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Histological changes in the liver of "Triton-induced" obesealbino rats (Light and electron microscopic study)

El-SayedGalal El-SayedKhe * and Ahmad Mohammad Abdel-Aleem Desoky.

Departments of Histology, Cairo* and Assiut Faculties of Medicine, Al-Azhar University

ABSTRACT: diets containing high amount of fats or cholesterol lead to both hyper-cholesterolemia and hyper-triglyceridemia which mostly induces oxidative stress, an important factor in the development of nonalcoholic fatty liver disease. Today, oxidative stress is one of the major threats to our survival; it became the nutritional and medical puzzle for the 21St century. Nonalcoholic fatty liver diseases the most common liver disorder in the world which caused by obesity, its incidence reaches 70-90%. The disease is characterized by the accumulation of triacylglycerols inside liver cells and the condition can progress into more serious liver disease, such as non-alcoholic steato-hepatitis, liver fibrosis, cirrhosis and more rarely, liver carcinoma.

Keywords: liver -triton -obese - albino rats.

INTRODUCTION: Obesity is a worldwide epidemic that currently affects both developed and developing

countries; it is the second leading cause of preventable death in the world (1).Worldwide, over 600 million were obese and count about 13% of the total population (11% of men and 15% of women), 42 million children under the age of 5 were overweight or obese in 2013 (2). Diets containing high amount of fats or cholesterol lead to both hyper-cholesterolemia and hyper-triglyceridemia which mostly induces oxidative stress, an important factor in the development of nonalcoholic fatty liver disease (3).Today, oxidative stress is one of the major threats to our survival, it became the nutritional and medical puzzle for the 21Stcentury(4). Nonalcoholic fatty liver diseases the most common liver disorder in the world which caused by obesity, its incidence reaches 70-90%(5). The disease is characterized by the accumulation of triacylglycerols inside liver cells and the condition can progress into more serious liver disease, such as non-alcoholic steato-hepatitis, liver fibrosis, cirrhosisand more rarely, liver carcinoma(6). The aim of this work is to study the light and electron microscopic changes in the liver induced by obesity in albino rats (that treated by intra-peritoneal injection of Triton "250 mg/kg" to induce obesity).

Vol. 5(4), pp. 144-159, July 2019 ISSN 2315-5396

DOI: 10.14412/SRI2019.159 Copyright© 2019

Author(s) retain the copyright of this article Available online at http://www.scienceparkjournals.org/SRI

Scientific Research

and Impact

MATERIAL AND METHODS: This study was carried out at the Faculty of Veterinary Medicine, Alexandria University. 60 male albino rats weighing 120 ± 5 grams and aged 4-5 weeks were used throughout this study. The rats were housed in six clean metallic cages with a metallic mish cover and dimension of 120 X 60X 60 cm, each cage contained 10 rats. The animals were fednormal laboratory dietwith liberal supply of water. All animals were housed under the mentioned environmental condition and the basal diet for one week before experiment for acclimatization to ensure normal growth and behavior. Weight of animals was recorded every week. The 60 male albino rats are divided into three groups (20 rats for each group):

Group I (Control group): 20 rats werefed normal laboratory diet for 4 weeks then sacrificed. This group

was used as a negative control group.

Group II (Obese group): 20 rats were treated by intra-peritoneal injection of Triton 250 mg/kg to induce

obesity and fed normal laboratory diet for 4 weeks then sacrificed. This group was used as a positive

control group.

Group III (Recovery group): 20 rats were similar to the obese group but left without treatment for

another 4 weeks (for spontaneous recovery) then sacrificed.

Healthy diet regimen with good fluid intake, as well as, increase exercise and daily activity should be

followed to avoid immobility, obesity and obesity related complication.

Group

Mean of

body weight

(grams)

Mean of

liver weight

(grams)

Mean of

ALT (U/l)

Mean of

AST (U/l)

Mean of

GGT (U/l)

Control 242 ± 04.37 c 04.54 ± 0.14

d 19.96 ± 1.38

c 030.00 ± 1.23

c 05.85 ± 0.59

c

Obesity 358 ± 10.70 a 11.90 ± 0.21

a 84.76 ± 3.56

a 172.10 ± 9.22

a 25.60 ± 1.55

a

Recovery 318 ± 08.15 b 07.04 ± 0.13

b 42.10 ± 2.55

b 072.70 ± 4.70

b 13.00 ± 0.70

b

Table (1): Comparison between means, standard errors and P value of body weight, liver weight and liver

enzymes in various groups:

Means with different superscript differ significantly (P<0.05).

(a) high value - (d) low value - (b) and (c) in between.

