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SITE SPECIFIC RESPONSE TO HIGH CHOLESTEROL DIET IN AORTA OF RABBIT.

Byline: Uzma Shahid, Shadab Ahmed Butt and Azhar Mubarik

Abstract

Objective: To compare the development of atherosclerotic changes in different parts of aorta in rabbits fed on high cholesterol diet.

Study design: Experimental study

Place and duration of study: The Anatomy department of Army Medical College, Rawalpindi; from March 2009 to February 2010.

Methodology: Twenty adult Newzealand white rabbits were randomly allocated to two equal study groups. Group I was given regular lab diet and group II was fed 2% high cholesterol diet. After six weeks, aorta was excised from each animal. Cross sections were taken from each aortic part for microscopic examination. Rest of aorta was opened longitudinally and stained with Sudan IV for gross estimation of lesion.

Results: In contrast to group I, atherosclerotic changes developed in all the animals of group II but of varying intensity and site specificity. In cholesterol-fed rabbits, sudanophilic lesion score was significantly higher in arch versus descending thoracic or abdominal parts. Intimal thickening and intima to media ratio maximally increased in ascending part, followed by arch and then substantially decreased with increasing distance from heart.

Conclusion: Ascending aorta and arch are the most atherosclerosis-susceptible aortic sites in cholesterol-fed rabbits.

INTRODUCTION

Text Box: Correspondence: Dr Uzma Shahid, Anatomy Dept, Army Medical College Rawalpindi Email: u.ahmer@yahoo.com Received: 24 March 2010; Accepted: 8 Jun 2010Atherosclerosis is the well-known global health problem with its incidence increasing rapidly in the young South-Asian population1 and hypercholesterolemia is considered to be the unique and sufficient underlying cause even in the absence of other risk factors2. Atherosclerotic changes occur randomly at specific arterial sites through various risk factors. For example: smoking selectively involves abdominal aorta but does not influence right coronary artery4 which is significantly affected by elevated serum glycohemoglobin levels sparing abdominal aorta5. Hypertension especially augments atherosclerosis in cerebral arteries6.

Nikolajewitsch Anitschkow established the cholesterol-fed Newzealand white rabbit (NZW) as a model for atherosclerosis research on account of being most susceptible and sensitive to present atheromatous lesions, quite similar to and with the involvement of same molecular mechanisms as in humans, through high cholesterol diet7. However, overwhelming data indicates that feeding high cholesterol diet results in regional variation of cholesterol accumulation8. A large number of studies in rabbits have mapped the distribution of atherosclerotic lesions both after short term cholesterol feeding of some days9 and after feeding cholesterol for several months10 but reported conflicting results, perhaps owing to their focus either on cross sectional histology or upon en face analysis of lipid stained surface.

En face methodology is preferable to estimate the lesion extent and to minimize the routinely impossible serial sectioning but unable to delineate the thickness and cellular composition of lesion which is only possible by cross sectional histology3. So the present study was designed by combining both the aforementioned methods so as to truly appreciate the atherosclerosis susceptibility of different segments of aorta.

Demarcation of atherosclerosis prone sites in laboratory mammals is vitally important for minimizing the variable response possessed by each vascular site to various dietary and pharmacological interventions.

Materials and methods

This experimental study was carried out in department of Anatomy, Army Medical College, Rawalpindi in collaboration with National Institute of Health, Islamabad (NIH) from March 2009 to February 2010. Twenty adult male Newzealand white rabbits, weighing 2.08 +- 0.03 Kg (mean +- SE) were kept at NIH. Baseline blood samples were drawn from the ear vein for the estimation of lipid profile. Rabbits were caged individually in a controlled environment. These were acclimatized to experimental conditions for seven days and each animal was given 100 g/day regular rabbit chow used at NIH. Rabbits were then randomly divided into two equal groups. Group I was maintained on the same diet while regular diet of group II was enriched with 2% cholesterol powder for the next six weeks (Applichem, Germany). By the end of experiment, after an overnight fast, animals were euthanized with ether anesthesia.

Blood samples were taken by cardiac puncture and serum cholesterol, triglycerides, LDL and HDL concentrations were measured by Pioneer diagnostic kits, USA. Aorta from each rabbit was excised together with heart from its origin till its bifurcation into common iliac arteries. After 48 hours of fixation in 10% formol calcium, aortic rings (approx 3-4 mm wide) from each part were taken according to the figure 1 for microscopic examination.

