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AGE-RELATED CHANGES IN THE RAT BRAIN UNDER ENVIRONMENTAL STRESS
DISSERTATION SUBMITTED FOR THE DEGREE OF
iHa^ter of ^titlosiapli;^ IN
ZOOLOGY
ASiss ACHX^A GUPTJL
INTERDISCIPLINARY BRAIN RESEARCH CENTER AND
DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA) 1 9 8 7
: ^ \ ;sr^j*.^."-'»!^
L OS/3 7 ^ ^ **^ •: , i . • » V ' .
DS1394
C B R T I F I C A T E
This i s to cert i fy that Mies Achla Gupta has completed
her research work under our supervision. The dissertation
ent i t led "Age-related changes in rat brain under environmen
t a l stress**, i s an origincJ. contribution and d i s t inc t addition
to the ex i s t ing knowledge on the subject . Sha i s allowed to
submit the work for the degree of Master of Philosophy in
Zoology under Interdiscipl inary Research of the Aligarh MusliBi
University, Aligarh.
(Prof. Dr. A,.-ffrSIDDIQUl) Ph.D.
Chairman Department of Zoology, Allgaih Muslim University, Aligeurh.
(Prof. Dr. MAHDI HASAH) M.S. (Hons), Ph.D., D.Sc, Director Interdisciplinary Brain
Researeh Centre, J.H. MediceJ. College, Aligarh Muslim University, kllgaTh.,
ACKH0VTJSDGEMENT3
No young s c h o l a r , h o w s o e v e r , keen i n p u r s u i t of kjaowledi-';e
and i o a r n i a g can o v e r a c h i e v e much u n l u B s l,ht; ;,';ui(3iaioe of a
s e a s o n e d s c h o l a r who h a s d e d i c a t e d h i s l i f e i n p u r s u i t of
imowledge t h r o u g h h a r d work , t o i l and n e v e r - e n d i n g r e s e a r c h ,
. i s a v a i l a b l e . I t i s i n d e e d my exrtreme good l u c k t h a t P r o f .
D r . Mahdi l l a a a n , D l r o c L o r , 1 u tor-dliic l | ) l i i j a fy Bi ' a in Ri-tiearch
C e n t r e , J . N . M e d i c a l C o l l e g e , A.M,.U., A l i g a r h , a c c e p t e d me a s
h i s d i s c i p l e and \ i n s t i n t i n g l y gave me a l l g u i d a n c e and a s s i s
t a n c e needed a t each s t a g e of my s t u d i e s . I h a v e no words t o
e x p r e s s my g r a t i t u d e t o him f o r a l l t h a t he h a s done f o r me.
I owe s p e c i a l g r a t i t u d e t o ray s u p e r v i s o r P r o f , ^icher
H u u a i a 3 i d ( l l q u i , ChnLrttiari, Du[)artni(>nt of Zoo logy , whose know-
l e d g o mid e x j i e r i e n c e i n r e s u a r c h waH a / / r e a t ai;;;et to mf- la
p u r s u i n g t h i s work .
I s h a l l n e v e r f o r g e t D r . S . Badi*ul Hasan and Mr. Ai'.i2
Khan, R e a d e r s i n t h e D e p a r t m e n t of Community M e d i c i n e , v/ho
e n c o u r a g e d and a s s i s t e d me i n ray scheme of r e s e a r c h v/ork and
s t a t l f j t l o a l work, ror.[)f;o t i vc I y .
D r . F a k h r u l I s l a m , L.-. S . Saleem H a i d e r and D r . ( M r s . )
Tayyaba Q a d r i r e n d e r e d g r e a t a s s iL^ tance and gave v a l u a b l e
s u g g e s t i o n s aiid g e n e r o u s h e l p i n t h e p r e p a r a t i o n of my r e
s e a r c h v/ork. I do n o t vnsh t o m i s s t h e o p p o r t u n i t y of c o n v e y -
ii
ing my sincere thanks to them.
I owe special thanks to Miss Prlti Vadhava and Mr. Naseem
Ahmad Khan for their cooperation, assistance and encouragement
in the preparation of this manuscript. Thanks are also due to
Dr. Yagana Bano and Mr. Masood Zaheer Naqvi for their help and
cooperation. I cannot forget to make special thankful mention
of Mrs. Amita Gupta, a research scholar in Biochemistry Deptt.
for her cooperation and assistance.
I am dutihouTid to thankfully mention the names of Mr.
Syed Mohammad o id Dr. (Mrs.) Madhu Chovdhary, artists in the
Department of Anatomy and Community Medicine, respectively,
who helped me a lot in the preparation of my figures and
charts included in my dissertation.
I shall fail in my moral obligation if do not thank
Mr. Auhfaq Ahmad, Deptt. of Applied Mathematics, Z.H. College
of Engineering and Technology for his help in calculating the
ANOVA and Mr. Syed Mujeer, Mr. Mansoor Ahmad and Mr. Wajid
Husain, Central Photography imd Audio-visual Section for
photop:raphs for my dissertation.
I am also thankful to all the members of the Laboratory
staff of the Anatomy Department, specially to Mr. Shaukat Ali
for their cooperative attitude.
