Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Histopathological Analysis of the Male White Mice Liver After Exposure to Halothane Anesthesia

Duaa Jasim Mohammad

National University of Science and Technology, Dhi Qar, Iraq.

Received | July 02, 2024; Accepted | September 29, 2024; Published | November 13, 2024

*Correspondence | Duaa Jasim Mohammad, National University of Science and Technology, Dhi Qar, Iraq; Email: [email protected]

Citation | Mohammad DJ (2024). Histopathological analysis of the male white mice liver after exposure to halothane anesthesia. J. Anim. Health Prod. 12(s1): 81-85.

DOI | http://dx.doi.org/10.17582/journal.jahp/2024/12.s1.81.85

ISSN (Online) | 2308-2801

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

An anesthetic is a chemical that diminishes sensitivity to pain or is a medicine used to produce anesthesia, which leads to a brief loss of feeling or awareness consciousness and blocks only the sense of unpleasant stimuli. After anesthsia administration, all modalities of sensation including unconsciousness are lost due to suppression of nerve functions. Halothane is one of the most common anesthesia and every year, over three million newborns, children, and adults in the United States receive anesthesia for medical surgeries, while many more people receive anesthesia for dentistry or imaging examination (Sun et al., 2012).

The anesthetic agents have been used for a long time, and it is critical that a suitable anesthetic agent holds qualities such as quick onset, offset of action, and predictable metabolism. Majority of anesthetic drugs are administered through inhalation to maintain general anesthesia in both animals and humans (Della-Rocca et al., 2001; Kleinsasser et al., 2001). However, every pharmaceutical carry adverse effects, including liver damage and the concomitant increased mortality (Njoku et al., 1997).

Anesthetic drugs should be eliminated without metabolic breakdown and regardless of liver or renal function. Thus, they should have the fewest unfavorable medication interactions, adverse effects or toxicity. However, anesthesia that is volatile are mostly eliminated through the lungs where a portion of inhaled gasses are processed. Intriguingly, some anesthetic compounds are metabolized in the liver, resulting in the formation of inorganic fluoride ions. It has been proposed that a high amount of inorganic fluoride in body tissues may cause nephrotoxicity (O’keeffe and Healy, 1999).

Currently used anesthetic drugs include halothane, enflurane, isoflurane, sevoflurane, and desflurane (Della Rocca et al., 2001). Mechanistically, volatile hydrocarbons with halogens have been transformed into trifluoroacetic acids. The latter reacts with liver proteins, triggers an immune reaction in vulnerable persons, and has a single hepatotoxic effect (Kitteringham et al., 1995). Since 1957, halothane (chemical composition: 2-bromo-2-chloro1,1,1 trifluoroethane) has been a commonly and regularly used volatile anesthetic agent in therapeutic settings (Kehlet, 1997; Topal et al., 2003; McLain et al., 1979). It is commonly known that the liver transforms halothane from a lipophilic xenobiotic to hepatotoxic mediators (Minoda and Kharasch, 2001). The halothane as anesthetic agent possesses numerous benefits including immediate action, ease of administration, noninflammability, great potency, and low cost, which led to its widespread usage in a short period of time (Rosenak et al., 1989).

This study was designed to assess any side effect of halothane on the liver in male white mice to highlight the useability, practicalities and implication in animal health.

Materials and Methods

A total of 40 albino male white mice from Drug and Healthy Center in Baghdad Province were used in this study. The experimental animals were kept in an animal house at a controlled temperature of 25-28 ºC and fed standard pellets. At the start of the experiment, animals weighted average 30gram per animal. All animals were divided into four groups: three (A, B, and C) were given varying amounts of halothane, whereas the last group served as a control group. Each experimental group consisted of 10 mice. each group exposure to halothane with inhalation for 15 minutes daily during thirty days. The number of mortalities was 28 animals after exposure to halothane in three different concentrations. The lethal concentration of halothane to the experimental animals was 4% after exposure for 30 minutes. The laboratory animals were placed in individually labeled cages. The concentration of halothane was determined by the use of vaporizer.

Group (A): treated with 1% concentration of halothane.

Group (B): treated with 2 % concentration of halothane.

Group (C): treated with 3 % concentration of halothane.

Tissue samples collection

The liver tissue samples were collected with a diameter of cm³ after slaughtering the experimental animals. The samples were processed for the histology procedure, which involved numerous phases.

Histological technique

Fixation: The liver sample was laid in labeled container contain 10% formalin for 48 hours, Wishing: The sample was washed by using the tap water for (1 h), Dehydration: The sample was done to remove all extractable water by dehydrated diffusing through the tissue, alcohol was used, Clearing: This stage is considered as a middle step, it is obligatory because the used alcohol for dehydration will not dissolve or mix with molten paraffin, Embedding (Blocking): Paraffin wax that used is hard (melting point 60-68°C), Cutting: cutting was done by using the rotary microtome. The thickness is (5 µm). the sample were put in hot water path with (52°C) for extending tissue, then the samples were carried on the slides which had a thin layer of Mayer egg albumin, the slide was put on the hot plate with (40°C) for overnight, Staining: Hematoxylin and Eosin stain: for demonstrating of the general structure of the tissue (Luna, 1968).

Results and Discussion

The histological profile of the liver

Control group

The histological findings of the liver in the control group revealed that the liver was composed of hepatic lobules, each lobule appeared to be separated by the septa. The tissue section showed that normal portal area with a central vein in the liver parenchyma (Figure 1). The histological result of the liver showed that the liver parenchyma was composed of elongated hepatic cords. These cords are composed of an aggregation of the hepatocytes. We identified that hepatocytes carried spherical pigmented black nuclei with acidophilic cytoplasm. The current analysis of liver tissue from the control group revealed a normal distribution of Kupffer’s cells between hepatic cords. The histological outcome of the liver in the control group appeared normal blood sinusoids with a normal endothelial layer (Figure 1).

