ORIGINAL�ARTICLE

ABSTRACT
Objective: To identify the effect of tcotrienols on adventitial changes induced by high cholesterol diet in 
descending thoracic aorta of rabbits.
Study Design: Laboratory based randomized control trial.
Place and Duration of Study: The Anatomy department of Army Medical College Rawalpindi in collaboration 

th th
with National Institute of Health, Islamabad. The study commenced on 10  March 2009 and completed on 10  
September 2009.  
Materials and Methods: Thirty adult male New Zealand White rabbits were randomly divided into three equal 
groups (I, II & III). Group-I consumed standard NIH diet while group-II was fed 2% high cholesterol diet. Group-III 
animals were given the same diet as to group II, however, tocotrienols 6 mg/kg body wt/day were also added to 
the diet. Following six weeks of experiment, aorta of every animal was dissected. Cross sections were taken 
from descending thoracic aorta and processed for light microscopic examination. In H&E and verhoeff stained 
slides, adventitial histomorphological changes were compared among the three groups.
Results: In rabbits on standard diet, tunica adventitia was a thin layer composed of loose network of collagen 
and elastic fibers which lacked lamellar pattern as that of media. Adventitial cells were relatively scanty. In 
contrast, aortic adventitia in group-II was thickened with increased number of inflammatory cells characterized 
by central round nucleus and foamy cytoplasm. Above mentioned histological changes were present in group-
III but were of lesser degree than group-II. Mean±SD thickness of adventitia and inflammatory cells score was 
significantly greater in group-II &III when either was compared with group-I. However, group-III showed 19% 
(p<0.05) reduction in adventitial thickening and & 36% (p<0.05) lesser inflammatory cells score versus group-II.
Conclusion: Tocotrienols decrease adventitial thickening and inflammation induced by high cholesterol diet in 
aorta of cholesterol-fed rabbits.

Key Words: Adventitia, High Cholesterol Diet, Rabbit Aorta, Tocotrienols.

4
event in the process of atherogenesis.   Majesky et al 
labeled adventitia as a dynamic interface harboring 
progenitor cells which respond to arterial injury and 

5
then migrate into tunica intima.  Gutterman 
proposed that future managements for prevention 
of atherosclerosis should focus on drug therapies 

6
targeting adventitia of vessels.  
Vitamin E includes eight chemically distinct 
substances: four tocopherols and four tocotrienols 
(alpha, beta, gamma and delta). Heath benefits of 
vitamin E in the prevention and treatment of 
atherosclerosis have been postulated with 
conflicting results but more than 95 per cent studies 
of vitamin E were directed towards tocopherols and 

7
tocotrienols remain poorly understood.  Moreover, 
available data regarding tocotrienols is mainly 
limited to their potent hypocholesterolemic, 

8,9
antioxidant and anti-inflammatory properties  and 
thus addresses a major void in evaluation of their 
effect on histomorphological aspects of the 
atherosclerosis.
Therefore, this experimental study was designed to 
investigate the effect of this lesser known form of 

Introduction
Atherosclerosis is considered to be the most 

1
common physical burden globally. Despite the use of 
dietary modifications and newer pharmacological 
approaches, very few effective measures exist for the 
prevention and treatment of this disease. Anti-
atherogenic effects of tocotrienols (members of 
vitamin E family) are also unclear as yet. 
Traditionally, atherosclerosis is considered as disease 
of intima with involvement of media in later stages of 
disease progression. Accordingly, intimal and medial 
thickening was accepted as the most appropriate 

2
predictor of atherosclerosis.  With the advancement 
of new era,  however, it was noted that features of 
atherosclerosis definitely exist in the tunica 

3
adventitia   and adventitial inflammation is an early 

Effect of Tocotrienols on Aortic Adventitia of Cholesterol-Fed Rabbits
Uzma Shahid, Asma Hafeez, Kaukab Anjum, Zubia Athar, Nomana Mahmood