I believe that the affectionate blessings, spoauaneous
Ill
and unstiated cooperation of my TAUJEE, Mr. S. P. Gupta and
my father, Mr. S. C. Gupta have borne fruits and enabled me
to bring out this small piece of research work. A special
word of thanks is also due to my younger brother, Mr. A. K.
Gupta, who helped me in my statistical calculations.
' Last but not the least I owe special gratitude to my
respected mother who gave me ample free time to pursue my
study and research work at the cost of her comfort. I cannot
forget my younger sister, Miss Archana Gupta who always ea~
couraged me through her letters.
Finally, the financial assistance from Indian Council
of Medical Research, New Delhi, in the form of Jiuiior n..nu?afch
Fellowship, is thankfully acknowledged.
ACHLA GUPTA
ABSTRACT
A l t e r a t i o n s i n l i p i d p r o f i l e s and i n t h e r r ; t e of li/.i'l
p e r o x i d a t i o n were s t u d i e d i n t h e d i f f e r e n t r e g i o n s of ON 3
of a g i n g r a t s f o l l o w i n g r e s t r a i n t s t r e s s f o r 24 h o u r s . In
t h e 6 months o l d r a t s , t h e c o n c e n t r a t i o n of t o t a l l i p i d s ,
c h o l e s t e r o l and p h o s p h o l i p i d s were i n c r e a s e d i n v a r i o u s
r e g i o n s of b r a i n but d e c r e a s e d i n t h e s p i n a l c o r d , Flowever,
t h e c o n t e n t s of g a n g l i o s i d e s and t h e r a t e o f l i p i d p e r o x i
d a t i o n were d e c r e a s e d i n c e r e b r u m , c e r e b e l l u m ajid b r a i n ster;.
a f t e r 24 h o u r s r e s t r a i n t s t r e s s . On t h e o t h e r h a n d , i n 12
months o l d r a t s t h e l e v e l s of t o t a l l i p i d s , c h o l e s t e r o l ai.d
g a n g l i o s i d e s were d e p l e t e d i n v a r i o u s r e g i o n s of CHS, excei: t
c e r e b e l l u m . I n t e r e s t i n g l y , t h e c o n t e n t of p h o s p h o l i p i d t:
were d e c r e a s e d i n a l l t h e r e g i o n s of CNS and t h e r a t e *'
l i p i d p e r o x i d a t i o n was i n c r e a s e d i n a l l t h e r e g i o n s of C ]:< S
a f t e r r e s t r : a n t s t i - e s s f o r ' 4 h o u r s . R e g i o n a l h e t oro,»-" :'!.C'' ;
was a p p a r e n t :.ot o n l y i n t h e c o n c e n t r a t i o n of l i o l d c : - :
l i p i d p e r o x i d a t i o n in t h e v a r i c j u s r>;,iTiiS ):' tr^' •.• . •
but a l s o f 'A i^ovi t.i:: r e : - t r a i n t s t r e s s .
LIST OP FIQURBS
Figure No. Page
1. Correlation between scientific developments, stress and its effect on aging
2. General adaptation syndrome (O-AS)
3. Various concepts of stress
4. Restraint cage lodging an albino rat, immobilized for 24 hours
5. Photograph showing dorsal view of the rat brain
6. Dissection of rat brain parts for biochemical studies
7. Calibration curve for the estimation of total lipids
8. Calibration curve for the estimation of phosphorus
9. Calibration curve for the estimation of cholesterol
10. Calibration curve for the estimation of gangliosides
11. (a) Control animal
(b) Effect of restraint stress on the animal
12. Gastric mucosal stxrface of rat stomach
(a) In control animals
(b) In stressed ones, multiple ulcers (arrow)
2
6
7
25
27
28
50
34
37
39
44
44
45
45
LIST o r TABLB3
Table No. ^ ^ e
1. Rest ra in t s t ress - induced changes in the l eve l s of t o t a l l i p i d s in d i f fe ren t bra in regions of 6 months old r a t s — 46
2. Res t ra in t s t ress - induced changes in the l e v e l s of t o t a l l i p i d s in d i f fe ren t brain regions of 12 months old r a t s — 47
3. Restraint stress-induced changes in the levels of phospholipids in different brain regions of 6 months old rats ... 49
4. Res t ra in t s t ress - induced changes in the l eve l s of phospholipids in d i f fe ren t bra in regions of 12 months old r a t s . . . 50
5. Res t ra in t s t ress - induced changes in the l eve l s of c h o l e s t e r o l in d i f fe ren t bra in regions of 6 months old r a t s . . . 51
6. Res t ra in t s t ress - induced changes in the l e v e l s of c h o l e s t e r o l in d i f f e ren t b ra in regions of 12 months old r a t s . . . 52
7. Res t ra in t s t ress - induced changes in the l e v e l s of gangl ios ides in d i f f e ren t b ra in reg ions of 6 months old r a t s . . . 53
8. Res t ra in t s t r ess - induced changes in the l e v e l s of gang l ios ides in d i f fe ren t b ra in regions of 12 months old r a t s . . . 54
9. Res t ra in t s t r ess - induced changes in the r a t e of l i p i d peroxidat ion in d i f fe ren t b ra in reg ions of 6 months old r a t s . . . 56
LIST or TABLES (continued)
Table No. Page
10. R e s t r a i n t s t r e s s - i n d u c e d changes in t h e r a t e of l i p i d p e r o x i d a t i o n i n d i f f e r e n t b r a i n r e g i o n s of 12 months old r a t s . . . 57