ALT = Alanine aminotransferase.

AST = Aspartate aminotransferase.

GGT = Gamma glutamyltransferase.

Figure (1): Comparison between means and P value of body weight, liver weight and liver enzymes in

Triton WR 1339 (Tyloxpole)® was purchased from Sigma-Aldrich U.S. Ain the form of vial 50 ml containing 50 grams of triton. Triton was dissolved in normal saline to make a solution at concentration of 100 mg/ml (1 ml of triton/9 ml of normal saline). Triton is given by intra-peritoneal (IP) injection at a dose of 250 mg/kg (i.e. 0.25 ml of the prepared solution/100 grams body weight) according to Walaa and Saad(2014)(1).Intra-peritoneal injection is the most frequently used parenteral route of administration in rats. The large surface area of the abdominal cavity and its abundant blood supply facilitate rapid absorption. Absorption from this route is usually one-half to one-quarter as rapid as that from the intravenous route(7). Intra-peritoneal injection of Triton is done as the following: Firstly we locate the point of entry for the needle by drawing an imaginary line across the abdomen just above the knees. The needle will be inserted along this line on the animal’s right side and close to the midline to avoid the cecum, which is a large fluid-filled organ on the left side of the abdomen. The small intestines (on the right side) are less likely to be punctured by the needle.Inserting the needle too far caudally or laterally from the insertion point mentioned above would risk making an injection into the near leg which would injure the muscle tissue.

242

358318

0

50

100

150

200

250

300

350

400

Control Obesity Recovery

Mean of body weight (grams)

4.54

11.9

7.04

0

2

4

6

8

10

12

14

Control Obesity Recovery

Mean of liver weight (grams)

0

20

40

60

80

100

120

140

160

180

200

Mean of ALT (U/l) Mean of AST (U/l) Mean of GGT (U/l)

Control

Obesity

Recovery

Mean of liver enzymes (U/l)

Secondly we perform an IP injection, were the rat was restrained so that it cannot move during the procedure. This avoids traumatizing the organs once the needle has entered the abdomen.The rat was tilt so that the head is facing downward and its abdomen is exposed.The needle was inserted into the abdomen at about a 30-degree angle after disinfecting injection site.The shaft of the needle should enter to a depth of about half a centimeter.We aspirate to be sure that the needle has not penetrated a blood vessel, the intestines or the urinary bladder (greenish brown aspirate indicates needle penetration into intestines while yellow aspirate indicates needle penetration into the bladder). If any fluid is aspirated, the solution is contaminated and must be discarded and the procedure repeated with a new syringe and needle.If no fluid is aspirated, we may inject.The needle was withdrawn and the mouse was return to its cage. Determination of liver enzymes: at the end of the experiment as well after overnight fasting, blood samples were drawn from the retro-orbital plexus of rats of all groups under diethyl ether anesthesia before sacrificing by decapitation. Blood samples were collected without anticoagulant in clean and dry Wassermann tubes and left in slope position to clot at room temperature. The tubes were centrifuged at 3000 rpm for 5 minutes and the non-hemolysis serum was carefully separated and transferred into clean dry epindorffs which kept frozen at -20 0C until used for biochemical analysis. Kits for liver function tests as (ALT, AST and GGT) were purchased from Vitro Science Company while histological stains were purchased from El-gomhria Company for chemicals, Mansoura, Egypt. They include Haematoxylin and eosin stain, Mallory trichrome stain and PAS stain.

Figure (2):(A) and (B): two photomicrographs of liver sections obtained from control group showing normal liver architecture with classic hepatic lobules containing central vein "C" and radiating cords of hepatocytes. Hepatocytes are polyhedral with large rounded euchromatic nuclei and prominent nucleoli "arrows". Blood sinusoids "S" present between hepatocytes are lined by flattened endothelial cells and Kupffer cells "arrow heads". Portal tract at the periphery of a classic hepatic lobule "PT" reveals a portal venule, a hepatic arteriole and a bile ductule(H and E stain - X 200 and 1000). (C) and (D): two photomicrographs of liver sections obtained from hyperlipidemic group showing highly disturbed portal area with marked dilatation and congestion of portal vein "PV" that contain haemolysed RBCs "double arrow". Hepatic artery "HA" and bile canaliculi"BC" are surrounded by many lymphocytic infiltrations "thick arrows" and fat cells "arrow heads". Most hepatocytes have vacuolated cytoplasm pushing their nuclei "thin arrows" with widening of space of Disse"stars"(H and E stain - X1000). (E) and (F): two photomicrographs of liver sections obtained from recovery group showingnormal histological structure with some fat vacuoles in most hepatocytes "arrows". Dilated blood sinusoid "S" and dilated congested central vein "CV" with haemolysed RBCs inside it were also noticed. Loss of cord architecture with wide spaces between hepatic cords "arrow heads" is also observed (H and E stain - X 200 and 1000).

