Skip to main content
NCBI home page
Comparative Medicine logoLink to Comparative Medicine
. 2008 Oct;58(5):465–480.

Pathologic Findings in Rabbit Models of Hereditary Hypertriglyceridemia and Hereditary Postprandial Hypertriglyceridemia

Yoko Mitsuguchi 1, Tsunekata Ito 2, Kazuo Ohwada 1,*
PMCID: PMC2707129  PMID: 19004373

Abstract

In recent years, the association between hyperlipidemia and the development of arteriosclerosis has been addressed in several studies. Rabbit models of hypertriglyceridemia (TGH) and postprandial hypertriglyceridemia (PHT) have been developed at the authors' institute. TGH rabbits manifest pathology similar to that of humans with TGH, such as xanthoma, in addition to atherosclerosis of arterioles. Furthermore, PHT rabbits show visceral obesity, insulin resistance, and impaired glucose tolerance, with pathologic features similar to those of the metabolic syndrome assumed to be the cause of human ischemic heart disease. This study was designed to investigate the histopathologic features of TGH and PHT rabbits. TGH rabbits showed advanced aortic atherosclerosis, accompanied by intimal thickening of coronary and renal arteries, fatty liver changes, and xanthoma. PHT rabbits demonstrated aortic intimal thickening and hepatic fatty degeneration. The results of this study suggest that TGH and PHT rabbits are useful animal models for studying human hyperlipidemia and metabolic syndrome and the cardiovascular diseases that result from these conditions.

Abbreviations: LDL, low-density lipoprotein; PHT, postprandial hypertriglyceridemia; TGH, high triglyceride

The 3 leading diseases causing death in humans currently are cancer, cardiac disease (ischemic heart disease), and cerebrovascular accidents. Because the total number of deaths attributed to ischemic heart disease and cerebrovascular accidents exceeds those due to cancer, seeking a remedy for cardiovascular disease is an important task in current medical research.

Cardiac disease and cerebrovascular accidents are caused by arteriosclerosis, and high cholesterol levels play an important role in the onset of this disease.44,46 However, not all patients affected by ischemic heart disease have high cholesterol levels, and hypertriglyceridemia recently has been reported to be associated with the onset of ischemic heart disease.2,12,18,26 Patients with visceral fat accumulation, hypertriglyceridemia, glucose intolerance, and hypertension have high risk for cardiovascular disease.32,41,47 These relationships indicate the importance of determining the mechanism that underlie of the onset of hypertriglyceridemia and to establish prophylactic and therapeutic modalities.

Many animal models, including WHHL rabbits,1,45 KHC rabbits,23 and mice deficient in receptors for low-density lipoproteins (LDL),4 used to study hyperlipemia have innately high cholesterol levels. Among rodent models of hyperlipemia, apoE-deficient mice29 and LDL receptor-deficient mice4 are well known. Rodents produce apoB48-containig lipoproteins in liver, they have high levels of high-density lipoprotein–cholesterol. However, unlike humans (who produce apoB100-containing very-low–density lipoproteins), rodents lack cholesteryl ester transfer protein, which plays a key role in the onset of atherosclerosis, and the underlying mechanism therefore differs from that of human lipoprotein metabolism.5 Therefore the usefulness of comparisons between humans and rodents in regard to lipid metabolism is limited.

In contrast to rodents, rabbits and humans are similar with regard to the chemical composition and content of lipoproteins (including apoB), production of lipoproteins (including apoB100) in the liver, and the presence of cholesteryl ester transfer protein.5 WHHL and KHC rabbits are well known as hypercholesterolemic rabbit models. WHHL rabbits also are prone to developing marked coronary atherosclerosis,37 and WHHLMI rabbits derived from WHHL have symptoms of spontaneous myocardial infarction.38 These models are used in various medical fields such as in the study of the mechanism of lesion onset and development of curative therapy.36,38 Two groups3,25 have developed the SMHL rabbit as a hypertriglyceridemia model. However, neither the triglyceride concentration nor the cholesterol level in the blood of these rabbits exceeded 300 mg/dl even if fatty foods were supplied, and both triglyceride and cholesterol undergo lipolysis by complex mechanisms.6 Therefore, the SMHL rabbit is unsuitable for confirming the influence of triglyceride in cardiac disease and atherosclerosis.

Scientists at the Yamagata University Faculty of Medicine have developed inbred rabbit models of both high hereditary triglyceridemia (the TGH model) and hereditary postprandial hypertriglyceridemic (the PHT rabbit) by selectively mating WHHL rabbits and normal Japanese White (JW) rabbits and screening blood triglycerides values14,15,43 (Figure 1).The hereditary type of TGH shows autosomal recessive inheritance,14,43 and TGH rabbits show blood cholesterol values in excess of 1000 to 1500 mg/dl and triglyceride values greater than 500 mg/dl. In addition, physical examination and necropsy of TGH rabbits often reveal xanthomas and lesions of aortic atherosclerosis similar to those in human disease.9,11

Figure 1.

Figure 1.

Open in a new tab

Breeding schema for TGH and PHT rabbits. TGH rabbits were bred with the progeny of a cross between JW and WHHL. TGH rabbits were generated by selectively crossbreeding animals with plasma triglyceride values of 500 mg/dl or more. In addition, progeny rabbits that showed plasma triglyceride levels of 50 to 100 mg/dl after 24 h of feed deprivation and 500 to 3000 mg/dl after 24 of continuous food supply obtained as a result of breeding TGH (F1) × TGH (F1) were separated into the PHT subpopulation.