1 1 . Trend of a n a l y s i s of v a r i a n c e (ANOVA) on t h e l e v e l s of totsuL l i p i d s f o l l o w i n g 24 hoxirs r e s t r a i n t s t r e s s i n d i f f e r e n t r e g i o n s of t h e 6 months o ld r a t b r a i n and s p i n a l cord . . . 59
12. Trend of a n a l y s i s of v a r i a n c e (ANOVA) on t h e l e v e l s of t o t a l l i p i d s f o l l o w ing 24 hours r e s t r a i n t s t r e s s i n d i f f e r e n t r e g i o n s of t h e 12 months o ld r a t b r a i n and s p i n a l cord . . . 60
13 . Trend of a n a l y s i s of v a r i a n c e (ANOVA) on t h e l e v e l s of p h o s p h o l i p i d s f o l l o w ing 24 hours r e s t r a i n t s t r e s s i n d i f f e r e n t r e g i o n s of t h e 6 months old r a t b r a i n and s p i n a l cord . . . 61
14. Trend of a n a l y s i s of v a r i a n c e (ANOVA) on t h e l e v e l s of p h o s p h o l i p i d s f o l l o w i n g 24 hou r s r e s t r a i n t s t r e s s i n d i f f e r e n t r e g i o n s of t h e 12 months old r a t b r a i n and s p i n a l cord . . . 62
15. Trend of a n a l y s i s of v a r i a n c e (ANOVA) on t h e l e v e l s of c h o l e s t e r o l f o l l o w i n g 24 hours r e s t r a i n t s t r e s s i n d i f f e r e n t r e g i o n s of t h e 6 months o ld r a t b r a i n and s p i n a l cord . . . 63
16. Trend of a n a l y s i s of v a r i a n c e (ANOVA) on t h e l e v e l s of c h o l e s t e r o l f o l l o w i n g 24 hour s r e s t r a i n t s t r e s s i n d i f f e r e n t r e g i o n s of t h e 12 months o ld r a t b r a i n and s p i n a l cord . . . 64
LIST OF TABLBS (continued)
Table No. Page
17. Trend of analysis of variance (AHOVA) on the levels of gangliosidee following 24 hours restraint stress in different regions of the 6 months old rat brain and spinal cord ... 65
18. Trend of analysis of variance (ANOVA) on the levels of gangliosides following 24 hours restraint stress in different regions of the 12 months old rat brain and spinal cord ... 66
19. Trend of analysis of variance (ANOVA) on the levels of rate of lipid peroxidation following 24 hours restraint stress in different regions of the 6 months old rat brain and spinal cord^ ... 67
20. Trend of analysis of variance (ANOVA) on the levels of rate of lipid peroxidation following 24 hours restraint stress in different regions of the 12 months old rat brain and spinal cord ... 68
C 0 M T B H T 8
ACKUOWLEDGEl NTS
ABSTRACT
LIST OF FIGURES
LIST OF TABLES
Page
I . INTRODUCTION
1.1 Ageing
1.2 S t r e s s
1.5 R e s t r a i n t s t r e s s
1.4 R e s t r a i n t or Immo"bili2iation used a s s t r e s s o r
1.5 Lipids
1.6 Lipid peroxidation
1.7 Aims and Objectives
II. MATERIALS AND METHODS
2.1 Animals
2.2 R e s t r a i n t s t r e s s
2 .3 D i s s e c t i o n and i s o l a t i o n of d i f f e r e n t p a r t s of "brain
2 .4 E x t r a c t i o n of l i p i d s from b r a i n
2 .5 E s t i m a t i o n of t o t a l l i p i d s
2 .6 E s t i m a t i o n of phospha te
2.7 E s t i m a t i o n of c h o l e s t e r o l
2 .8 E s t i m a t i o n of g a n g l i o s i d e s
1
3
4
8
9
14
20
22
24
24
24
24
26
29
31
35
38
Page
2.9 Determination of rate of lipid peroxidation
2.10 S t a t i s t i c a l analysis
2.11 Ana lys i s of v a r i a n c e (ANOVA)
40
42
42
I I I . RESULTS
3.1 Physical signs
3.2 Effect of r e s t r a in t s t r e s s on regional brain l ip id levels in aged ra te
3.3 Effect of restrednt s t ress on the ra te of l ip id peroxidation
3.4 Analysis of variance
43
43
43
55
58
17, DISCUSSION
BIBLIOGRAPHY
LIST OF PUBLICATION AND PRESENTATIONS
69
76
91
INTRODUCTION
I. INTRODUCTION
God created living beings but made them mortal. Thus
birth presupposes death. In between birth and death are
various stages of life, viz., infancy, childhood, youth and
old age. This gradual process from birth till natural death
is called ageing. Ageing involves sloir and gradual change
both in physical appearance and in the various regions of
the brain. The latter is the product of several factors,
both internal and external. The advancement of modem
sciences ushered in an era entirely unkno\m to our fore
fathers. Thus began the industrial revolution. Mass pro
duction of things started. Manual labour became uneconomical
and was replaced by machinery. In this process many lost
their source of livelihood. Some of them migrated to the
centres of industrial and commercial activities. This led
to urbanization which posed problems of its own. Carefree
life received a rude shock. Stress and strain of every day
life posed a big threat to the peace and tranquility hitherto
enjoyed by man. This has since engaged the attention of
scientists who have accepted the challenge to study carefully
and analytically the whole problem confronting the mankind.