Figure (3):(G) and (H): two photomicrographs of liver sections obtained from control group showing normal

distribution of collagen fibers in the form of fine collagen bundles supporting central veins )arrows) “Mallory's

trichrome stain” )X 400). On the other side, normal glycogen distribution in the central and portal areas of liver tissue

are seen around central vein )CV) and portal tract )PT) respectively “PAS stain” )X 200). (I) and (J): two

photomicrographs of liver sections obtained from hyperlipidemic group showing increased collagen fibers in most of

liver tissue condensed around dilated central vein (double arrow) and extending between cords of hepatocytes

)arrows) “Mallory's trichrome stain” )X 400). On the other side, hepatocytes are depleted from glycogen )indicated by

highly reduced stain) specially around central veins (CV). Hepatocytes showed variable amounts of cytoplasmic

vacuolations )arrows) while central vein showed endothelial detachment )arrow head) “PAS stain” )X 200). (K) and

(L): two photomicrographs of liver sections obtained from recovery group showing collagen fibers around dilated

congested central veins (CV) extending between hepatic cords (arrows). Hepatocytes shoed small cytoplasmic fat

vacuoles )arrow heads) “Mallory's trichrome stain” )X 400). On the other side, hepatocytes showed moderate stain

affinity of glycogen specially around centerl vein )arrows) “PAS stain” )X 400).

Group

Median of

Collagen area %

Median of

Optical density (μm)

Median Inter-quartile range Median Inter-quartile range

Control 1.80 a 2.45 0.241

a 0.057

Obese 2.33 a 3.77 0.138

c 0.021

Recovery 1.85 a 3.51 0.205

b 0.018

Table (2): Comparison between median, P value and inter-quartile range of collagen area and optical

density of hepatocytes in varous groups.

Groups with different superscript differ significantly (P<0.05).

(a) high value - (d) low value - (b) and (c) in between.

Analysis and comparisons based on non-parametric methods.

Figure (4): Comparison between medins and P value of collagen area (detected by Mallory trichrom stain)

and optical density (detected by PAS stain) in different groups.

1.80

2.33

1.85

0

0.5

1

1.5

2

2.5

Control Obese Recovery

Median of Collagen area %

0.241

0.138

0.205

0

0.1

0.2

0.3

Control Obese Recovery

Median of Optical density (μm)

Figure (5):(L) and (M): Magnified electron micrographs of hepatocytes obtained from control group showing normal ultrastructure of the nucleus "N" and its nuclear membrane "short arrows" that contains nucleopores. Rough endoplasmic reticulum "double arrows", mitochondria "M" and glycogen granules "arrow head" are clearly appeared. On the other side, the liver ultrathin section shows space of Disse"long arrows" between hepatocytes containing microvilli of hepatocytes with blood sinusoids containing Kupffer cell "Kc" (x 15,000 and x 12,000). (N) and (O): Magnified electron micrographs of liver ultrathin sections obtained from obese group showing nucleus with apparent nucleolus "N" and condensed chromatin. Some mitochondria are surrounded by vacuoles "arrows". Many lipid droplets with variable size and shape are seen within blood sinusoid and hepatic cytoplasm "arrow heads". On the other side, enlarged highly