The PHT rabbit model was established from the F2 generation of TGH × JW crosses and shows increased postprandial blood levels of cholesterol (100 to 150 mg/dl) and triglyceride (500 to 3000 mg/dl), despite fasting levels similar to those in JW rabbits (cholesterol, 50 to 100 mg/dl; triglyceride, 50 to 100 mg/dl). In addition, the PHT rabbit model shows visceral fat accumulation,14,19 glucose intolerance,13-15,19 insulin resistance,13,14,16,19 and a syndrome very similar to the metabolic syndrome8,47 that is considered to be the origin of human ischemic heart disease. Therefore, the TGH and PHT rabbit models are considered to be extremely useful in studies of lifestyle-related diseases such as hyperlipidemia, obesity, glucose intolerance, and insulin resistance and to identify prophylactic treatments as well as remedies for such conditions.

Thus far, studies of the PHT and TGH rabbit models have been conducted mainly from the viewpoint of clinical and blood biochemistry.13-17,19 This study therefore was designed to investigate the histopathologic findings in the main internal organs of TGH and PHT rabbits.

Materials and Methods

Animals and husbandry.

To investigate the tissue changes during aging of TGH and PHT rabbit models (Oryctolagus cuniculus var. domesticus), this study used male PHT rabbits that were 6, 12, 24, 36, and 48 mo old (n = 1 each), male TGH rabbits that were 6, 12, and 36 mo old (n = 1 each), and, as the control group, male JW rabbits that were 6, 12, and 24 mo old (n = 1 each).

All the animals used for the experiment were bred and maintained under conventional housing conditions and were clinically healthy. The animal room was controlled for temperature at 22 ± 2 °C, humidity at 55% ± 15%, and a 12:12-h light:dark cycle (lights on at 0600), and each animal was housed in a cage measuring 48 cm (width) × 61 cm (length) × 37 cm (height). The animals each received 120 g commercial solid feed (Labo R Grower, Nosan Corporation, Tokyo) at 1230 daily. The nutritional composition and energy value of the feed were: moisture, 7.4% moisture; crude protein, 17.1%; crude fat, 5.4%; crude fiber, 17.1%; crude ash, 9.6%; crude nitrogen-free extract, 43.5%; and metabolizable energy, 2087 kcal/kg. Water was supplied ad libitum by an automatic watering system.

After weaning of the rabbits, cholesterol and triglyceride values were measured once when the animals were 5 to 10 mo old, except for the 48-mo-old PHT rabbit, which was used to determine whether the genotype maintained its phenotypic characteristics. The lipid levels of the PHT rabbit that was maintained until 48 mo of age were measured when it was 19 mo old.

Pathologic specimens were obtained at 6, 12, and 24 mo of age in the JW rabbit; 6, 12, and 36 mo of age in the TGH rabbit; and 6, 12, 24, 36, and 48 mo of age in the PHT rabbit.

This study was carried out with the approval of the Committee on Laboratory Animal Care and Use, Yamagata University Faculty of Medicine.

Measurement of blood cholesterol and triglyceride values.

The plasma cholesterol and triglyceride values of the rabbits used to produce pathology specimens were measured for each animal at only 1 time point between the ages of 5 and 10 mo, except for the 48-mo-old PHT rabbit. The plasma cholesterol and triglyceride values of the PHT rabbit euthanized at 48 mo of age were measured when it was 19 mo old. Blood (1 ml) was collected from ear arteries of conscious, restrained TGH and JW rabbits after 24 h of feed deprivation. Blood samples similarly were obtained from conscious, restrained PHT rabbits after 24 h of feed deprivation and after continuously supplying food for 24 h. To clearly show that the diet fed does not influence the lipid levels of TGH and JW rabbits, they were measured only after 24 h of feed deprivation, although the concentrations do not appear to change regardless of 24 h of feed deprivation or continuously supplying food for 24 h.14 Blood was separated on a cooling centrifuge at 956 × g for 15 min. The cholesterol and triglyceride values of the resulting plasma were measured by using an automated clinical chemistry analyzer (Spotchem EZ SP-4430, Arkray, Kyoto, Japan).

Histopathology.

Before necropsy, the animals were anesthetized with pentobarbital sodium (40 mg/kg), after which the abdominal midline was incised, and the animals were terminally bled from the iliac vein and artery. The desired organs were removed and immediately immersed in 10% neutral buffered formalin, after which various samples were excised (Table 1). Histopathologic specimens were produced from the artery, heart, lungs, liver, kidney, pancreas, and skin of the 11 animals euthanized. Sections of the aortic arch, thoracic aorta, and pulmonary artery were inspected grossly for the presence of atherosclerotic plaques before processing for histopathology. The tissue samples were embedded in paraffin according to established methods and cut into 3-μm slices. All sections were stained with hematoxylin and eosin; sections of blood vessels also were stained with elastica–Goldner to differentially stain elastic fibers for detailed examination.

Table 1.

Samples obtained from JW, TGH, and PHT rabbits used in this study

Organ Sample obtained
Aortic arch Right common carotid artery branch
Thoracic aorta Fifth intercostal artery branch
Abdominal aorta Region including central portion of diaphragm and the branch to the iliac artery
Heart Myocardium and origin and terminus the left coronary artery
Lung Pulmonary lobe and origin of the left pulmonary artery
Liver Upper portion of the right medial lobe
Kidney Region surrounding the renal hilus, including the renal artery, cortex, and medulla
Pancreas Central region
Skin Left hindlimb crus
Open in a new tab

Results

Cholesterol and triglyceride levels in blood.