Correspondence:
Dr. Uzma Shahid
Associate Professor, Anatomy
Wah Medical College, Wah Cantt

Department of Anatomy
Wah Medical College, Wah Cantt

Funding Source: NIL; Conflict of Interest: NIL
Received: Nov 02, 2016; Revised: Feb 08, 2017
Accepted: May 01, 2017

Effect of Tocotrienols on Aortic AdventitiaJIIMC 2017 Vol. 12, No.2  

87



scored accordoing to the following criteria: 0 = No 
inflammatory cells, 1 = inflammatory cells present in 

≥  25 to < 50 per cent circumference, 2 = 
inflammatory cells present in ≥50 to < 75 per cent 
circumference, 3 = inflammatory cells present in ≥ 
75 per cent circumference. Inflammation was 

defined as presence of ≥ 25 mononuclear round 
cells with foamy cytoplasm per field with 40X 

13,14
magnification objective.  
Parametric data was analyzed using SPSS (Statistical 
package for social sciences) windows version 20. 
Quantitative data was expressed as Mean ± S.D. For 
each variable, group differences were compared by 
one way analysis of variance (ANOVA) followed by 
post hoc tukey  test for intergroup comparison of 
parameters. All the results were considered 
statistically significant at a p-value less than 0.05.

Results
In rabbits on standard NIH diet, tunica adventitia was 
a thin layer composed of loose network of collagen 
and elastic fibers which lack lamellar pattern as that 
of media. Adventitial cells were relatively scanty and 
mainly suggestive of fibroblasts (Figure 1-a). Vasa 
vasora and nervi vascularis were also scattered 
amongst the fibers. In contrast, aortic adventitia in 
group-II appeared to be thickened especially 
beneath the intimal lesions (Figure 1-b). This 
thickening was associated with increased number of 
mononuclear round cells with central nucleus and 
foamy cytoplasm. These cells were forming 
a g g re ga t e s  i n  a d v e n t i t i a  a n d  p e n e t ra t i n g  
medioadventitial interface (Figure 1-d). Above 
mentioned changes were present in group-III (Figure 
1-c) but were of lesser degree than group-II 
 Mean±SD thickness of adventitia and inflammatory 
cells score was significantly greater in group-II &III 
when either was compared with group-I. However, 
group-III showed 19 % reduction in adventitial 
thickening and & 36% lesser inflammatory cells score 
versus group-II (Table-II).

vitamin E (tocotrienols) on aortic adventitia of 
rabbits fed high cholesterol diet. 

Materials and Methods 
The present study was a laboratory based 
randomized control trial conducted in the Anatomy 
department of Army Medical College Rawalpindi in 
collaboration with National Institute of Health (NIH), 
Islamabad. All experimental procedures were 
approved by the institutional animal ethical 

th
committee. The study commenced on 10  March 

th
2009 and completed on 10  September 2009. 
Inclusion criteria were thirty adult male New Zealand 
White (NZW) rabbits, 10-18 months old and 
weighing 1.5 to 2.5 kg. Female rabbits and animals 
with any evident pathology were excluded from the 
study. Each rabbit was kept in a separate cage, at a 
standard temperature of 21 ± 20C with 12 hour 
light/dark cycle at NIH. Each animal was given 
100g/day standard NIH diet. Water was available ad 
libitum. After one week of acclimatization to 
experimental conditions, thirty rabbits were divided 
into three equal study groups (I, II&III) by using non-
probability convenient sampling technique. Group-I 
continued standard NIH diet (100g / head/day) while 
each animal in group-II was given 2% high cholesterol 
diet {2g cholesterol powder (Applichem, Germany) 

10
mixed with 100g standard NIH diet/ head/day.  
Group-III animals were fed the same diet as to group 
II, however, tocotrienols {mixture of 90% delta & 10% 
gamma (American River Nutrition, Inc. Hadley, MA. 