The aortic rings were further processed for paraffin embedding. Approximately 4-5 um thick cross sections were cut. H and E staining was performed for histomorphological study and Verhoeff staining was executed for quantitative analysis. The section showing maximum atherosclerotic changes was selected from each aortic part of every aorta. At 400X magnification, thickness of intima (in um) from endothelial margin to the internal elastic lamina was measured at three areas of maximum intimal lesion11. At the same points, thickness of media from internal to external elastic lamina was also taken in um to calculate intima-media ratio12. Mean of the three values was taken for each parameter in each section.

Rest of aortic strips from arch, descending thoracic and abdominal parts were opened longitudinally, and stained with Sudan IV according to the method described by Ling et al.13. Classification of lesions was done on gross examination, according to scoring criteria14: 0=Nil lesion, 1=One to two fatty streaks, 2=Three to five fatty streaks, 3 = More than five fatty streaks, 4=Atheromatous plaque.

Statistical Analysis: Data were entered in a database using SPSS windows version 15. Values are expressed as mean+-SE. Comparison between means of initial and final lipid profile within each group was carried out through paired sample 't' test. Independent sample 't' test was applied for comparison between the two groups. Values of four parts of aorta were compared by one way ANOVA. All the results were considered statistically significant at a P value less than 0.05.

RESULTS

Biochemical analysis: Baseline levels of serum total cholesterol, triglycerides, LDL and HDL were statistically insignificant between the groups. Comparison of means between initial (at week 0) and final (at week 6) values of serum cholesterol (1.994 +- 0.100 versus 1.967 +- 0.120, P greater than 0.05), triglycerides (0.847 +- 0.037 versus 0.883 +- 0.026, P greater than 0.05), LDL (1.178 +- 0.138 versus 1.074 +- 0.117, P greater than 0.05) and HDL (0.488 +- 0.029 versus 0.486 +- 0.011, P greater than 0.05) was insignificant in group I while significantly greater increases in serum total cholesterol 1.908 +- 0.101 versus 32.708 +- 2.475, P = 0.000), triglycerides (0.815 +- 0.055 versus 1.718 +- 0.237, P = 0.000), LDL (1.071 +- 0.085 versus 29.290 +- 2.411, P = 0.000) and HDL (0.461 +- 0.017 versus 2.632 +- 0.170, P = 0.000) concentrations were detected in group II (Table 1).

versus 2.632 +- 0.170, P = 0.000) concentrations were detected in group II (Table 1).

Table - 1: Sirum lipid parameters of the rabbits fed regular lab diet (group I) and 2% high cholesterol diet (group II) for 6 Weeks.

###Serum lipid parameters (mmol/L)

Groups###Cholesterol###Triglycerides###LDL###HDL

###Week 0###Week 6###Week 0###Week 6###Week 0###Week 6###Week 0###Week 6

I###1.994+-###1.967+-###0.847+-###0.883+-###1.178+-###1.074+-###0.488+-###0.486+-###

###0.100a###0.120a###0.037a###0.026a###0.138a###0.117a###0.029a###0.011a

II###1.908+-###32.708+-###0.815+-###1.718+-###1.071+-###29.290+-###0.461+-###2.632+-

###0.101a###2.475b###0.055a###0.237b###0.085a###2.411b###0.017a###0.170b

Data are expressed as mean +- SE. Values for each parameter with different letter in a row or column are statistically significant (P less than 0.05) while those with similar ones are statistically insignificant (P greater than 0.05).

Gross examination: Sudan IV-stained luminal aspect in all the aortic strips from group I showed smooth unstained surface while those from group II revealed multiple macroscopically visible atheromatous plaques mainly in arch of aorta and fatty streaks in descending thoracic and abdominal segments, more marked around the branching orifices and with a decreasing gradient towards aortic bifurcation (Figure 2). Mean +- SE lesion score was significantly greater in arch versus descending thoracic (3 +- 0.33 versus 1.60 +- 0.22, P = 0.006) and abdominal part (3 +- 0.33 versus 1.50 +- 0.30, P = 0.003) but the difference of means between descending thoracic and abdominal parts was statistically insignificant (P = 0.968).