Cholesterol and triglyceride values were obtained after 24 h of feed deprivation from all study animals at 5 to 19 mo of age; these parameters also were measured in PHT rabbits after 24 h of continuous feed supply. Compared with their JW counterparts, the TGH rabbits euthanized at 6 and 12 mo of age showed remarkably high cholesterol and triglyceride values (Table 2). All of the TGH rabbits used in this study met the screening criteria for TGH (plasma triglyceride level, 500 mg/dl or higher). Blood triglyceride value of TGH rabbits used in this study was 1121 to 2270 mg/dl (Table 2). The fasting cholesterol and triglyceride values of the 24-mo-old PHT rabbit were higher than those of the age-matched JW rabbit. However, fasting cholesterol and triglyceride values did not differ between JW rabbits and any other PHT rabbits (Table 2).

Table 2.

Plasma lipid values of 5- to 19-mo-old rabbits and the tissue changes present at necropsy

Age at necropsy (mo) and strain Cholesterol (mg/dl)
 Triglyceride (mg/dl)
 Tissue changes at necropsy
Weight (kg) After feed deprivation After continuous feed supply After feed deprivation After continuous feed supply Ar AT AA CA L PA RA P S
6 JW 2.85 50 not done 44 not done — — — — — — — — —
12 JW 3.84 21 not done 35 not done — — — — — — — — —
24 JW 4.07 26 not done 43 not done — — — — — — — — —
6 TGH 2.53 1134 not done 1121 not done A A FS IT FL — — — X
12 TGH 2.53 1545 not done 2270 not done A A IT — FL A — — X
36 TGH 2.87 1465 not done 1905 not done A A IT IT — A IT — X
6 PHT 2.87 59 60 45 541 LD — LM — FD — — — —
12 PHT 3.12 46 134 70 1055 — LD LM — — — — — —
24 PHT 3.71 126 256 261 2360 — — IT — — — — — —
36 PHT 3.46 28 76 85 682 FC IT LM — — — — — —
48 PHT 3.43 26 54 45 537 IT IT IT — — — — IL —
Open in a new tab

—, no tissue changes; A, atheroslerosis; Ar, aortic arch; AT, aorta (thoracic); AA, aorta (abdominal); CA, coronary artery; FC, fibrotic change; FD, fatty degeration; FL, fatty liver; FS, fat spot; IL, enlargement of islets of Langerhans; IT, intimal thickening; L, liver; LD, lipid deposit; LM, lipid deposit or mucoid denaturation; P, pancreas; PA, pulmonary artery; RA, renal artery; S, skin; X, xanthoma

See Figures 2 through 10 for examples of histopathology of tissue changes included here.

Pathologic findings in TGH and PHT rabbits.

On gross inspection, artherosclerotic plaques were present in the aortic arch and thoracic aorta of all of the TGH rabbits and in pulmonary artery of 12 and 36 mo of TGH rabbits, and xanthomas were present the skin of all of the TGH rabbits. All PHT and JW rabbits and remaining sampled organs of TGH rabbits were free of lesions on gross postmortem examination.

Aortic arch (Figure 2).

Figure 2.

Figure 2.

Open in a new tab

Pathologic findings in the aortic arch of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. White arrow, smooth muscle cells; black arrow, macrophages; gray arrowhead, lipid core; C, calcification; *, internal elastic lamina; gray arrow, lipid deposit; black arrowhead, fragmentation or disappearance of the internal elastic lamina; and white arrowhead, fibroid change. Elastica Goldner stain; bar = 100 μm (A through C, G through K), 200 μm (D through F).

The TGH rabbit euthanized at 6 mo of age showed foam cells, accumulations of smooth muscle cells in the intima, calcification, and fragmentation of the internal elastic lamina of the aortic arch. The TGH rabbit necropsied at 12 mo of age showed smooth muscle cells, foam cells, and a lipid core in the intima, in addition to lesions of atherosclerosis. The TGH rabbit sampled at 36 mo also showed smooth muscle cells, foam cells, and a lipid core in the intima, calcification and fragmentation of the internal elastic lamina, and lesions of atherosclerosis.

The 6-mo PHT rabbit showed lipid deposition under the endothelium, and 36-mo PHT animal showed fibrotic changes in the intima. The PHT rabbit necropsied at 48 mo showed an increase in smooth muscle cells vertically from the lumen of the vessel toward the tunica media as well as intimal thickening was observed.

None of the JW rabbits (euthanized at 6, 12, and 24 mo of age) showed any abnormal histologic changes in the aortic arch.

Thoracic aorta (Figure 3).

Figure 3.

Figure 3.

Open in a new tab

Pathologic findings in the thoracic aorta of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. White arrow, smooth muscle cells; black arrow, macrophages; gray arrowhead, lipid core; C, calcification; *, internal elastic lamina; gray arrow, lipid deposit; black arrowhead, fragmentation or disappearance of the internal elastic lamina; and white arrowhead, fibroid change. Elastica Goldner stain; bar = 100 μm (A through C, G through K), 200 μm (D through F).

The TGH rabbit necropsied at 6 mo old showed accumulations of foam cells in intima, calcification, and fragmentation of the internal elastic lamina of the thoracic aorta, whereas TGH rabbits euthanized at 12 and 36 mo had foam cells, a lipid core, and smooth muscle cells in the intima and lesions of atherosclerosis.

The PHT rabbit euthanized at 12 mo old had tissue changes considered to be lipid deposition, and the animal necropsied at 36 mo old showed multilayered elastic fibers, which were parallel to the internal elastic lamina, smooth muscle cells in the intima, and intimal thickening. The 48-mo PHT rabbit showed multilayered elastic fibers and smooth muscle cells in the intima and intimal thickening.

Abdominal aorta (Figure 4).

Figure 4.

Figure 4.