disturbed Kupffer cell "Kc" is seen with irregular compressed nucleus and multiple vacuole aggregation. Pyknotic nucleus of one hepatocyte is also seen "double arrows" (x 10,000 and x 4,000). (P) amd (Q): Magnified electron micrographs of liver ultrathin sections obtained from recovery group showing variable sized mitochondria with cristae "thick arrows". Secondary lysosome "arrow heads" and dilated space of Disse"double arrows" containing disturbed microvilli are also seen. On the other side, blood sinusoid contains two large fat vacuoles "thin arrows" between RBCs whileKupffer cell "Kc" is enlarged and show aggregation of fat vacuoles "stars" (x 15,000 and x 6,000). Electron microscopic study: after anaesthia, blood sampling and decapitation; the abdomen and chest are opened longitudinally, then while the heart is still beating a 12 gauge canola is inserted in the right ventricle and a small incision in the right atrium is done using scalpel, to infuse a solution of normal saline containing 2.5% glutraldehyde and one ampule of heparin directly in the heart to infuse the rat tissue with the fixative to avoid any destruction or changes in the ultra-structure of the cell. Immediately after perfusion of the fixative, liver is excised and a small piece from liver is cute in to a very small multiple cubic about 1×1×1 mm in a dish containing 2.5% glutraldehyde solution, then putted in a small plastic pipes filled with 2.5% glutraldehyde. According to Mark et al.(2014)(8), tissue is transferred to osmium tetroxide. Osmium tetroxide reacts strongly with membrane lipids and also with proteins. Osmium is a heavy enough metal to confer some electron density to the membranes and other cellular structures to which it binds. Sample is then embedded by infiltrating it with resins that can be polymerized into a hard plastic suitable for thin sectioning. After imbedding tissue is transferred in to a tissue block or capsule that is ready for cutting and then staining for EM examination. Toluidine blue staining of thick (semi-thin 0.5 μm or 1.0 μm.) sections are used as guideline to the area of interest and further trimming (reagents: 1% Toluidine Blue and 2% borate in distilled water).Ultrathin sections, 40-100 nm (regularly 60 nm for TEM) are spread mostly on 200 or 300 mesh copper grids and stained with uranyl acetate followed by lead citrate to enhance contrast and is easily done by floating the grids, sections side down, on droplets of stain, followed by water rinses. Light microscopic study: after decapitation evisceration under di-ethyl ether anesthesia, liver was removed and washed by normal saline to remove the blood and then fixed in 10% formol saline. After putting the tissues in the fixative (10% formol saline) for 24 hours, the fixed tissues is washed in running tap water to remove the fixative from them, dehydration was done gradually in ascending grades of alcohol by putting the tissues in 50% alcohol then in 70% alcohol and finally in 100% alcohol. Clearing of the tissues was done to remove alcohol and to allow the fixed tissues to be miscible with xylol. The cleared fixed tissues were put in melted soft paraffin in an oven at 50 C° for 24 hour then transferred into melted hard paraffin in an oven at 55 C° for one hour. The specimens were then transferred to casts filled with melted hard paraffin. The casts with their contents were cooled in ice until the paraffin is completely solidified forming blocks of hard paraffin with the tissues in its centers. Each block of hard paraffin was cut into thin sections (5 micrometer) by rotatory microtome. Paraffin sections representing all groups were then placed on clean glass slides smeared with albumin glycerin, allowing few drops of albumin glycerin to flow beneath the section. The slides were warmed on a hot plate then leave them for several hours in the incubator to dry(9) then stained by:Haematoxylin and eosin stain (to study the general structure of the liver), Mallory trichrome stain(to study distribution of collagen fibers in the liver) and PAS stain (to study glycogen content of the liver).Image analysis was done using Optima’s (6.2.1) program to measure percentage of collagen area outside hepatpcytes and optical density of glycogen inside hepatocytes.

Statistical analysis: after measuring means, standard errors and P-value of the following parameters:

Body weight and liver weight. Liver function tests (ALT, AST and GGT). Percentage of collagen area outside hepatpcytes and optical density of glycogen inside

hepatocytes. One-way analysis of variance (ANOVA) was used to assess the effect of obesity on liver enzyme variables with Turkey post-hoc test for multiple comparisons. Collagen area and optical density variables either raw or transformed have violated the assumptions necessary to run the analysis of variance (normality of data and homogeneity of variances). Therefore, the alternative non-parametric Kruskal-Wallis test, followed by pair wise comparison using Mann Whitney U test with Bonferroni correction were used. All analyses were run using SPSS software (IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp.) A probability value of 0.05 was considered significant.

RESULTS: 1. Behavior and appetite: during period of Triron WR 1339 injection rats were calm, easy to handle without injuries or deaths. Appetite was increased in obese group more than in control group, this indicated by increased amount of food needed for each group (about 20 grams/ rat/ day for obese group and 14 grams/ rat/ day for control group). Appetite was lower in recovery group (about 17 grams/ rat/ day). No changes occurred in the skin or hair. 2. Body weight and liver weight: Obese group increased in weight by (up to 70 grams per week) while control group showed lower rate of weight gain (about 30 grams per week). After stopping triton, recovery group started to lose weight (about 10 grams per week). The results observed in table (1) and figure (1) cleared that there is highly significant increase in body weight and liver weight of rats in obese group when compared to the control one. Also, there is a significant reduction of body weight and liver weight in recovery group compared to obese group. 3. Biochemical results: the results observed in table (1) and figure (1) cleared that there is a highly significant increase in liver enzymes (ALT, AST and GGT) in obese group when compared with control group. As regard to recovery group, there is a significant improvement in the level of these enzymes when compared with obese group. 4. Light microscopic results: examination of Haematoxylin and Eosin (H&E) stained sections obtained from liver of control group animals showed classic hepatic lobules with roughly hexagonal shape. Central veinform a central axis and rows of liver cells are arranged in the form of radiating cords. At the angles, there were portal areas containing connective tissue stoma and portal triads consisted of a terminal branch of the hepatic artery and a small branch of the portal vein as well as bile ductule. Hepatocytesare arranged as anatomizing cords, radiating from the center towards the periphery and separated from each other by narrow blood sinusoids which found as a network between the plates of hepatocytes converging towards the central vein. They were lined by two types of cells, endothelial and Kupffer cells. The endothelial cells appeared flattened forming discontinuous layer with darkly stained flat nuclei while Kupffer cells appeared scattered among the endothelial cells with large rounded nuclei. The cytoplasm of hepatocytes appeared acidophilic with scattered fine basophilic granules. The hepatocytes were