Text Box: Figure 1: Showing sites of tissue sampling from each part of rabbit aorta for microscopic examination (1, 2 in a; 3, 4 in b; 5 to 8 in c). CA: Celiac artery, SMA: Superior mesenteric artery. Table-1: Serum lipid parameters of the rabbits fed regular lab diet (group I) and 2% high cholesterol diet (group II) for 6 weeks. Groups Serum lipid parameters (mmol/L) Cholesterol Triglycerides LDL HDL Week 0 Week 6 Week 0 Week 6 Week 0 Week 6 Week 0 Week 6 I 1.994 +- 0.100a 1.967 +- 0.120a 0.847 +- 0.037a 0.883 +- 0.026a 1.178 +- 0.138a 1.074 +- 0.117a 0.488 +- 0.029a 0.486 +- 0.011a II 1.908 +- 0.101a 32.708 +- 2.475b 0.815 +- 0.055a 1.718 +- 0.237b 1.071 +- 0.085a 29.290 +- 2.411b 0.461 +- 0.017a 2.632 +- 0.170b Data are expressed as mean +- SE. Values for each parameter with different letter in a row or column are statistically significant (P less than 0.05) while those with similar ones are statistically insignificant (P greater than 0.05).

Microscopic examination: In group I, intima appeared as a single continuous layer of flat and elonga ed endothelial cells in close approximation with internal elastic lamina. Histomorphological examination of arch of and ascending aorta in group II revealed multiple foci of extensive intimal thickening characterized by lipid deposition and extracellular matrix accumulation. Intima was laden with foam cells in the form of raised and wide fatty streaks. Advanced atherosclerotic changes like disrupted endothelium, fragmented internal elastic lamina, multiple large coalescing pools of extracellular lipid, thinning of media and focal areas of calcifications were also discernible. In descending thoracic aorta, only early atherosclerotic changes were found. Changes were minimal in abdominal aorta and only in the form of mild intimal thickening with small and sparse foam cells (Fig.3).

Histomorphometric analysis revealed significantly greater thickness of intima in each part of aorta of group II (P= 0.000) as compared with their equivalent parts in group I. In group II, maximum intimal thickening was found in ascending aorta, with a decreasing gradient towards abdominal aorta. Difference of means between ascending versus arch and descending thoracic versus abdominal part were insignificant (P = 0.976, P = 0.822, respectively) whereas it was highly significant (P = 0.000) when ascending or arch was compared with descending thoracic or abdominal part. Similarly intima to media ratio increased maximally in ascending aorta in response to high cholesterol diet.

Differences of means between ascending versus arch and descending thoracic versus abdominal part were insignificant (P = 0.461, P = 0.995, respectively) while it was highly significant (P = 0.000) when ascending aorta was compared with descending thoracic or abdominal part and significant when arch was compared with descending thoracic (P = 0.003) or abdominal parts (P = 0.002), (Table-2).

Table 2: Comparision of histomorphometric findings between diffrent aortic parts of the same group and between the same part of the two groups; fed regular lab diet (group II) for 6 Weeks.

###Parameters

Part of aorta###Thikness of intima###Intima media ratio

###group I###group II###group I###group II

Ascending###5.582+-0.278a 177.66+-20.35b###0.028+-0.001a###0.81+-0.31b###

Arch###6.167+-0.659a 168.41+-22.82b###0.030+-0.003a###0.62+-0.11b

Descending thoracic###4.751+-0.374a###36.49+-7.25c###0.027+-0.002a###0.14+-0.02c

Abdominal###4.500+-0.356a###17.16+-2.97c###0.037+-0.004a###0.11+-0.01c

DISCUSSION

Text Box: Figure 2: Showing luminal aspect of descending thoracic aorta with smooth unstained surface in group I (a) and red stained Sudan positive lesions in group II (b). Arrow indicates proximal (P) to distal (D) directionThe principal finding of the present study is that different segments of aorta respond differently to high cholesterol diet and vulnerability to develop atherosclerosis is more in proximal thoracic aorta.