Open in a new tab

Pathologic findings in the abdominal aorta of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. White arrow, smooth muscle cells; black arrow, macrophages; *, internal elastic lamina; gray arrow, lipid deposit; and white arrowhead, fibroid change. Elastica Goldner stain; bar, 100 μm.

The TGH rabbit euthanized when 6 mo old showed foam cells, and the one necropsied at 12 mo showed multilayered elastic fibers in the intima and intimal thickening. The TGH rabbit euthanized at 36 mo demonstrated multilayered elastic fibers, smooth muscle cells in the intima, and intimal thickening; however, no lesions of atherosclerosis were found in the aortic arch or thoracic aorta in any rabbit.

The PHT rabbits necropsied at 6, 12, and 36 mo showed tissue changes considered to be lipid deposition or mucoid denaturation, and the PHT rabbits euthanized at 24 and 48 mo of age showed increased smooth muscle cells in the intima and intimal thickening.

JW rabbits lacked noteworthy changes in the abdominal aorta.

Left coronary artery and myocardium (Figure 5).

Figure 5.

Figure 5.

Open in a new tab

Pathologic findings in the heart (coronary arteries) of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. White arrow, smooth muscle cells; and *, internal elastic lamina. Elastica Goldner stain; bar, 100 μm.

No coronary artery lesions were present in the TGH rabbit evaluated at 12 mo of age, but the 6- and 36-mo TGH rabbits showed smooth muscle cells in the intima and intimal thickening, although less than that in the aorta. The coronary artery tissues of all PHT and JW rabbits lacked noteworthy changes. None of the JW, PHT, and TGH animals had any myocardial changes (data not shown).

Pulmonary artery and lungs (Figure 6).

Figure 6.

Figure 6.

Open in a new tab

Pathologic findings in the pulmonary artery of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. Black arrow, macrophages; gray arrowhead, lipid core; C, calcification; and *, internal elastic lamina. Elastica Goldner stain; bar = 100 μm (A through C, G through K), 200 μm (D through F).

The TGH rabbit evaluated at 12 mo of age showed accumulations of atherosclerotic lesions, foam cells, and a lipid core in the proximal pulmonary artery. The 36-mo TGH rabbit showed lesions of atherosclerosis, including calcification, foam cells, and a lipid core in part of the proximal section of the pulmonary artery.

Samples of the pulmonary artery from all PHT and JW rabbits were free of histologic change. None of the JW, TGH, or PHT animals showed any alveolar changes (data not shown).

Liver (Figure 7).

Figure 7.

Figure 7.

Open in a new tab

Pathologic findings in the liver of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. White arrow, lipid droplet. Hematoxylin and eosin stain; bar, 100 μm.

The 6-mo TGH rabbit showed diffuse fatty degeneration throughout the liver. The TGH rabbit euthanized at 12 mo of age had more small and large lipid droplets in hepatocytes than those seen in 6-mo sample. The TGH rabbit euthanized at 12 mo of age also had advanced fatty liver, but the 36-mo TGH rabbit showed no hepatic changes.

The PHT rabbit necropsied when 6 mo old showed lipid droplets in hepatocytes, and the cell nucleus was compressed by lipid droplets. Neither the JW rabbit nor the PHT rabbit at 12 mo of age or older showed any tissue changes.

Renal artery (Figure 8).

Figure 8.

Figure 8.

Open in a new tab

Pathologic findings in the renal artery of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. Black arrow, smooth muscle cells; *, internal elastic lamina; and gray arrow, lipid deposit. Elastica Goldner stain; bar, 100 μm.

The TGH rabbit evaluated at 36 mo showed lipid deposition and smooth muscle cells in the intima of the renal artery and intimal thickening; no other TGH, JW, or PHT animal had any histologic change in the renal artery.

Pancreas (Figure 9).

Figure 9.

Figure 9.

Open in a new tab

Pathologic findings in the pancreas (islet cells) of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. Hematoxylin and eosin stain; bar, 200 μm.

The only animal with any observable difference in pancreas was the 48-mo PHT rabbit, which showed partially enlarged islets of Langerhans; all other PHT, JW, and TGH animals were free of histologic change.

Skin (Figure 10).

Figure 10.

Figure 10.

Open in a new tab

Pathologic findings in the skin of JW, TGH, and PHT rabbits. (A) JW, 6 mo old. (B) JW, 12 mo old. (C) JW, 24 mo old. (D) TGH, 6 mo old. (E) TGH, 12 mo old. (F) TGH, 36 mo old. (G) PHT, 6 mo old. (H) PHT, 12 mo old. (I) PHT, 24 mo old. (J) PHT, 36 mo old. (K) PHT, 48 mo old. Black arrow, macrophages. Hematoxylin and eosin stain; bar, 200 μm.

The TGH rabbits euthanized at 6, 12, and 36 mo of age showed accumulations of foam cells in the dermis. PHT and JW rabbits had no dermal lesions.

Table 2 summarizes the tissue changes detected by histology according to the animal from which the samples were obtained.

Discussion

The study of lipidosis in humans has used animal models with characteristics of hypercholesterolemia.28 In recent years, the relationship between hypertriglyceridemia and the onset of human cardiovascular disease has been demonstrated strongly;2,12,18,26 however, no hypertriglyceridemic animal model suitable for use in basic research was available.