polygonal in shape containing rounded euchromatic nuclei mostly large, rounded and of the same size with prominent one or more nucleoli"figure (2) A and B".On the other hand, examination of sections obtained from liver of obese group revealed several histological changes in hepatocytes characterized by loss of cellular details and hyper-eosinophilic cytoplasm. Marked dilatation and congestion of central veins, portal veins and blood sinusoids with rupture of endothelial lining of the blood vessels are reported. In addition, cellular infiltrations are seen around central veins, around and in between components of portal tract and even between hepatocytes. Hemolysis of RBCs inside blood vessels, increased Kupffer and fatty cells are also reported. Most of hepatocytes exhibited marked enlargement by multiple variable-sized vacuoles (most probably lipid inclusion) that distend the cytoplasm, some contained multiple small vacuoles and others showed one large vacuole, the later appeared ballooned with peripheral nuclei. In addition, some nuclear changes were observed in the form of darkly stained (pyknotic) nuclei, fragmented nuclei (karyorhexisis) and even lysed nuclei (karyolysis) "figure (2) C and D". Histological sections of recovery group showed slightly disturbed hepatic lobules with loss of cord architecture as well as presence of wide space between cords filled with erythrocytes and eosinophilic materials.Hepatocytes with multiple small and discrete fat vacuoles in hepatocytes are seen in different zones of hepatic lobules in addition to hyper-eosinophilic cytoplasm and pyknotic nuclei in some cases. Moderate dilatation and congestion of central veins, portal veins and blood sinusoids are also reported with focal areas of mononuclear cellular aggregation (mainly lymphocytes and macrophages). However, endothelial lining of central vein is intact with large flat nuclei "figure (2) E and F". Examination of Mallory’s trichrome stained liver sections of control rats showed normal distribution of fine collagen fibers in the wall of central veins and in portal tracts at the periphery of hepatic lobules. Collagen fibers are lightblue, nuclei are red while smooth musculature is violet. On the other side, examination of Periodic acid-Schiff (PAS)stained liver sections of control rats showed normal distribution of glycogen content in the central and portal areas of liver tissue with increase in stain affinity.Glycogen appears as magenta red or acidophilic and basement membranes appear also red "figure (3) G and H" On the other side, examination of Mallory’s trichrome stained sections obtained from liver of obese group revealed slight increase in the amount of collagen fibers around central veins, blood sinusoids as well as around and in-between component of portal tracts. On the other side, examination of PAS stained liver sections of this group showed highly depleted glycogen distribution from hepatocytes in all zones of hepatic lobules with highly reduced stain affinity of the liver tissue "figure (3) I and J" However, examination of Mallory’s trichrome stained sections obtained from liver of recovery group showed very slight increase in the amount of collagen fibers around central veins, blood sinusoids as well as around and in-between component of portal tractswith increase in the amount of cytoplasmic small fat vacuoles and pyknotic nuclei.On the other side, examination of PAS stained liver sections obtained from this group showed moderate glycogen content in hepatocytes at portal and central areas with moderate stain affinity to glycogen in hepatocytes" figure (3) K and L" The results observed in (Table 2) and (Figure 4) showed non-significant increase of collagen area in the liver tissue of obese group when compared with the control one. In addition, there is also non-significant reduction of collagen area in the liver tissue of recovery group when compared to obese group.On the other side, there isa highly significant decrease in optical density of glycogen in obese group when compared to the control one. In addition, there is also a significant increase in optical density of glycogen in recovery group when compared to obese group, however, the optical density in recovery group still lower than that of the control one.

