Planimetric and Histological Study of the Aortae in Atherosclerotic Chickens Treated with Nifedipine, Verapamil and Diltiazem
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Histol Histopathol (2003) 18: 1027-1033 Histology and http://www.hh.um.es Histopathology Cellular and Molecular Biology Planimetric and histological study of the aortae in atherosclerotic chickens treated with nifedipine, verapamil and diltiazem B. García Pérez1, I. Ayala2, M.T. Castells3, J.F. Madrid3, M.R. Ortega1, J.V. Ortega4, J. Ballesta,3 J. Fernández Pardo5 and M. Valdes1 1Clinical Universitary Hospital, Virgen de la Arrixaca, 2Department of Veterinary Medicine, College of Veterinary Medicine, University of Murcia, Campus de Espinardo, 3Department of Cell Biology, Medical School, University of Murcia, 4Los Arcos Hospital, Santiago de la Ribera and 5General Hospital, Murcia, Spain Summary. Calcium appears to be involved in many of Introduction the cellular events which are thought to be important in atherogenesis. Calcium channel blockers have been Atherosclerosis and its consequences continue to be shown to reduce arterial lipid accumulation in animals the major cause of death in Europe and the United States without altering serum cholesterol. Avian models of (Sans et al., 1997; Kesteloot et al., 2002). It appears to atherosclerosis offer economic and technical advantages develop over much of the lifetime of a patient, and is over mammalian models. related to well-known risk factors, especially heredity, In this study, we examine the effects of nifedipine, hyperlipidemia, hypertension, cigarette smoking and verapamil and diltiazem at clinical and higher doses, on diabetes mellitus (Peeters et al., 2002). the extent of atherosclerosis of egg-fed chickens. In Animals have been used as experimental models in order to assess the extent of atherosclerosis atherosclerosis-related research since the turn of the past quantitatively, the aortic lesions of the thoracic and century (Narayanaswamy et al., 2000). Avian models of abdominal aorta, aortic arch and supraaortic regions atherosclerosis helped pioneer the study of vascular were measured by planimetry. Atherosclerotic lesions biology, and offer economic and technical advantages were evaluated histologically. Statistically significant over mammalian models (Wang et al., 1999). The egg- reductions in the lipid deposition of the aorta were found fed chicken is a good animal model for the study of in all the treated groups. The extent and distribution of atherosclerosis research (Siller, 1961; Gosling et al., atherosclerotic lesions were decreased in a significant 1969; Valdés, 1976; García Pérez et al., 2002). The way by verapamil, nifedipine and diltiazem. The higher chicken is small and suitable for prolonged laboratory the dosage used, the higher the regression of the investigation; able to develop spontaneous atherosclerotic lesions. At clinical dosage, nifedipine atherosclerosis; capable of producing atherosclerosis showed the highest decrease of the lesions. In addition, after cholesterol feeding with elevated the chicken atherosclerosis model has proved itself hypercholesterolemia and there is no essential difference useful and very suitable for in vivo drug intervention between vascular lesions seen in chickens as a result of studies. cholesterol diet and that of atherosclerosis observed in man (Wong, 1975). The rabbit, also free of spontaneous Key words: Atherosclerosis, Nifedipine, Verapamil, atherosclerosis, is extremely sensitive to lipid-rich diets, Diltiazem, Chicken but the lesions induced resemble more a xanthomatosis than an atherosclerosis (Hadjiisky et al., 1991). The monkey and pig, which are phylogenetically close to man, develop spontaneous atherosclerosis exacerbated by lipid-rich diets or other procedures, but the cost and problems of upkeep make these two models inaccessible to most laboratories (Hadjiisky et al., 1991). The utility Offprint requests to: Dr. Ignacio Ayala, Department of Veterinary of transgenic mouse models of atherosclerosis, such as Medicine, College of Veterinary Medicine, University of Murcia, Campus the apolipoprotein E (apoE)-deficient mouse and the low de Espinardo s/n, 30100 Murcia, Spain. Fax: 34-968-364147. e-mail: density lipoprotein (LDL) receptor-negative mouse, for [email protected] the evaluation of antiatherosclerotic agents has yet to be 1028 Effect of calcium antagonists on chicken atherosclerosis determined. Paradoxical observations raise an important given ad libitum. issue relating to interpretation of the results of drug Each of the 8 groups consisted of 10 animals which intervention studies in genetically-derived mouse models were treated as follows. Group A: normal diet. Group B: (Bocan, 1998). atherogenic diet. Group C: atherogenic diet and Calcium appears to be involved in many of the nifedipine at clinical doses (3 mg/kg/day in two doses). cellular events, which are thought to be important in Group D: atherogenic diet and nifedipine at high doses atherogenesis (Rouleau et al., 1983; Catapano, 1997; (30 mg/kg/day in two doses). Group E: atherogenic diet Miller, 2001; Pepine and Handberg, 2001). Compounds and verapamil at clinical doses (4 mg/kg/day in two which decrease intracellular calcium by reducing doses). Group F: atherogenic diet and verapamil at high calcium influx have been shown to suppress doses (40 mg/kg/day in two doses). Group G: atherogenesis in experimental atherosclerosis (Rouleau atherogenic diet and diltiazem at clinical doses (6 et al. 1983, Ginsburg et al. 1983; Catapano, 1997). mg/kg/day in two doses). Group H: atherogenic diet and Hypocalcemic agents, calcium channel blockers and diltiazem at high doses (60 mg/kg/day in two doses). other calcium antagonists have been shown to reduce For all experiments, the calcium entry blockers arterial lipid accumulation in several animals without nifedipine, verapamil and diltiazem were prepared altering serum cholesterol, blood pressure or heart rate weekly. The clinical doses were equivalent to the (Kramsch et al. 1980). Calcium antagonists have standard doses of each drug for adult humans. The high protective effects against ischaemia and antiatherogenic doses were 10 times the clinical doses. Animals were potential (Zannad, 2000), and may have beneficial weighed weekly, in order to calculate the doses. Water effects on the development of atherosclerosis, such as was used for the solution of verapamil and diltiazem; the inhibition of vascular smooth muscle cell polyethyleneglycol for nifedipine. An ultrasound bath proliferation and migration, inhibition of calcium influx was needed for the complete solution of diltiazem. into the vascular wall, reduction of extracellular matrix Nifedipine was protected from light in dark bottles. synthesis, promotion of uptake and breakdown of low- Medications (1 ml) were administered (force-fed) density lipoproteins, protection of lipoproteins from daily at 7 a.m. and 7 p.m. for two months (at the end the oxidative modification, maintenance of endothelial cell chickens were 81 days old). Polyethylenglycol was function, inhibition of platelet activation and reduction administered to the control groups as placebo. of blood pressure (Schachter, 1997). They also appear to have antioxidant effects in addition to their potent Tissue preparation vasorelaxant properties (Sobala et al., 2001). However, there are very few experimental studies The chickens were sacrificed after two months of comparing different kinds of calcium entry blockers and treatment. Traumatic damage to the arterial endothelium most of them use much higher doses than the clinical and intima was avoided by reducing the duration of the ones (Henry and Bentley, 1981; Ginsburg et al., 1983; animals’ dissection to less than 15 minutes. The aortae Catapano, 1997). Besides, there are no serial studies of five animals were removed, cleaned of surrounding which focus on the use of calcium entry blockers in tissue, opened and stained for lipid with Sudan III as avian models of atherosclerosis. described by Holman et al. (1958). Briefly, after washing In this work, we examine the effects of three calcium in 70% alcohol for 2 hours, samples were incubated in entry blockers (nifedipine, verapamil and diltiazem), at 0.3% Sudan III in 70% alcohol for 2 days at 37 ºC. human clinical and higher doses, on the extent of Dissected aortas were then washed and the luminal atherosclerosis of chickens fed a high-cholesterol diet. surface of the aortae was photographed by means of a Therefore, new histological and planimetric data Reprovit (Leitz Wetzlar, Germany) and Gold 100-2- concerning reduction in the lipid deposition of the aorta Kodak 5095 film. The atherosclerotic involvement was by the use of several calcium channel blockers in evaluated in the thoracic and abdominal aortae, aortic chickens are provided in this study. arch and supraaortic regions by computerized planimetry (Hewlett-Packard 85-B computer and HP 9111-A digital Materials and methods ultrasonic tracer). The percentage of the sudanophilic area was quantified (sudanophilic area/ aortic area). Experimental procedure Samples of five animals of each group were fixed for light and electron microscopy. For light microscopy, Eighty male 3-week-old White Leghorn chickens samples were fixed in 10% formalin in 0.1M phosphate- were housed under controlled conditions and randomly buffered saline, pH 7.4 (PBS), for 12 hours. After assigned to 8 groups and 2 kinds of diet (during the first fixation, they were processed for inclusion in paraffin 3 weeks of life all of them received a standard growing wax and 5 µm-thick paraffin sections were cut. Sections diet). Diet A (normal): a standard grower mash. The were stained