Medical scientists contributed a lot in the advancement of
medical science whereby life span increased but the impact of
stress on hvunan life, especially due to industrialization and
INCREASE IN THE POPULATION OF THE AGED.
Problems?
G E R O N T O L Q ^
LIFE-EXPECTANCY increased
I ADVANCEMENT
IN VMedicalSciences 2.Science and
Technology
INDUSTRIALIZATION AND
URBANIZATION
R ^ . l CORRELATION BETWEEN SCIENTIFIC DEVALOPMENTS,STRESS AND ITS EFFECT ON AGING.
urbanization, has created problems for them which require
deep study in the field of age-related changes in the various
regions of brain due to environmental stress (Fig. 1).
1.1 AGEIMG
Ageing is a universal and inevitable scientific and
social challenge confronting hiamanity. The lives of all
multicellular organisms begin with conception, extend through
development, maturity, senescence and end in death. Accord
ing to G. H. Hunt (1959), "the term ageing process, as
applied to living organisms, is the genetically determined
progressive and essentially irreversible diminution with the
passage of time of the ability of an organism or of one of
its parts to adapt to its environment, manifested as dimi
nution of its capacity to withstand the stresses to which it
is subjected, and culminating in the death of the organism".
Comfort (19 5^ said that ageing is an increased liability to
die or an increasing chronological age or the passage of the
life cycle. Ageing processes have been defined by Ma3mard
Smith (1959) as those which render individuals more suscep
tible, as they grow older, to the vsurious factors, intrinsic
or extrinsic, which may cause death.
Recently, Backmann and coworkers (1984) are of the view
that ageing is a differentiation process which is caused by
a phase-specific genetic information and by changes during
the processing of the primary transcript to the functional
mRNA. These changes influence the functional accuracy of
enzyme systems vhich are involved in gene realization. This
means that with ageing of an individual the error load
increases steadily to reach a threshold value. This causes
a collapse of the dynamic and thermodynamically controlled
interplay of the metabolic events.
1.2 STRESS
The literal meaning of the word 'stress' is 'condition
causing hardship'. In the language of life science, the
meaning of 'stress' is "an intense force, strain, agent or
mentaJ. condition which produces a defense reaction, which if
continued or intensified may lead to pathological lesions".
In other words, stress denotes the 'rate of wear and tear in
the body*. According to Selye (1956), stress is the nonspeci
fic response of the organism to any demand made upon it but
this definition would not be satisfactory any more, because
it is so wide that any physiological response and any exter
nal stimulus could be brought under this concept. Stress is
a very divergent phenomenon. It was generally felt that there
is a natural tendency to speak of a stress situation only when
the demand exceeds a certain level and is experienced as un
desirable or unacceptable. In the opinion of Tuwiler (1971),
'stress' like the word love, is broadly understood but poorly
defined. He considered the life as a series of micro- and
macro-adaptations to a constantly changing internal and exter
nal environment; and emphasized that s tress was an everpresent
condition. Rabkin and Struening (1976) mentioned that s tress
l ike anxiety, was a broad and general concept describing
organisms' reactions to environmental demands, Selye (1979)
further emphasized that s tress causes certain changes in the
structure and chemical composition of the body. Some of
these changes are merely signs of damage, others are manifes
tat ion of the body's adaptive reactions, i t s mechanism of
defense against s t re s s . The t o t a l i t y of these changes, the
s tress syndrome, i s called the general adaptation syndrome
( G . A . S . ) which develops in three stages, (1) the alarm reac
t ion , (2) the stage of res istance, and (3) the stage of
exhaustion (Fig. 2 ) .
Stress has different concepts in different branches of
science (Pig. 3 ) . Any environmental factor that can s i g n i f i
cantly modify an animal's b io log ica l responses result ing into
s tress i s cal led a s tressor .
Rabkin and Struening (1976) remarked that "social
stressor* refers to personal l i f e changes, such as bereave
ment, marriage, or l o s s of job, which a l ter the individual 's
socisJ. s e t t ing . A more speci f ic def in i t ion was proposed by
Holmes and Rahe (1967) who defined as soc ia l s tressors , any
set of circumstances the advent of which s i g n i f i e s a required
change in the individual 's on-going l i f e pattern.
{iiija (S - E)
(ii) (S - A)
(Ui)b {S - E)
Fig.2 GENERAL ADAPTATION SYNDROME (G.A.S.)