5. Electron microscopic results: Examination of ultrathin liver sections obtained from the control group revealed normal electron microscopic features of the liver. Hepatocytes were polyhedral in shape containing rounded nuclei with prominent nucleoli and distinct nuclear envelope. The nucleolus showed aggregations of euchromatin and heterochromatin granules.The cytoplasm of hepatocytes contains abundant cell organelles, mainly mitochondria which appeared rounded or oval with well-developed cristae. Mitochondria are slightly variable in size and distributed throughout the cytoplasm in association with endoplasmic reticulum, and secondary lysosomes. Glycogen appeared as electron dense particles dispersed as single minute granules. Kupffer cells with their characteristic nuclei were observed in the spaces of Disse between hepatocytes and blood sinusoids. Blood sinusoid and space of Disse appeared normal" figure (5) M and N". On the other side, examination of ultrathin liver sections obtained from obese group revealed marked changes in hepatocytes in the form of numerous intra-cytoplasmic fat globules of various sizes, some hepatocytes appeared swollen with compressed nuclei. Nuclei of most hepatocytes showed irregularity with condensed chromatin as well as indentation and widening of the peri-nuclear space. Dilated small stacks of fragmented rough endoplasmic reticulum cisternae associated with lysosomes are also seen. The structure of mitochondria was changed and show enlargement, swallowing, complete or partial destruction in of their cristae and most of them were surrounded by fat vacuoles.The blood sinusoids showed hypertrophied endothelial cells and Von Kupffer cells containing numerous RBCs. Von Kupffer cells showed numerous membranous vacuoles and irregular condensed nucleus which was compressed by vacuole. Disse’s spaces was dilated and contained fragments of distorted microvilli" figure (5) O and P". However, examination of ultrathin liver sections obtained from recovery group revealed mild change in hexagonal structure of hepatocyte. Moderate ultra structure changes were seen in hepatocytes in the form of intra-cytoplasmic fat globules of various sizes, primary and secondary of lysosomes. Nucleus showed irregular nuclear membrane, condensed chromatin and widening of the peri-nuclear space. The mitochondria showed mild changes in the form of enlargement and swallowing. Some of mitochondria preserved their cristae, others showed destruction in their cristae. Disse’s spaces of this group appeared dilated with moderate destruction in microvilli. Glycogen appeared as electron dense particles. Blood sinusoids showed moderate hypertrophic Von Kupffer cells that contained multiple membranous fat vacuoles and irregular condensed nucleus compressed by these vacuole" figure (5) Q and R". DISCUSSION: Obesity is a disorder characterized by chronic energy imbalance, whereby energy expenditure is consistently lower than energy intake, necessitating the expansion of adipose tissue to allow the storage of excess energy(10). Obesity is increasing in prevalence at a fast rate due to the rapidly increasing adoption of diets rich in saturated fats and simple carbohydrates, in combination with a sedentary lifestyle(11). Obesity is associated with a spectrum of liver abnormalities, known as nonalcoholic fatty liver disease (NAFLD), characterized by an increase in intrahepatic triglyceride (IHTG) content (i.e. steatosis) with or without inflammation and fibrosis (i.e. steato-hepatitis). NAFLD has become an important public health problem because of its high prevalence, potential progression to severe liver disease and association with serious metabolic abnormalities, including type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD) (12). The pathogenesis of NAFLD and nonalcoholic steato-hepatitis (NASH) is not fully understood. It was explained as prolonged over-nutrition that leads to accumulation of free fatty acids and triglycerides within the liver (steatosis) and progression of NAFLD to NASH that is associated with other factors such as oxidant stress, mitochondrial injury, fatty acids lipotoxicity, innate immunity and inflammatory

cytokines. Steatosis is a characteristic histological feature of NAFLD that results from increased fatty acid influx or impaired fatty acid utilization in the hepatocytes (13). The aim of this work is to study the light and electron microscopic changes, as well as, enzymatic changes in the liver induced by obesity in 60 albino rats (120 ± 5 grams and 5 weeks old) treated by intra-peritoneal injection of Triton "250 mg/kg" for 4 weeks to induce obesity. We use rat module in this study because the great similarity and homology between the genomes of rodents and humans that make these animal models important in the experimental studies. In addition, the animals enable us to obtain answers in a short time, since 10 days in the life of a rat are approximately 1 year in humans when comparing changes in body weight (14). Moreover, Sprague-Dawley and Wistar rats are popular strains to study obesity as they readily gain weight (15).