11
USA)} 6 mg/kg body wt/day  were also added to the 
diet. Composition of diet per animal in each group is 
given in table-I. After six weeks of experiment, 
rabbits were euthanized. Cross sections were taken 
from descending thoracic aorta and placed in 10% 
formol calcium. After 48 hours, tissues were 
processed for light microscopic examination. H&E 
sta i n i n g  wa s  d o n e  fo r  h i sto m o r p h o l o g i ca l  
examination. For morphometry, verhoeff van-
Geisson stain was used for the delineation of 
collagen and elastin. Under 40X objective, at three 
point of maximal luminal narrowing, adventitial 
thickness (AT) was measured from media-adventitia 
interface (external elastic lamina) to the adventitia-
periadventitia interface (outer edge of collagen 

12
containing dense fibrous tissue).  Mean of the three 
values was calculated for each cross section. 
Inflammation in adventitia was semiquantitatively 

Table I: Showing composi�on of diet per animal per day
in each group

Effect of Tocotrienols on Aortic AdventitiaJIIMC 2017 Vol. 12, No.2  

88



which in turn produce increase amount of 
extracellular matrix. These fibrobasts also generate 
an inflammatory response by releasing cytokines 

16,17 
and chemokines. Growth of vasa vasora further 
modulates arterial wall structure by acting as conduit 

16
for inflammatory and progenitor cells.  Adventitial 
thickening and inflammation, in response to high 
cholesterol diet, as observed in the current 
experiment, supports the findings of Gradus-Pizlo et 

17
al  who found significantly greater thickness of 
adventitia in patients with coronary atherosclerosis 
than those with normal arteries. This adventitial 
thickening was associated with enhanced number of 
mononuclear vacuolated cells especially in the outer 

18
half of vessel. According to dushkin,  these cells are 
considered as an attribute to generate inflammation. 

19
Maillaro & Taylor  reported that population of these 
inflammatory cells include lymphocytes, monocytes, 
macrophages and fibroblasts which work in concert 
to elicit an inflammatory response that progresses 
towards tunica intima. In reviewing the immune and 
i n f l a m m a t o r y  m e c h a n i s m s  r e g a r d i n g  

20
atherosclerosis, Galkina and Ley  stated that these 
cells are found in normal adventitia but their number 
expands in atherosclerotic lesions. Contrary to our 

21
findings, Deopujari and Dixit  reported that basic 
pathological changes occur in tunica intima and 
media and tunica adventitia is not affected in 
coronary artery disease. Tocotrienols mediated 
substantial reduction in adventitial thickening is 

22
quite close to the findings of Qureshi et al  whose 
striking results highlighted the atheroprotective 
properties of novel tocotrienols of rice bran by 
substantial reduction of 57 per cent, 33 per cent and 
47 per cent in growth of atheromatous plaque in 
three genotypes of mice. Comparable results were 
seen in a recent local study which showed that 
tocotrienols significantly decrease atheromatous 

23
changes in diet induced diabetic BALB/c mice.  
However, our findings are not consistent with Ismail 

2 4
et al  who found no beneficial effect on 
atherosclerosis development in six rabbits given 
palm tocotrienols plus 2 per cent cholesterol for 10 
weeks compared with the six rabbits given 
cholesterol alone and six rabbits on regular diet.  The 
difference can be accredited to relatively small 
sample size in their study and more severe nature of 
the disease. 

Discussion
The present experimental study evaluated the effect 
of tcotrienols (mixture of 90% delta & 10% gamma) 
on adventitial atherosclerotic changes induced by 
high cholesterol diet in aorta of rabbits. Our results 
suggested that tocotrienols supplementation 
significantly decrease adventitial thickening and 
inflammation. Adventitial thickness displays a strong 
correlation with atherosclerosis risk factors 

12,15
especially dyslipidemias.  Atherogenic stimuli 
excites adventitial cells particularly fibroblasts, 

Table II: Showing comparison of adven��al thickness 
and inflammatory cells score between rabbits fed 
standard NIH diet (Group-I), 2% high cholesterol diet 
(Group-II), 2% high cholesterol diet+ tocotrienols 
(Group-III) for 6 weeks