PHYSICAL • LOAD • PRESSURE .•TEMPERATURE
BIOCHEMICAL • METABOLIC
CHANGES • ENZYMES • HORMONES.
PSYCHOLOGICAL • DEPRESSION (STRESS
GUILT FRUSTRATlOt
PHYSIOLOGICAL • BEHAVIOUR • SITTING
YAWNING
SOCIOLOGICAL BEREAVEMENT MARRIAGE
^ LOSS OF JOB
MEDICAL • RATE OF
WEAR AND TEAR
Fig.3 VARIOUS CONCEPTS OF STRESS
8
Stress plays a role in such diverse maaifestations of
l i f e as ageing, the development of indiv idual i ty , the need
for s e l f expression, and the formulation of man's ultimate
aims. Psychological s tress ar ises in man due to the effect
of various factors on the neocortex. Experiments in animals
have shown that deep and prolonged psychosomatic disturbances
arise from psychological s t re s s .
Environmental s tress invokes compensatory metabolic
changes through modification of the quality and quantity of
enzymes that are normally rate- l imit ing or under fine con
t r o l or inducible by hormones (Ramasarma, 1978). The pro
cesses of adaptation at the ce l lu lar l e v e l to chronic s tress
seem to occur by sequential changes in hormones, enzymes and
metabolites leading to anew steady-state . Ramasarma (1960)
emphasized that those changes which are benef ic ia l to the
organism's need are promoted and accelerated. On long expo
sure, the changed pattern (due to stress) s e t t l e s to a stage
representing the de l icate balance of ce l lular constituents
best suited to the metabolic demands of the organism.
1.5 RESTRAINT STRESS
When an animal is kept under control in such a way that
it cannot move its body at its will it is said to be under
restraint stress. For example, when a rat is kept in a mini-
cage with the result that its movement is restricted, it is
said to be under restraint stress.
In the works of Renaud (1959), Baker et al., (1962),
GaniB (1962), Thompeon (1966), and Girardet (1974) details
of many types of restraint devices, cages and other appara
tus for rats, which are commercially available for general
or specific needs, may be seen.
1.4 RESTRAINT OR IMMOBILIZATIOM USED A3 A STRESSOR
Restraint or immobilization is one of the commonly used
laboratory stressors which has the apparent advanteige of be
ing relatively simple. It was further observed that this
particular stressor is compounded of fear, isometric muscular
activity, and muscle cramps due to specific positioning.
According to Tuwiler (1971), immobilization poses some prob
lems since the magnitude of the stressor varies with exper
ience and, more subtly, with differences in positioning and
handling the animals between experiments and experimeutors.
Seegal (1981) advocated that immobilization is usually
selected as a stressor or as a means of inducing stress
because of its being simple and also due to the large n\imber
of stuaies that have employed this stressor (Du Ruisseau et
al., 1979; Kawakami et al., 1979; Kobayashi et al., 1976;
Reigle and Meites, 1976; Kventnansky and MikulaJ, 1970;
Krigger et al., 1979; and Zholkute and Udupa, 1978, 1979).
After chronic restraint (continuous restraint for 24 to
48 hours) involution of the thymus and adrenal, hypertrophy
10
was reported by Selye (1936). Chronic restraint, of course,
is also complicated by concomitant voluntary or involuntary
fasting, which varies both with accessibility of food and
with the emotional state of the animal. Therefore, in experi
ments using immobilization as a stressor, the control (un
stressed) animals should also be kept deprived of any food
for the same period as the immobilized ones.
The production of gastric ulcers after chronic immobili
zation was noticed by Rossi et al., (1956). Brodie and Hanson
(i960) observed that the degree of ulceration and ulcer loca
tion varies species to species. They reported that mice and
rats show a high ulcer incidence, guinea pigs have a lower
incidence, and hamsters a very low incidence of ulceration.
They further noted that rabbits and monkeys ordinarily have
little or no ulceration upon restraint. Parisio and Clementi
(1976) noted stress-induced ulceration in gastric mucosa in
albino rats.
Liberson et al., (1964) detected a decrease in serotonin
in cerebral cortex and hippocampus after forced positioning
of animals without direct physical restraint other than conti
nually replacing the animal in position. An increase in the
serotonin concentration after long and short intervals of
immobilization was observed by De Schaepdryver et al., (1969).
Bliss and Zwanziger (1966) noted that restraint resulted in a
slight increase in the levels of GABA (gamma amino butyric acid)
11
An increase in tyrosine amino-transferase activity due
to chronic immobilization was observed by Hanninen and Har-
tiala (1967). They noted that this increase is dependent
upon the adrenals and only occurs in animals showing develop
ment of ulcers. These workers also reported increased
tryptophan oxygenase activity two-fold by 6 hours of restraint
which was somewhat more after 24 hours.
Welch and Welch (1968) detected an increase in brain
norepinephrine levels in mice, housed singly in isolation
cages after short restraint (7 minutes). Bliss and Zwanziger
(1966), Corrodi et al., (1968), on the other hand, noted 10
to 20 per cent decrease in brain norepinephrine levels of
mice, guinea pigs, or rats restrained for two or more hours.