Our results cleared that there is highly significant increase in body weight, liver weight and liver enzymes (ALT, AST and GGT) of rats in obese group when compared to the control one, this may be due to fat cell deposition in various organs and lipid droplet deposition in hepatocytes. These results agree with those of Ferguson et al. (2016) who reported that one of the major mechanisms of obesity is adipose tissue hyperplasia (increase in number of adipocytes) which is facilitated by increased adipogenesis. Adipogenesis is the process of conversion of pre-adipocytes into adipocytes (16). In addition, the results agree also with those of Cui et al. (2011) who stated that liver weight in hyperlipidemic groups were heavier than those of normal diet group. He explained these finding by presence of predominant microvesicular and focal macrovasicular fatty changes(17).This also explains the presence of significant improvement in body weight and liver weight of recovery group when compared with obese group. Moreover, the increase of liver enzymes were explained by Noha et al. (2017)who stated that intravenous injection of Triton WR 1339 cause fat infiltration in hepatocytes which is responsible for their damage that indicated by high levels of liver enzymes AST and ALT (18).Excess fat accumulation in hepatocytes may lead to hepatocellular injury via direct cellular cytotoxicity mediated by free fatty acids (FFAs), oxidative stress, lipid peroxidation, mitochondrial impairment and cytokine-induced hepatotoxicity leading to liver disturbed function(19).This also explains the presence of significant reduction in liver enzymes (ALT, AST and GGT)of recovery group when compared with obese group. Light microscopic study cleared that many histological changes in the liver tissue of obese group and recovery group are observed, however, these changes are more prominent in obese group. These changes include vacuolated hepatocytes with intra-cytoplasmic fat droplets. The hepatic portal areas lost their normal architecture and contained highly distorted blood vessels and bile canaliculi. These finding are explained by Brunt and Tiniakos (2010) who reported that, the adipocytes in fatty liver are considered as active cells that secrete multiple immune modulator factors in the form of pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrotic factor (TNF) with reactive oxygen species (ROS), all these factors contribute to the chronic inflammatory condition and the hepatocyte injury(20). So, increased lymphocytic infiltration and hemolysed RBCs were detected in the portal area. There is also a non-significant increase of collagen area in liver tissue of obese group and recovery group when compared with the control one. Collagen fibers are observed more around hepatocytes and walls of blood vessels leading to hepatocellular injury. These results were explained by Horn et al. (1985)who reported that the presence of collagen in the peri-sinusoidal spaces might affect the blood supply to liver cells and would reduce the exchange of metabolites may cause hepatocellular dysfunction and necrosis(21).

On the other side, there isa highly significant decrease in optical density of glycogen in obese group when compared to the control one. In addition, there is also a significant increase in optical density of glycogen in recovery group when compared to obese group, however, the optical density in recovery group still lower than that of the control one. These results agree with those of Han et al. (2007) that reported highly affected glycogen content in hepatocytes of obese group and explain this by altered insulin levels and insulin sensitivity or insulin resistance due to obesity(22). Electron microscopic study showed many ultra structure changes in the form of lipid inclusions in hepatocytes of obese group and recovery group. Some lipid inclusions were macro-vesicular pushing the nucleus to the periphery and giving the characteristic signet ring cell appearance. Other lipid inclusions were micro-vesicular giving multiple lipid vacuoles around central nucleus. Some mitochondria were engulfed by lysosomes. Massive fat vacuoles were also observed in blood sinusoid withKupffer cell indicating hyperlipidemia. These results agree with Brunt and Tiniakos (2010) who reported vacillations in hepatocytes as microvesicular and macrovesicularsteatosis(23). The macro-vesicular steatosis is explained as abnormalities in the synthesis, metabolism and export of lipids. Ballooned hepatocytes can be attributed to microtubular disruption and severe cell injury. However, microvesicularsteatosis which is the hallmark of liver diseases is associated with defective beta-oxidation of fatty acids, indicating mitochondrial abnormality(24). Furthermore, cytoplasmic vacuolation were attributed to lipid per-oxidation because of oxidative stress damage cell membrane as well as membranes of cell organelles leading to increase in their permeability and disturbance of the ions concentrations in the cytoplasm and cell organelles(25).The hepatocellular injury in obese group was explained by two major mechanisms: the direct one involves direct cytotoxicity of the fatty acids on hepatocytes as a result of excessive intracellular fatty acid accumulation, while the indirect mechanism involves cytotoxic effects of lipid peroxidation of fatty acids (26). We also detected small and shrunken nuclei with irregular torn nuclear membrane. Some of them showed alteration in chromatin distribution in the form of imagination and clumping, this may be attributed to morphological injury in the nucleus due to obesity. The mitochondria of obese group showed morphological disruption, where the sizes are increased, the aristae are disrupted and their matrixes are hypo-dense compared to control group. These mitochondrial dysfunctions are explained by Wei et al., (2008) who stated that changes in the morphology of mitochondria including increase in its size, loss of the aristae and hypo-density of the matrix, in NAFLD might attributed to disturbances in DNA and in lipid oxidation function(27). Noticeable signs of improvement were detected in the liver tissue of recovery group. Space of Disuse was dilated with few disturbed microvilli. Kupffer cell still enlarged with aggregation of few fat vacuoles. However, increased lymphocytic infiltration and haemolysed RBCs were reported. Hepatocytes showed normal histological hexagonal shape with few fat vacuoles and many secondary lysosomes. SUMMARY: This work was performed to study the light and electron microscopic changes, as well as, enzymatic changes in the liver induced by obesity in male albino rats that fed normal laboratory diet treated byintra-peritoneal injection of Triton WR 1339 "250 mg/kg" for 4 weeks to induce obesity. Sixty male adult albino rats were divided into three groups of 20 rats each. Group I (control group) received normal diet only. Group II (obese group) fed normal diet and received intra-peritoneal Triton WR 1339 for four weeks. Group III (recovery group) as group 2but left another 4 weeks for spontaneous recovery. At the end of the study rat weighed, its serum liver enzymes were assessed and the histological changes in the liver were studied morphologically by light and electron microscopes. The obtained results were analyzed statistically. The obtained results revealed that, rats treated with triton WR 1339 for duration of 4 weeks tended to exhibit obesity features characterized by an increase in the body weight gain, elevated serum liver enzymes and features of NAFLD.The results cleared that there is a highly significant

