Hyperlipidemia is accompanied by elevated serum TC, TG, LDL-C and VLDL-C and decreased HDL-C levels¹. It is associated with cardiovascular diseases (CVD) including coronary heart disease and stroke and is the leading causes of mortality in both developed and developing countries, accounting to 30% of all worldwide deaths per year². Current reports suggest that by the year 2020, India will have the largest cardio vascular disease burden in the world³. Hyperlipidemia is considered as the primary mediator of a cascade of atherosclerosis⁴, pancreatitis, renal injury⁵ and hereditary familial hypercholesterolemia⁶. Athero-sclerosis is a cardio vascular and fibro proliferative inflammatory disease commonly associated with age and ‘dietary- related factors’ in humans. In animals, atherosclerosis is rarely noticed. Understanding of lipid profile, tissue antioxidants and alterations of tissue changes by microscopic methods in induced hyperlipidemia and atherosclerosis helps in the formulation of various therapeutic agents against hyperlipidemia and its associated conditions. Hence the present study has been carried out to evaluate the clinical, serum biochemical, tissue antioxidant and histopathological changes in experimentally induced hyperlipidemia and atherosclerosis in male Wistar rats.

Aorta from atherogenic diet fed group revealed moderate initiation of atherosclerotic lesions with degeneration of endothelial cells, sub intimal lipid laden macrophages (foam cells), slight thickening of the tunica intima with proliferation of few smooth muscle cells were observed in in group II rats (Fig. 1). Neo vascular channels formation and extra vascular fat cells were observed in few cases and in some other sub endothelial thickening of aortic valve along with vacuolation and inflammatory cell were noticed. Dilatation of sub endothelial space by extracellular network containing connective tissue fibers were also seen in few rats. Microthrombus attaching to the endothelium of aorta was observed in few rats by 45^th day of study (Fig. 2). Moderate to severe endothelial degeneration, subendothelial lipid laden macrophages and thickening of tunica intima were observed in majority of atherogenic diet fed rats by the end of 90^th day of study. Disruption of elastic lamina of tunica media with structural and directional changes in the myocytes were also seen in few cases. Atheromatous plaque consists of foam cells, SMCs were observed in the aorta of few rats and in some rats thrombus formation in the aorta was also evidenced by the end of 90th day of study in the same group (Fig. 3).

Hyperlipidemic diet fed rats (Group II) revealed grossly pale and enlarged liver in majority of the rats (Fig. 4). Microscopically, microvesicular hepatic steatosis was observed predominently and macrovesicular fatty change in few rats¹⁷ (Figs. 5, 6). Liver steatosis was absent in control group rats. The spleen of the hyperlipidemic diet fed group II was enlarged and microscopically revealed low red to white pulp ratios compared to the control group I (Fig. 7). Small fat vacuoles were observed in the tubular epithelium of kidney of group II rats fed on hyperlipidemic diet compared to normal control group I (Fig. 8).

Hyperlipidemia induces the obesity with significant increase in body weight and sluggishness with poor hair coat in group II rats¹⁵,¹⁶,¹⁷. It might be due to inclusion of 1% cholesterol and 15% saturated oil present in the hyperlipidemic diet control compared to standard normal rat chew control. Except TLC, all other clinico pathological parameters like TEC, PCV and Hb% were normal in group II. Increased WBC and lymphocytes levels in rabbits that were fed with high cholesterol diet has been reported¹⁸. Increased leukocytes count in the present study might be due to increased level of LDL cholesterol which is responsible for increased viscosity of the blood and thereby resulted into highest TLC¹⁹.