TGH rabbits have high plasma cholesterol and triglyceride levels and show lesions of atherosclerosis and dermal xanthoma. PHT rabbits show hypertriglyceridemia, visceral fat accumulation, and insulin resistance, which are features of metabolic syndrome. Arteriosclerosis, fatty liver, xanthoma, obesity, and insulin resistance have been reported to be the complications of hyperlipemia in humans.8,9,11,47 Therefore the same changes of fatty liver, arteriosclerosis, intimal thickening, and xanthoma shown by TGH and PHT rabbits in this study develop in human hyperlipidemia and metabolic syndrome and contribute to cardiovascular disease.8,9,11,47 Therefore we consider that TGH and PHT rabbits are extremely useful animal models for human hyperlipidemia, metabolic syndrome, and subsequent cardiovascular disease.

Atherosclerosis forms a complex plaque from a fatty streak or clump of intimal cells. After formation of a complex plaque, a thrombus forms, vascular closure is initiated, and vascular disease (myocardial infarction, cerebral infarction) occurs. Calcification in the inner fibrotic membrane and atheroma are characteristics of a complex plaque.10 Because they have smooth muscle cells, lipid, and calcification in the vascular intima (that is, atheromas) of the aortic arch (Figure 2) as well as atherosclerotic lesions and intimal thickening in the pulmonary artery (Figure 6) and renal artery (Figure 8), TGH rabbits are considered to show advanced lesions of atherosclerosis. Further, the tissue changes in the TGH rabbits were similar to those found on histology of samples from WHHL rabbits with high cholesterol.30,31,39 The organizing region of atherosclerosis in the aorta of TGH rabbits was similar to that in WHHL rabbits.20,30,39

Lesions of atherosclerosis progress to advanced lesions with fatty streaks and fibrous changes and subsequent calcification and thrombus formation, 10thus causing constriction or occlusion of blood vessels. Lesions result from a process whereby cholesterol, as LDL, is released into the blood; LDL in the blood undergoes oxidative stress, denatured oxidized LDL leads to the formation of foam cells from macrophages, and foam cells are deposited as a layer of the lipid streak. Because oxidized LDL causes endothelial dysfunction,22 cholesterol is considered to play an important role in the pathogenesis of arteriosclerosis. In addition, hyperlipidemia is a key factor in the transition from macrophage to foam cell.34,35 In the current study, progressive lesions of atherosclerosis were present in the intima of the aortic arch and thoracic aorta in the TGH rabbit ; in contrast, PHT rabbits with high postprandial triglyceride levels lacked progressive lesions with prominent foam cells. We therefore consider that TGH rabbits mimic features of high cholesterol conditions in humans.

Atherosclerotic plaques have a thick fibrous cap consisting of many collagen fibers and smooth muscle cells. This structure is stable and does not readily form an occluding thrombus. However, when a large lipid core and thinning of the fibrous cap are accompanied by many macrophages and other inflammatory cells just under or over the cap surface, the plaque becomes unstable, cracks or bursts, and thus forms an occluding thrombus.42 In particular, macrophages secrete a protein catabolic enzyme that renders the plaque brittle.33 The lesions of atherosclerosis in TGH rabbits likely also represent the influence of high blood pressure, turbulent bloodflow, and so forth. Because smooth muscle cells were plentiful in the lower layer of the intima but sparse in the upper layer and because accumulations of foam cells were present in the upper layer of the intima, we suspect that TGH rabbits tend to form unstable plaques. We propose that the high cholesterol and triglyceride levels of TGH rabbits are important factors of the atherosclerosis in these animals.

Increased triglyceride levels and decreased levels of high-density lipoprotein–cholesterol are also the defining components of the dyslipidemia in fatty liver: if the lipid level in the blood is elevated, fat will be stored in the liver. This phenomenon is involved in human hyperlipidemia.9,47 Because TGH rabbits are hypercholesterolemic and hypertriglyceridemic, we speculate that advanced fatty liver (Figure 7) results because these lipid levels exceed the metabolic capacity of the liver and thus results in lipid accumulation in the liver. TGH rabbits of 6 to 12 mo old have high lipid levels in the blood (cholesterol, 1000 to 1500 mg/dl; triglyceride, 1000 to 10,000 mg/dl);14,15 these levels gradually decrease with age, such that the TGH rabbit euthanized when 36 mo old did not show any hepatic changes. We therefore hypothesize that, although lipid that cannot be metabolized forms lipid droplets in liver cells in young rabbits with a high blood lipid level, the age-associated decrease in serum lipid decreases the amount of uptake of lipid by the liver, and the lipid droplets in hepatic cells decreases as the accumulated lipid is metabolized gradually by the liver. Therefore, we assume that the age-associated difference in the blood lipid level is related to the accumulation of lipid in liver cells.

Foam cells accumulated as partial xanthomas in the skin of TGH rabbit models (Figure 10). Mating WHHL rabbits showing high serum cholesterol due to a deficient LDL receptor 37 with normal JW rabbits yielded the TGH rabbit (Figure 1), whose high cholesterol level likewise is considered to be mediated by a deficient LDL receptor. In contrast, PHT rabbits, which have lower serum cholesterol levels than do TGH rabbits, do not develop xanthoma. We surmise that xanthomas form after macrophages, which acquire LDL from the blood thus causing foaming, permeate into the dermis. Therefore, the propensity of TGH rabbits to develop xanthomas is strongly influenced by their high serum cholesterol concentrations.

The vascular lesions of TGH rabbits contained foam cells, whereas those of PHT rabbits did not. The findings from the TGH rabbits lead us to speculate that their high cholesterol levels play a greater role in the formation of lesions as they do in the WHHL rabbit model.30,31,39 Because of the findings from the PHT rabbits, we speculate that their postprandial hypertriglyceridemia plays an important role in the vascular changes seen because these animals also showed fatty liver and intimal thickening, although the contributions of insulin resistance and visceral obesity to lesion formation in PHT rabbits cannot be discounted.