According to Bliss and Zwanziger (1966), these changes were
most marked in cerebellum and hypothalamic areas. A histo-
chemical depletion of central noradrenergic neurons after two
or more hours of immobilization was noted by Corrodi et al.,
(1968).
In acute experiments with this stressor, plasma (De
Schaeperyver et al., 1968) and adrenal (Zimmerman and Critchlow,
1967) corticoids are elevated, and in contrast to stressors
such as hunger or shock, some degree of adaptation to exper
ience occurs after immobilization stress.
Kvetnansky and Mikulaj (1970) reported a decrease of adre
nal catecholamine content and a marked elevation of urinary
1?
catecholamine excretion after forced immobilization, as well
as many other stresses. Interestingly, increased dopamine
levels were observed after acute restraint by Welch and Welch
(1968), while chronic restraint led to their fall l>De Schaep-
dryver et al., 1969).
Rudeen and Morgan (1975) showed that immobilization
stress affected the serotoninergic system differently in
five areas (cerebral cortex, brain stem, diencephalon, hippo-
campal formation, and corpus striatum) of the brain in rats
which were immobilized for the time intervals of 30, 60, 120,
180 and 300 minutes. They suggested that the decrease in the
elevation of brain 5-hydroxyindoleacetic acid (5-HIAA) with
continued stress might reflect physical exhaustion or adap
tation to the stress or might indicate an increase in the
excretion of 5-HlAA from the brain.
Kvetnansky et al., V1978) implicated hypothalamic cate
cholamines in neuroendocrine processes; as evidenced by dec
rease in the catecholamine contents of specific nuclei and
areas of the rat hypothalamus. They suggested that adrenaline-
forming neurons in the anterior hypothalamic region were
involved in the complex central regulatory pathways for the
integration of tne peripheral adrenergic responses.
Furthermore, immobilization stress was found to be
associated with significant increase in plasma ACTH and corti-
1'
coeteroid concentrations and decrease in anterior pituitary
ACTH concentration (Krigger et al., 1979). These workers
observed no significant change in medial basal hypothalamic
or median eminence ACTH concentrations and suggested the
involvement of different systems in the origin and regulation
of brain and pituitary ACTH. Selye (1976) stated that the
inhibitory effect of stress on plasma testosterone levels was
not confined to rodents but had also been observed in humans.
Evaluating the neuroendocrine mechanisms involved in
chronic intermittent immobilization stress-induced testo
sterone suppression in adult male rats, Tache et al. (1980)
noted that in intact animals, restraint applied six hours
daily for six days Increased plasma corticosterone, decreased
significantly testosterone and, to a lesser degree, LH levsls.
Recently, Seegal (1981) noted alterations in the metabo
lism of central catecholamines by selective activation of
tuberoinfxindibular dopaminergic neurons in the chronically
restraint rats. Earlier, Kvetnansky et al. (1976) detected
decreased steady-state concentrations of dopamine and norepi
nephrine in the arcuate nucleus and nucleus dorsomedialie
ventralis after 20 minutes of restraint. Also Palkovits et
al. (1975) and Kobayashi et al. (1976) reported similar find
ings after 3 hours of immobilization.
14
1.5 LIPIDS
Neural membranes contain three major categories of
lipids: cholesterol, sphingolipids, and glycerophospholipids.
These lipids are highly enriched in the central nervous system
of vertebrates and they play a vital role in both structure
and function of neural membranes. Other lipids, such as
triglycerides, free fatty acids, and cholesterol esters,
which are abundant in systemic organs, are minor constituents
of the central nervous system. However, some of these lipids
are metabolically very active and thus their functional sig
nificance should not be overlooked.
It is well recognized that the brain undergoes distinct
changes in lipid composition during growth and development
that reflect the structural differentiation of the nervous
system. As a result, specific patterns are formed during
development of specific brain regions and subcellular mem
branes, particularly the myelin. Although a voluminous amount
of information has already appeared to dociiment these changes
during development, little detailed information is available
on lipid changes and aging.
The studies by Rouser and Yamamoto (1968, 1969) on male
human brain still remain one of the most comprehensive studies
regarding quantitative changes in all the major lipid classes.
They showed that lipid composition changes continuously
throughout development and aging, with individual lipid
15
c lasses having different rates of change at d i f fe ren t ages.
Whole-brain t o t a l l i p i d increases rapidly upto about 2 years
of age, cont inues to increase l e s s rapidly to a peak at
50-40 yea r s , and then dec l ines at a steady r a t e a f t e r the
age of 50 (Rouser et a l . , 1972). The changes in the i nd iv i
dual l i p i d c l a s se s have been summarized by Horrocks et a l .
(1975,1981). They used the equations of Rouser and Yamamoto
(1968,1959). The g rea tes t losses ( 1 2 - 1 ^ ) in l i p i d s betweea
40 and 70 years of age are fo\ind in c h o l e s t e r o l , cerebros ides ,
cerebroside su lpha tes , ethanolamine plasmalogens and sphingo
myelins. All of these l i p i d s are r e l a t i v e l y enriched in
myelia. These da ta are supported by Ber le t and Volk (1980),
who quant i f ied myelin from white matter of htunan bra ins be t
ween the age of 17 and 89 years and found a s ign i f ican t dec
rease in myelin content in the aged b ra in s .