increase in body weight, liver weight and liver enzymes (ALT, AST and GGT) of rats in obese group when compared to the control one. Also, there is a significant reduction in body weight, liver weight and liver enzymes of recovery group compared to obese group. Our results showed also non-significant increase of collagen area in the liver tissue of obese group when compared with the control one. In addition, there is also non-significant reduction of collagen area in the liver tissue of recovery group when compared to obese group. On the other side, there isa highly significant decrease in optical density of glycogen in obese group when compared to the control one. In addition, there is also a significant increase in optical density of glycogen in recovery group when compared to obese group, however, the optical density in recovery group still lower than that of the control one. Aim of the work: The aim of this work is to study the light and electron microscopic changes, as well as, enzymatic changes in the liver induced by obesity in 60 albino rats (120 ± 5 grams and 5 weeks old) treated by intra-peritoneal injection of Triton "250 mg/kg" for 4 weeks to induce obesity. Materials and methods: this study was carried out at the Faculty of Veterinary Medicine, Alexandria University. 60 male albino rats weighing 120 ± 5 grams and aged 4-5 weeks were divided into three groups (20 rats for each group): Group I (control group)were fed normal laboratory diet for 4 weeks then sacrificed.Group II (obese group)were treated by intra-peritoneal injection of Triton 250 mg/kg to induce obesity and fed normal laboratory diet for 4 weeks then sacrificed. Group III (recovery group) issimilar to the obese group but left without treatment for another 4 weeks (for spontaneous recovery) then sacrificed. Weight of animals was recorded every week. Intra-peritoneal injection of Triton WR 1339 (Tyloxpole)® is done according to Walaa and Saad (2014) (28). At the end of the experiment, blood samples were drawn for liver function tests (ALT, AST and GGT).Rats were eviscerated after decapitation; liver was removed and washed by normal saline then weighted. Small piece were fixed in glutraldehyde for EM examination, the remaining part was fixed in 10% formol saline and sent for histological staining (by H&E, Mallory trichrome and PAS stains) and for LM examination. Results: the results cleared that there is highly significant increase in body weight, liver weight and liver enzymes (ALT, AST and GGT) of rats in obese group when compared to the control one. Also, there is a significant reduction in body weight, liver weight and liver enzymes of recovery group compared to obese group. Our results showed also non-significant increase of collagen area in the liver tissue of obese group when compared with the control one. In addition, there is also non-significant reduction of collagen area in the liver tissue of recovery group when compared to obese group. On the other side, there isa highly significant decrease in optical density of glycogen in obese group when compared to the control one. In addition, there is also a significant increase in optical density of glycogen in recovery group when compared to obese group, however, the optical density in recovery group still lower than that of the control one. Conclusion: there are several stages of NAFLD natural history, ranging from simple steatosis to steatohepatitis (NASH), liver cirrhosis, and finally carcinoma of the liver. The first stage, which is steatosis, is characterized by the presence of lipid inclusion in the liver. In NASH, steatosis is accompanied by inflammatory cells, ballooning of the hepatocytes, and often elevation of liver enzymes. Fibrosis is present when cirrhosis develops and is due to liver cells death.

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biochemistry department, Faculty of Medicine, Tanta University, El-Geish Street, Tanta, El-Gharbia, Egypt

Journal of Food Biochemistry ISSN 1745-4514