High cholesterol is thought to be associated with the onset of human cardiovascular disease.44,46 In recent years, however, it has become clear that high triglyceride also plays an important role in the onset of cardiovascular disease.2,12,18,26,41,47 Therefore, clarifying the characteristics of lipid metabolism and the basic histopathology of PHT rabbit models will facilitate the elucidation of the morbid state of cardiovascular disease caused by abnormal lipid metabolism in humans.

In humans intimal thickening and lipid deposition in blood vessels increases with age,40 and the tissue changes in the PHT rabbit model demonstrated a similar age-associated pattern. For example, smooth muscle cells increased in the intimal thickening of the aorta in the PHT rabbit model, similar to the early lesions of human atherosclerosis.24 Metabolic syndrome is associated with carotid intimal and medial wall thickness.27 Because the tissue changes occurred in PHT rabbits showing postprandial hypertriglyceridemia, insulin resistance, and visceral obesity, we suspect that these conditions contribute to the early changes of arteriosclerosis in the intima of the vessel walls of PHT rabbits.

Insulin resistance plays an important role in the accumulation of triglyceride in liver cells, and visceral fat is a risk factor for fatty liver.7 PHT rabbits show glucose intolerance, insulin resistance, and visceral obesity, as well as high postprandial triglyceride levels in blood in PHT rabbits of 6 or 9 mo old (approximately 1400 to 1800 mg/dl).14,43 The fatty changes in the hepatic tissue of PHT rabbits necropsied at 6 mo of age likely reflect these influences. However, blood triglyceride levels in PHT rabbits decrease gradually with age,14 consistent with the current finding that PHT rabbits 12 mo or older at necropsy showed no histologic changes in liver samples. We therefore hypothesize that the age-associated decrease in the blood lipid level of PHT rabbits decreases lipid assimilation in the liver, with gradual metabolism of lipid droplets accumulated earlier in life.

This study has demonstrated that the lesions of atherosclerosis that develop in the main arteries of TGH rabbits are histologically similar to those in humans. In addition, the increase of smooth muscle cells in the intima of the main arteries, which was observed in PHT rabbit models, is similar to the changes associated with early lesions of atherosclerosis in humans. 24Although both TGH and PHT rabbits showed intimal thickening and arteriosclerosis in the main arteries, these changes were not present in the coronary and renal arteries of the rabbits as they are in humans. 24Likewise, the fatty degeneration of the liver seen in the TGH and PHT models and the dermal xanthoma of the TGH model are similar to tissue changes found in human hyperlipidemia. In addition, the blood lipid level decreases gradually with age in humans;21 similarly, the high blood lipid levels of TGH and PHT rabbits decreased gradually after peaking at 6 to 12 mo and 6 to 9 mo, respectively.

Based on our experience of these lines, the lifespans of the PHT and TGH rabbits are approximately 7 y and 3 to 4 y, respectively; therefore, these animals can be used for long-term research. Because the TGH and PHT genotypes and phenotypes are inbred, researchers can select rabbits of the appropriate age for observing pathologic changes, lipid values in blood, and so on, in accordance with their research purpose.

Acknowledgments

We would like to thank Professor M Yamakawa, H Saito, and Mr K Suzuki (First Department of Pathology, Yamagata University Faculty of Medicine), who instructed us in the production of pathology specimens and the diagnosis of pathologic lesions.

We also thank all the staff of the Laboratory Animal Center (Yamagata University Faculty of Medicine), who instructed and helped with the breeding and selection of animals.