Lipid changes in rodent brain d i f f e r from those in human
b ra in . Increase in phospholipid, c h o l e s t e r o l , and cerebro
side content of aging b ra in of mouse, Peromyscus leucopus
( long- l ived f i e l d mouse) and ra t have been reported by Sun
and Samorajski (1972), Horrocks et a l . (1981) and Palo et a l .
(1964) and Bonet t i et a l , (1983), r e spec t i ve ly . These in
creases c o r r e l a t e well with reported increases in myelin
content of aging rodent b ra ins (Sun et a l . , 1972; Rawlin
et a l . , 1971; Norton et a l . , 1975; Malone et a l . , 1982).
These inc reases also c o r r e l a t e with spec i f ic increases of
16
60-809 in myelin galactolipids and cholesterol in mouse brain
(Sun and Samorajski, 1972).
Phospholipids are of particular interest in aging
studies because they are an integral part of the membrane
lipid bilayer. Changes in composition may result in altera
tion of the membrane protein activities. Sun and Faudin
(1984) have recently reviewed the changes in phospholipid
composition and metabolism during development and aging.
In addition to the changes in ethanolamine plasmalogens and
sphingomyelin mentioned earlier, Rouser and Yamamoto (1968,
1969) found small decreases in phosphatidylcholines, phoapha-
tidylethanolamines, and phosphatidylserines after the age of
50 and little or no change in other phospholipids. Hawthrone
et al. (1983) recently reported a 67% decrease in total lipid
inositol in aged human brain (90 years old) along with a two
fold decrease in free inositol. The decrease in lipid inosi
tol may be related to a change in the polyphosphoinositide
levels. The study of Svennerholm (1968) indicated pronounced
changes in phospholipid composition of hximan cerebrum during
growth of a person from birth till 26 years of age. However,
there ie little change after maturation between the age of 26
and 82 years. Svennerholm (1968) also reported both tissue-
specific and age-dependent fatty acid patterns in the indivi
dual phospholipids. The phospholipids in cerebral white
matter are more enriched in monoenoic acyl groups, whereas
17
polytmeaturated acyl groups are more abiindant in the cortex.
With increasing age, the proportion of (n-6) and (n-3) poly-
xinsatUrated fatty acids indicated an overall decline.
At the subcellular level, myelin lipid compoBition in
ageing brain has received greatest attention because this
membrane is relatively easy to purify and pathological loss
of myelin may be associated with loss of neuronal function.
Myelin phospholipids have been examined in some detail in
ageing brain of mouse (Sun and Samorajski, 1972), rat (Rawlins
et al., 1971; Norton and Poduslo, 1973; Malone and Szoke, 1982;
Norton, 1971; Smith, 1973) and human (Horrocks et al. , 1981;
Svennerholm et al., 1978). Age-related changes in the indivi
dual phospholipids were found. In rat and mouse (Norton and
Poduslo, 1973; Horrocks, 1968) phosphotidylchollnes decreased
and ethanolamine plasmalogens increased during maturation,
but little change was seen in the proportion of any phospho
lipids during ageing. Among the acyl groups of mouse brain
ethanolamine phosphoglycerides, there was an increase in the
proportion of 20:1 with a concomitant decrease in the poly
unsaturated fatty acids (Sun and Sun, 1972).
Changes in ganglioside concentration and composition
during ageing have been reported in rodents (Horrocks et al.,
1981; Rahmann, 1980) and human (Rouser and Yamamoto, 1968,
1969; Cherayil, 1969; Berra and Bnmngraber, 1974; Fredman
18
et a l . , 1980; Segler-Stahl et a l . , 1983). In human brain,
the concentration of ganglioside-boxmd s i a l i c acid drops to
475 (from 1100/ig/g t i ssue to 570 JUg/g) from 25 to 48 years
of age, reaches a plateau un t i l the inid-70s, and then begins
to drop again to a low level of 400 /ig/g at the age of 85
years (Segler-Stahl et a l . , 1983). In par t icular , GMI and
GDIa decrease rapidly, so that the t o t a l ganglioside composi
tion i s shifted to a more polar pat tern. Fredman and coworkers
(1980) reported that the brain of a 65-year-old man contained
more t e t r a - and t r is ia logangl ios ides but less di-s ialoganglio-
sides than the brain of a 3-month-old infant. In contrast ,
the s i a l i c acid level of rodents declines only s l ight ly during
senescence (Rahmann, 1980). The decline of ganglioside con
centration during ageing i s probably related to degeneration
of the ganglioside-rich components such as neuronal membrane.
However, the cause-effect relat ionship has not been delineated
(Segler-Stahl et a l . , 1983).