The histological results of the mice liver following exposed to 1% halothane concentration

After treatment of 1% halothane, the liver showed aggregation of inflammatory cells, high distribution of Kupffer’s cells in the liver parenchyma, and lipidosis or fatty changes characterized by harmful amounts of fats or lipids accumulate in some of the cells in the liver. with a similar histolological picture was revealed earlier where fatty liver disease was characterized by fatt cells carrying much larger cytoplasmic vacuoles and nuclei that are located at the

peripherally of the cells or not visible (Curran and Crocker, 2005) (Figure 2A, B) detailed histological analysis showed hepatic granuloma formed by small abnormal clumps of macrophages and more proliferating inflammatory cells that followed antigen exposure (Figure 2C). These results further justify the finding reported earlier, granulomas were aggregates of modified macrophages and other inflammatory cells that accumulate after chronic exposure to antigens (Ferrell, 1990). Post 1% halothane treatment, a very wide cystic dilation in the liver parenchyma filled with blood was observed. Hepatic sinusoidal dilation marked by enlargement of the hepatic capillary may be caused by blood congestion in the liver parenchyma. These results agree with the finding of (provide author names), (Brancatelli et al., 2018). They have stated that the swelling of the hepatic capillaries was referred to as hepatic sinusoidal dilatation. Hepatic venous outflow blockage, which causes vascular stasis and congestion of the hepatic parenchyma, the most common cause of this syndrome. The histological result of the liver after treatment with 1% halothane showed high degeneration of liver parenchyma, disappeared the structure of the hepatic cord, and high aggregation of inflammatory cells in different locations of the liver parenchyma. The Kupffer cells showed prominent oval nuclei and were highly distributed in the liver parenchyma (Figure 2A, C).

The histological results of the liver following exposed to 2% halothane concentration

The tissue section of the liver showed severe hepatocyte degeneration with very wide sinusoidal dilation, hepatic necrosis, and cystic dilation filled with inflammatory cells. The results showed bile accumulation, and the bile plugs were observed in the canaliculi (Figure 3A). A similar picture was projected earlier, where cholestasis was characterized by distended bile canaliculi, each containing a plug of very dark brown bile (Curran and Crocker, 2005). This appearance was characterized by very marked centrilobular cholestasis. The present result showed large Kupffer cell aggregation in different locations of liver parenchyma. The histological result noted high numbers of inflammatory cells present commonly around the portal area (Figure 3B). The high halothane concentration may increase the tissue damage of liver tissue, which leads to an increase in the inflammatory cell aggregation. These results confirmed previous findings (Bourdi et al., 2001), which halothane, compared with sevoflurane, revealed higher indices of morphological changes. These changes include liver inflammation, and destruction in the hepatocytes, and massive, predominantly centrilobular hepatic necrosis. The results showed cystic dilation rounded in shape and filled with inflammatory cells. The results showed most hepatocytes have destruction nuclei with many cytoplasmic vacuoles. The results showed very wide dilation and irregular in shape of cystic dilation because of severe degeneration of hepatocytes. Large aggregation of Kupffer cells with clusters of inflammatory cells inside dilated sinusoid

(Figure 3C). The results showed hepatic granuloma formed by small abnormal clumps of macrophages and other inflammatory cells that accumulate after exposure to antigens (Figure 3D). These results are similar a previous study (Brancatelli et al., 2018), where authors have identified granulomas aggregates of modified macrophages and other inflammatory cells that accumulate after chronic exposure to antigens (Figure 3). These unclear changes may be because the halothane toxicity reached to nuclei and affected the chromatic materials, leading to the destruction of chromatic mass as reported earlier (Soubhia et al., 2011). It has been previously shown that the genotoxic effect of inhalation anesthetics demonstrates (halothane) provoke DNA injury. However, the anesthetic does not interact directly with DNA Also, it has been found that halothane can irreversibly damage cells genome at 1.5 mM concentration which explain the observed toxicity.

The histological results of the liver following exposed to 3% halothane concentration

The histological result of the liver after exposure to a 3 % concentration of halothane showed abnormal central vein with destruction of endothelial layer in the central vein and aggregation of inflammatory cells in this region (Figure 4C) as reported before (Bourdi et al., 2001). In this study it has been identified that trifluoroacetic acid may bind covalently to hepatocyte macromolecules and phospholipids, producing trifluoroacetate-protein compound (TFA-protein compound), covalent binding to liver proteins may be the preliminary step in acute halothane hepatitis. The histological results showed hepatic granuloma formed by small abnormal clumps of macrophages and other inflammatory cells that accumulate after exposure to antigens. A similar observation has been made earlier (Ferrell, 1990), where granulomas were aggregates of modified macrophages and other inflammatory cells that accumulate after chronic exposure to antigens. We observed bile accumulates and the bile plugs in the canaliculi (Figure 4B). The current result of the liver after treatment with 3% halothane led to aggregation of inflammatory cells, high distribution of Kupffer cells in the liver parenchyma, and lipidosis or fatty changes characterized by harmful amounts of fats or lipids accumulate in some of the cells in the liver. It has previously been reported that fatty liver disease is characterized by fatty cells having much larger cytoplasmic vacuoles and nuclei that are located at the peripherally of the cells or not visible (Curran and Crocker, 2005) (Figure 4A, C). The tissue section of the liver revealed that hepatocyte nuclei were big in size and rounded in shape with fragmented chromatic materials inside the nuclei and noted wide spaces between fragmented chromatic material. Most of the enlargement of nuclei were characterized by distributed chromatic materials peripherally at locations with very prominent enlargement nuclei (Figure 4A).

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