and are rare in circumvallate papillae. In contrast, T1R2 are poorly expressed in FuP but are expressed in all circumvallate papillae. T1R3 are expressed in both papillae types. We did not observe T1R3-immunoreactive cells, which would mediate the sweet taste, in the cirrhosis group. In 2003, Kim et al.

Compared the signalling activity of circumvallate papillae and FuP and determined that FuP can play the same role in responding to sweet stimuli. In our study, using various techniques, we observed thinning of FiP in the cirrhosis group, which would impair the salty taste. The salty taste is detected directly by Na + permeability in the apical region of the papilla, causing cell depolarisation to perceive the taste.

When there is a thinning of the papillae or they have a decreased diameter, sodium comes into contact with other structures in the dorsal region of the tongue, causing a gradual loss of papillary structure because of a continuous imbalance. Sweet taste aversion and salty taste intolerance are the two symptoms related to taste that are commonly mentioned by cirrhotic patients, and the changes in the gustatory papillae of cirrhotic animals may play a role in dysgeusia. CONCLUSION The experimental cirrhosis model induced by the intraperitoneal injection of CCl 4 mimicked the biochemical and histological parameters of human cirrhosis. However, we know that xenobiotics may alter the conformity of some cells, these may be the taste buds. In the present study, it was possible to determine that cirrhotic rats present significant alterations in the gustative papillae mainly related to sweet and salt taste. Further studies are needed, such as cirrhosis by ligation of bile duct, to associate this like change to cirrhosis.