References

  • 1.Aliev G, Burnstock G. 1998. Watanabe rabbits with heritable hyper-cholesterolaemia: a model of atherosclerosis. Histol Histopathol 13:797–817 [DOI] [PubMed] [Google Scholar]
  • 2.Assmann G, Schulte H, von Eckardstein A. 1996. Hypertriglyceridemia and elevated lipoprotein (a) are risk factors for major coronary events in middle-aged men. Am J Cardiol 77:1179–1184 [DOI] [PubMed] [Google Scholar]
  • 3.Beaty TH, Prenger VL, Virgil DG, Lewis B, Kwiterovich PO, Bachorik PS. 1992. A genetic model for control of hypertriglyceridemia and apolipoprotein B levels in the Johns Hopkins colony of St. Thomas Hospital rabbits. Genetics 132:1095–1104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bird DA, Tangirala RK, Fruebis J, Steinberg D, Witztum JL, Palinski W. 1998. Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice. J Lipid Res 39:1079–1090 [PubMed] [Google Scholar]
  • 5.Brousseau ME, Hoeg JM. 1999. Transgenic rabbits as models for atherosclerosis research. J Lipid Res 40:365–375 [PubMed] [Google Scholar]
  • 6.De Roos B, Caslake MJ, Milliner K, Martin GB, Suckling KE, Packard CJ. 2005. Characterisation of the lipoprotein structure in the St. Thomas' Mixed Hyperlipidaemic (SMHL) rabbit. Atherosclerosis 181:63–68 [DOI] [PubMed] [Google Scholar]
  • 7.Festi D, Colecchia A, Sacco T, Bondi M, Roda E, Marchesini G. 2004. Hepatic steatosis in obese patients: clinical aspects and prognostic significance. Obes Rev 5:27–42 [DOI] [PubMed] [Google Scholar]
  • 8.Grundy SM, Brewer HB, Jr, Cleeman JI, Smith SC, Jr, Lenfant C. 2004. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition. Arterioscler Thromb Vasc Biol 24:e13–e18 [DOI] [PubMed] [Google Scholar]
  • 9.Harada-Shiba M, Takagi A, Miyamoto Y, Tsushima M, Ikeda Y, Yokoyama S, Yamamoto A. 2003. Clinical features and genetic analysis of autosomal recessive hypercholesterolemia. J Clin Endocrinol Metab 88:2541–2547 [DOI] [PubMed] [Google Scholar]
  • 10.Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. 1995. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol 15:1512–1531 [DOI] [PubMed] [Google Scholar]
  • 11.Hodis HN, Mack WJ, Azen SP, Alaupovic P, Pogoda JM, LaBree L, Hemphill LC, Kramsch DM, Blankenhorn DH. 1994. Triglyceride- and cholesterol-rich lipoproteins have a differential effect on mild/moderate and severe lesion progression as assessed by quantitative coronary angiography in a controlled trial of lovastatin. Circulation 90:42–49 [DOI] [PubMed] [Google Scholar]
  • 12.Hokanson JE, Austin MA. 1996. Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies. J Cardiovasc Risk 3:213–219 [PubMed] [Google Scholar]
  • 13.Ito T, Ohwada K, Tomoike H. 2005. A hereditary postprandial hypertriglyceridemic (PHT) rabbit model. The 1st International Conference on Transgenic Rabbits; Jun 18; Tsukuba Japan: The International Conference on Transgenic Rabbits; c2005. Abstracts 8 [DOI] [PubMed] [Google Scholar]
  • 14.Ito T, Ohwada K, Endo J, Tomoike H. 2005. [A hereditary postprandial hypertriglyceridemic (PHT) rabbit model. Nippon Yakurigaku Zasshi]. 125:301–306 [DOI] [PubMed] [Google Scholar]
  • 15.Ito T, Ohwada K, Endo J, Kamimura E, Tomoike H. 2003. [Postprandial hypertriglyceridemic rabbit as a new lipidosis model]. New Development of Experimental Rabbit to Contribute to Medical Treatment; Mar 3; Saga Japan: New Development of Experimental Rabbit to Contribute to Medical Treatment; Abstracts 12-13 [Google Scholar]
  • 16.Ito T, Hata M, Mitsuguchi Y, Ohwada K, Shiba M, Tomoike H. 2005. Usual plasma lipid and insulin level of hereditary postprandial hypertriglyceridemic (PHT) rabbit. The 39th meeting of Japanese Association for Experimental Animal Technologists; Jun 25; Kanazawa Japan: JAEAT; c2005. Abstracts 113. [Google Scholar]
  • 17.Ito T, Hata M, Mitsuguchi Y, Ohwada K, Tomoike H. 2005. [Basic examination of TG production and disassembly on postprandial hypertriglyceridemic (PHT) rabbit model]. The 52nd Annual Meeting of the Japanese Association for Laboratory Animal Science; May 20; Tokyo Japan: JALAS; c2005. Abstracts 122. [Google Scholar]
  • 18.Jeppesen J, Hein HO, Suadicani P, Gyntelberg F. 1998. Triglyceride concentration and ischemic heart disease: an eight-year follow-up in the Copenhagen Male Study. Circulation 97:1029–1036 [DOI] [PubMed] [Google Scholar]
  • 19.Kawai T, Ito T, Ohwada K, Mera Y, Matsushita M, Tomoike H. 2006. Hereditary postprandial hypertriglyceridemic rabbit exhibits insulin resistance and central obesity. A novel model of metabolic syndrome. Arterioscler Thromb Vasc Biol 26:2752–2757 [DOI] [PubMed] [Google Scholar]
  • 20.Koike T, Liang J, Wang X, Ichikawa T, Shiomi M, Sun H, Watanabe T, Liu G, Fan J. 2005. Enhanced aortic atherosclerosis in transgenic Watanabe heritable hyperlipidemic rabbits expressing lipoprotein lipase. Cardiovasc Res 65:524–534 [DOI] [PubMed] [Google Scholar]
  • 21.Kozaki K. 2007. [Management of hyperlipidemia in the elderly] Nippon Ronen Igakkai Zasshi 44:181–184 [DOI] [PubMed] [Google Scholar]
  • 22.Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD. 1990. Impairment of endothelium-dependent arterial relaxation by lysoleci- thin in modified low-density lipoproteins. Nature 344:160–162 [DOI] [PubMed] [Google Scholar]
  • 23.Kurosawa T, Kusanagi M, Yamasaki Y, Senga Y, Yamamoto T. 1995. New mutant rabbit strain with hypercholesterolemia and atherosclerotic lesions produced by serial inbreeding. Lab Anim Sci 45:385–392 [PubMed] [Google Scholar]