Hyperlipidemia significantly increased the TC, TG, LDL-C and VLDL-C. On the other hand it significantly decreased the HDL-C compared with control rats¹ and it might be due to inclusion of 1% cholesterol and 15% saturated oil to the diet. Low density lipoprotein is a lipoprotein that transports lipids from the liver to the peripheral (extra heaptic) and is often called “bad” cholesterol and constitutes a half to two thirds of cholesterol and high levels of LDLs are highly atherogenic lipoproteins²⁰. High density lipoprotein (HDL) cholesterol is often called “good' because it is a lipoprotein that transports lipids from the periphery to the liver because the HDL can pass through the vascular endothelial cells and into the intima to bring back the accumulated cholesterol in macrophages²¹. HDL particles enhance the net removal of cholesterol from a variety of cells, such as smooth muscle cells, fibroblasts and cholesterol laden macrophages²². HDLs also prevent the oxidation of LDL by virtue of its antioxidant and anti-inflammatory properties²³. The low levels of HDL in the blood will increase the risk of atherosclerosis and coronary heart disease²⁴. Increased atherogenic index indicates increased proportion of bad cholesterol over good cholesterol¹,²⁵.

Increase in TBARS levels in both liver and heart (includes aorta) of group II compared to control rats, may indeed indicates an increased amount of oxidative stress in these rats¹⁶. Hypercholesterolemia induces oxidative stress by causing a reduction in the tissue defense antioxidant enzymes, leading to acceleration of lipid peroxidation, cellular injury, atherosclerosis and heart disease²⁶,²⁷. Hyperlipidemic diet in the present study induced the hypercholesterolemia indicated by increased serum lipid profile and which might have increased the thiobarbutric reacting substances (TBARS) and oxygen radicals in the aorta resulting in endothelial cell injury, modulation of cell adhesion molecules and eventually it mighthave initiated the atherosclerosis and which is evidenced microscopically and ultra-structurally. All other antioxidant enzymes (CAT, SOD, GPx, Reduced glutathione and Glutathione S transferase) activity was reduced in hyperlipidimic diet fed group compared to control group fed on standard diet¹⁵.

Hyperlipidimic diet induced the initiation of atherosclerosis in the aorta with degeneration of endothelial cells, sub intimal lipid laden macrophages (foam cells), slight thickening of the tunica intima with proliferation of few SMCs were observed in group II rats¹,¹⁶. It might be due to oxidation of high level of serum low density lipoprotein - cholesterol (LDL-C). Low density lipoproteins leads to altered gene expression of endothelial cells and transformation of SMCs into proinflammatory intimal layer, there by initiation of atherosclerotic lesions²⁸. Microthrombus attaching to the endothelium of aorta was observed in few rats by 45^th day of study²⁹. Thrombus formation in the aorta of humans described as type VI atherosclerotic lesion³⁰. No atherosclerotic changes was observed in group I rats.

Liver was enlarged and pale grossly with micro and macro vesicular hepatic steatosis¹⁶. Spleen was also enlarged and lymphoid hyperplasia was observed due to low red to white pulp ratio. Hypercholesterolemia increases the viscosity of the blood and also causes activation of the immune system¹⁸ Small fat vacuoles in the epithelium of kidney tubules were observed³¹. All these changes might be due to inclusion of 1% cholesterol and 15% saturated fat to the rat diet.

In conclusion, hyperlipidemic diet of present study was established the hyperlipidemia evidenced by increased TC, TG, LDL-C, VLDL-C and low HDL-C and elevated the total leukocyte count and tissue TBARS levels along with decrease in other cellular anti-oxidant enzymes. Hepatic steatosis and degenerated endothelium with initiated atherosclerotic lesions were evidenced microscopically. Other organs revealed minimal or no changes. Further studies with fully established atherosclerosis in laboratory models will helps in the formulation of various treatment protocols to counter the hyperlipidemia and associated atherosclerosis as this is the leading cause of morbidity and mortality in human beings.

Author is thankful to the authorities of College of Veterinary Science, Tirupati and Sri Venkateswara Veterinary University, Tirupati for extending facilities for carrying experiment.

Mean values with different superscripts differ significantly (P < 0.05)

Mean values with different superscripts differ significantly (P < 0.05)

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