Fig 1: Aor�c cross sec�ons of rabbit fed standard NIH 
diet (1-a), 2% high cholesterol diet (1-b, 1-d, ) & 2% high 
cholesterol diet+ tocotrienols (1-c) for 6 weeks (1-a, 1-b, 
1-c: Magnifica�on X400, Verhoeff Van Geisson stain; 1-d: 
arrow indicates inflammatory cells, H&E stain). 
TI: Tunica in�ma, TM: Tunica media. TA: Tunica adven��a

Effect of Tocotrienols on Aortic AdventitiaJIIMC 2017 Vol. 12, No.2  

89



the other half of the natural vitamin E family. Mol Aspects 
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8. Aggarwal BB, Sundaram C, Prasad S, Kannappan R. 
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9. Qureshi AA, Khan DA, Mahjabeen W, Trias AM, Silswal N, 
Qureshi N. Impact of δ Tocotrienol on Inflammatory 
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15. Skilton MR, Sullivan TR, Ayer JG, Harmer JA, Toelle BG, 
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16. Stenmark KR, Frid MG, Yeager M, Li M, Riddle S, McKinsey T, 
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17. Gradus Pizlo I, Bigelow B, Mahomed Y, Sawada SG, Rieger K, 
Feigenbaum H. Left anterior descending coronary artery 
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American journal of cardiology. 2003; 91: 27-32.

18. Dushkin M. Macrophage/foam cell is an attribute of 
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role. Biochemistry (Moscow). 2012; 77: 327-38.

19. Maiellaro K , Taylor WR. The role of the adventitia in 
vascular inflammation. Cardiovascular Research. 2007; 75: 
640–8.

20. Galkina E, Ley K. Immune and Inflammatory Mechanisms of 
Atherosclerosis. Annu Rev Immunol. 2009; 27: 165–97.

21. Deopujari R, Dixit A. The study of age related changes in 
coronary arteries and its relevance to the atherosclerosis. J 
Anat Soc India. 2010; 59: 192-6.

22. Qureshi AA, Salser WA, Parmar R, Emeson EE. Novel 
Tocotrienols of rice bran inhibit atherosclerotic lesions in 
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23. Kiani MRB, Butt SA, Arshad M. Histological Effects of 
Tocotrienol on Intima Thickness In Aorta Of Diabetic Mice. 
Pakistan Armed Forces Medical Journal. 2011: 61: 555-60.

As inflammation is the hallmark of atherosclerotic 
25

lesions.  Reduction in adventitial inflammatory cells, 
26

in our study, is in supportive context with Wu et al  
who concluded that tocotrienols possess potent 
anti-inflammatory activity by suppressing the 
expression of inflammatory mediators in human 
monocytic cells. Many researchers have compared 
anti-inflammatory properties of various tocotrienols 
and tocopherols and found delta tocotrienol as the 

2 7 , 2 8
m o s t  p o t e n t  i s o f o r m .  I n  a  s t u d y  o n  
hypercholesterolemic subjects,  delta tocotrienol 
was given in doses of 125, 250, 500, 750 mg/day for 4 
weeks. In each concentration, delta tocotrienol was 
therapeutically effective in reducing biomarkers of 
oxidative stress and inflammation including serum 
nitric oxide, C-reactive protein, malondialdehyde, 

9
and δ-glutamyl-transferase.   
In conclusion, our experimental data suggests that 
tocotrienols exhibit significant potential in lowering 
adventitial atherosclerotic changes induced by high 
cholesterol diet in aorta of rabbits. The study had 
been carried out in an experimental model of 
atherosclerosis that differs from chronic lesions 
observed in human cases. Therefore, human trials 
should be considered necessary for final elucidation 
of tocotrienols as atherosuppressive agents. 
Moreover, mechanisms involved in suppression of 
a t h e r o s c l e r o t i c  c h a n g e s  w a r ra n t s  f u r t h e r  
investigations. 

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