  • 24.Lavezzi AM, Ottaviani G, Matturri L. 2005. Biology of the smooth muscle cells in human. Atherosclerosis. APMIS 113:112–121 [DOI] [PubMed] [Google Scholar]
  • 25.La Ville A, Turner PR, Pittilo RM, Martini S, Marenah CB, Rowles PM, Morris G, Thomson GA, Woolf N, Lewis B. 1987. Hereditary hyperlipidemia in the rabbit due to overproduction of lipoproteins. I. Biochemical studies. Arteriosclerosis 7:105–112 [DOI] [PubMed] [Google Scholar]
  • 26.Malloy MJ, Kane JP. 2001. A risk factor for atherosclerosis: triglyceride-rich lipoproteins. Adv Intern Med 47:111–136 [PubMed] [Google Scholar]
  • 27.McNeill AM, Rosamond WD, Girman CJ, Heiss G, Golden SH, Duncan BB, East HE, Ballantyne C. 2004. Prevalence of coronary heart disease and carotid arterial thickening in patients with the metabolic syndrome (The ARIC Study). Am J Cardiol 94:1249–1254 [DOI] [PubMed] [Google Scholar]
  • 28.Moghadasian MH. 2002. Experimental atherosclerosis: a historical overview. Life Sci 70:855–865 [DOI] [PubMed] [Google Scholar]
  • 29.Moghadasian MH, McManus BM, Godin DV, Rodrigues B, Frohlich JJ. 1999. Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E-deficient mice: possible mechanisms of action. Circulation 99:1733–1739 [DOI] [PubMed] [Google Scholar]
  • 30.Murakami S, Kondo Y, Sakurai T, Kitajima H, Negate T. 2002. Taurine suppresses development of atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbits. Atherosclerosis 163:79–87 [DOI] [PubMed] [Google Scholar]
  • 31.Ogawa M, Ishino S, Mukai T, Asano D, Teramoto N, Watabe H, Kudomi M, Shiomi M, Magata Y, Iida H, Saji H. 2004. (18)F-FDG accumulation in atherosclerotic plaques: Immunohistochemical and PET imaging study. J Nucl Med 45:1245–1250 [PubMed] [Google Scholar]
  • 32.Olijhoek JK, Martens FM, Banga JD, Visseren FL. 2005. [The metabolic syndrome: a cluster of vascular risk factors] Ned Tijdschr Geneeskd 149:859–865 [PubMed] [Google Scholar]
  • 33.Pakala R, Leborgne L, Cheneau E, Chan RC, Yazdi H, Fournadjiev J, Weber D, Hellinga D, Kolodgie F, Virmani R, Waksma R. 2003. Radiation induced atherosclerotic plaque progression in a hypercholesterolemic rabbit: a prospective vulnerable plaque model? Cardiovasc Radiat Med 4:146–151 [DOI] [PubMed] [Google Scholar]
  • 34.Schwartz CJ, Kelley JL, Nerem RM, Sprague EA, Rozek MM, Valente AJ, Edwards EH, Prasad AR, Kerbacher JJ, Logan SA. 1989. Pathophysiology of the atherogenic process. Am J Cardiol 64:23G–30G [DOI] [PubMed] [Google Scholar]
  • 35.Schwartz CJ, Valente AJ, Sprague EA, Kelley JL, Nerem RM. 1991. The pathogenesis of atherosclerosis: an overview. Clin Cardiol 14 Suppl 1:I1–I16 [DOI] [PubMed] [Google Scholar]
  • 36.Shiomi M, Yamada S, Ito T. 2005. Atheroma stabilizing effects of simvastatin due to depression of macrophages or lipid accumulation in the atheromatous plaques of coronary plaque-prone WHHL rabbits. Atherosclerosis 178:287–294 [DOI] [PubMed] [Google Scholar]
  • 37.Shiomi M, Ito T, Shiraishi M, Watanabe Y. 1992. Inheritability of atherosclerosis and the role of lipoproteins as risk factors in the development of atherosclerosis in WHHL rabbits: risk factors related to coronary atherosclerosis are different from those related to aortic atherosclerosis. Atherosclerosis 96:43–52 [DOI] [PubMed] [Google Scholar]
  • 38.Shiomi M, Ito T, Yamada S, Kawashima S, Fan J. 2003. Development of an animal model for spontaneous myocardial infarction (WHHLMI rabbit). Arterioscler Thromb Vasc Biol 23:1239–1244 [DOI] [PubMed] [Google Scholar]
  • 39.Shiomi M, Ito T, Tsukada T, Yata T, Watanabe Y, Tsujita Y, Fukami M, Fukushige J, Hosokawa T, Tamura A. 1995. Reduction of serum cholesterol levels alters lesional composition of atherosclerotic plaques. Effect of pravastatin sodium on atherosclerosis in mature WHHL rabbits. Arterioscler Thromb Vasc Biol 15:1938–1944 [DOI] [PubMed] [Google Scholar]
  • 40.Sumiyoshi A, Kurozumi T. 1980. Atherosclerosis in the pediatric age with special reference to initiation, progression and regression of atherosclerotic lesions. Jpn Circ J 44:39–45 [DOI] [PubMed] [Google Scholar]
  • 41.Tada N, Yoshida H. 2007. [Clinical significance of triglyceride-rich lipoproteins in metabolic syndrome] Rinsho Byori 55:439–446 [PubMed] [Google Scholar]
  • 42.Teramoto T. 2003. Hyperlipidaemia. Teramoto T,editors Inflammation and arteriosclerosis. JMEDJ,Inc. P26-27 [Google Scholar]
  • 43.Tomoike H, Ohwada K, Ito T.inventorsTomoike H, Ohwada K, Ito T.patent holder 2003. Hereditary postprandial hyper-triglyceridemic rabbit model. United States patent US 6,515,196 B2. [Google Scholar]
  • 44.Toshima S, Hasegawa A, Kurabayashi M, Itabe H, Takano T, Sugano J, Shimamura K, Kimura J, Michishita I, Suzuki T, Nagai R. 2000. Circulating oxidized low density lipoprotein levels. A biochemical risk marker for coronary heart disease. Arterioscler Thromb Vasc Biol 20:2243–2247 [DOI] [PubMed] [Google Scholar]
  • 45.Watanabe Y. 1980. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit). Atherosclerosis 36:261–268 [DOI] [PubMed] [Google Scholar]
  • 46.Witztum JL. 1993. Role of oxidised low density lipoprotein in atherogenesis. Br Heart J 69(1 Suppl:S12–S18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Yuan G, Al-Shali KZ, Hegele RA. 2007. Hypertriglyceridemia: Its etiology, effects and treatment. CMAJ 176:1113–1120 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Comparative Medicine are provided here courtesy of American Association for Laboratory Animal Science

RESOURCES