Text Box: Figure 3: Cross sectional histomorphology of aorta (ascending = a, b; descending thoracic = c) in rabbits fed the regular chow (a) and 2% high cholesterol diet (b, c) for six weeks. I = Intima, M = Media, FC = Foam cells, ECL = Extracellular lipid. a, b = H and E stain; c = Verhoeff stain. Approx X400. Bar = 50 mm. Table 2: Comparison of histomorphometric findings between different aortic parts of the same group and between the same part of the two groups; fed regular lab diet (group I) and 2% high cholesterol diet (group II ) for 6 weeks. Part of aorta Parameters Thickness of intima Intima media ratio Group I Group II Group I Group II Ascending 5.582 +- 0.278 a 177.66 +- 20.35 b 0.028 +- 0.001a 0.81 +- 0.13 b Arch 6.167 +- 0.659 a 168.41 +- 22.82 b 0.030 +- 0.003a 0.62 +- 0.11 b Descending thoracic 4.751 +- 0.374 a 36.49 +- 7.25 c 0.027 +- 0.002a 0.14 +- 0.02 c Abdominal 4.500 +- 0.356 a 17. 16 +- 2.97 c 0.037 +- 0.004a 0.11 +- 0.01 c Data are expressed as mean +- SE.

Values for each parameter with different letter n a row or column are statistically significant (P less than 0.05) while those with similar ones are statistically insignificant (P greater than 0.05).As expected, high cholesterol diet significantly increased serum cholesterol, LDL, HDL and triglyceride concentrations15. Significantly higher prevalence of sudanophilic lesions in arch, followed by the descending thoracic and abdominal part, with more obvious distribution around the branching orifices in our study is consistent with many researchers before15,16. Despite an insignificant difference of lesion development between the thoracic and abdominal parts10, relatively more involvement of thoracic than abdominal part in our study was not in association with Rong et al.17 who found abdominal segment more vulnerable than the thoracic part in cholesterol oxidation products injected NWZ rabbits. The reason might be the different route and method of lesion induction for this disparity.

Notably, McGill et al5 claimed abdominal aorta to be the most vulnerable aortic part for sudanophilic deposits, but in humans.

Intimal thickening is the hallmark of atherosclerotic lesions. As the intima increases in thickness due to cellular replication and lipid deposition, intima to media ratio also increases18. Histomorphologic examination revealing advanced atherosclerotic changes in ascending and arch and early changes in descending thoracic and abdominal segments was in a supportive context with Thiery et al.19 who demonstrated that ascending and arch of aorta were maximally responsive to high cholesterol diet in NWZ rabbits. Thickness of intima and intima to media ratio maximally increased in ascending part followed by arch of aorta and then substantially decreased towards abdominal aorta.

This gradient of lesion development in our group II rabbits can be explained by the study of Nielson et al.8 who showed a close positive connection between the LDL permeability and development of fatty streaks and stated that LDL permeability, being an important predictor of arterial cholesterol accumulation, was found to be decreased as we move away from aortic origin towards its bifurcation in chole terol fed rabbits. Moreover, atheroprotective properties like greater laminar shear stress, lower infiltration of monocytes/ macrophages, and increased mRNA expression of glutathione peroxides were detected in rabbit descending thoracic and abdominal aorta16. Andersen and Stender20 described the regional variation of cholesterol accumulation on the basis of endothelial nitric oxide synthetase (eNOS) activity which in the optimum amount is considered atheroprotective. They demonstrated lowest eNOS activity in proximal thoracic aorta which was gradually increasing towards abdominal aorta in NZW rabbits.

Conversel , Truskey et al.9 found highest densities of macrophages and maximum focal increases of vascular cell adhesion molecules in abdominal aorta as compared with other parts after feeding NZW rabbits a 0.25% high cholesterol diet for 2 weeks. Short duration and relatively lower amount of cholesterol feeding might account for this difference. Worthy to mention, atherosclerosis prone vascular beds analyzed in humans are coronary arteries and abdominal aorta as compared to thoracic aorta and differ from laboratory mammals depending on their hemodynamics and other risk factors including smoking, hypertension, immune status, genetics and oxidative stress3.

CONCLUSION

We conclude that ascending and arch of aorta are atherosclerosis prone and descending thoracic and abdominal segments are relatively atherosclerosis resistant parts in Newzealand white rabbits on feeding high cholesterol diet. Localizing these sites instigate a pathway for further exploration of the possible atherogenic mechanisms involved in modulation of a distinct site with reference to a specific risk factor.

Reference

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Correspondence: Maj Vaqar Ilahi Paracha, Dept of Cardiac Surgery, AFIC Rawalpindi Email: vparacha@yahoo.com Received: 19 April 2010; Accepted: 17 June 2010
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Author:Shahid, Uzma; Butt, Shadab Ahmed; Mubarik, Azhar
Publication:Pakistan Armed Forces Medical Journal
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2010
Words:3448
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