L i t t l e information i s available on the stress-induced
a l te ra t ions in the brain l i p id s . I t i s reasonably well- esta
blished tha t , with few exceptions, the fully mature brain i s
refractive to marked changes in the d i e t , including malnutri
tion (Ramsey and Nicholas, 1972). The neonatal and developing
brains, however, are known to be highly susceptible to the
nut r i t ion of the mother and dietary r e s t r i c t ions during the
period of active myelination. I t i s now amply substantiated
19
that irreversible changes in brain lipids occur in animals
deprived of proper nutrition neonatally or during the period
of active myelination. Bass and Netsky (1969) reported defi
ciencies, such as arrest of postnatal glial cell migration,
as a consequence of improper nutrition. Also, the dictum
"earlier the malnutrition, the more severe the effect and
the less the chance of recovery" has been described by Bass
et al. (1970).
A report on the stress-condition induced by inflammatory
processes causing inhibition of ketosis and decrement in
serxim fatty acids was given by Weufeld et al. (1972). These
workers showed that some of the inflammation-induced changes
decreased when nonseptic stress was superimposed. Cavenese
et al. (1978) observed the effect of radiation on the mamma
lian nervous system. An increase in the lipid deposition in
almost all the parts of the brain of Heteropneustes fossilis,
was reported by Saxena and Tyagi (1981), after X-irradiation
of the fish,
A very good piece of work from this laboratory by Dhanraj
Singh (1982) has shown that immobilization or restraint stress
produced modification of the gastric mucosa leading to focal
areas of partial surface erosions, followed by a detachment
of the superficial cells resulting into gastric ulcers or
variable shapes and sizes. On the other hand, the contents
of total lipids, phospholipids and cholesterol decreased in
?0
various regions of rat brain after restraint stress. Interest
ingly, however, lipid peroxidation increased in the different
regions of the rat brain.
1.6 LIPID PEROXIDATION
The investigation of lipid peroxidation is a recently
applied approach to the study of neurotoxicology (WHO, 1986).
Lipid peroxidation appears to be a phenomenon that reflects
free radical events associated with biological membranes,
which contain most of the polyunsaturated fatty acids contain
ing lipids in animal tissues. In addition, biological mem
branes are filled with active catalysts of oxidation of fatty
acids by oxygen with peroxide formation (lipo-peroxidation)
such as hemoproteins and nonheme iron, copper and manganese
complexes. The consequences of oxidation of even a small por
tion of unsaturated fatty acids in the phospholipid bilayer
are obviously dramatic deterioration of barrier and matrix
function of the lipid bilayer, enzyme inactivation, toxic
effects on cell division etc. Apparently, the formation of
membrane in primary cells proceeded in reduced atmosphere,
and later the cells developed specialized systems to protect
their membrane substance against oxidation by free oxygen
(Tappel, 1970).
Brain has been reported to show considerably high degree
of peroxidation among different tissues from normal rates
(Kartha and Krishnamurthy, 1978). This might be due to the
2]
fact that brain has the largest amount of lipids in compari
son to all other body organs. With aging lipid peroxidation
was increased and it is marked by the age marker 'lipofuscin'.
Lipofuscin deposition exhibits a special structural, topietic
and qualitative appearance as well as a special development
course in the process of the nervous tissue. The increasing
intraneuronal accumulation of lipofuscin pigment is consi
dered to be one of the consistent cytological alterations
correlated with mammalian aging (Hasan and Glees, 1972).
Lipofuscin accumulation has been traditionally ascribed to
the wear and tear process. Lipofuscin appears to be one of
the morphologically observable end-products of lipid peroxi
dation. Free radicals have long been suspected as inter
mediates of biological oxidative processes. They are highly
reactive transient chemical intermediates, the concentration
of which is increased by high energy irradiation and by mole
cular oxygen (Kormendy and Bender, 1971). The rates of many
free radical reactions in cells are known to be enhanced by
molecular oxygen and by metal ions such as copper, iron and
manganese. Also, thallium, nickel and cobalt have been shown
to increase the rate of lipid peroxidation, thereby to cause
significant accumulation of lipofuscin (Hasan and Ali, 1981).
Lipid peroxidation 'in vivo* has been claimed to be of basic
importance in aging, in the damage to cells by air pollution,
heavy metals and organophosphate pesticide intoxication
(Tappel, 1973; Hasan and Ali, 1981; Haider and Hasan, 1984;
22
Haider et a l . , 1981; Tayyaba and Hasan, 1985). Attempts have
recen t ly been made to i nh ib i t l ipofuscin accumulation or to
induce removal of l ipofuscin granules by adminis ter ing a n t i
oxidants (Vitamin E) or dimethyl aminoethyl p-chlorphenoxy
ace ta te (Hi l f e rg in , centrophenoxin or l u c i d r i l ) . Dissolut ion
of neuronal l ipofusc in as well as the t r anspor t and removal
of a l t e red pigment by phagocytic c e l l s and by cap i l l a ry endo
thelium following adminis t ra t ion of centrophenoxine ' i n v ivo '
and ' i n v ivo ' has been c l ea r ly demonstrated (Hasan et a l . ,
1974; Spoerri and Glees, 1975).
Res t r a in t has been used as a s t r e s s o r because i t simu
l a t e s the s t a t e of r e s t r i c t e d a c t i v i t y commonly observed in
the aged ind iv idua l s and transmission e lec t ron microscopy
revealed an increased incidence of l ipofuscin granules in the
neurons of the brain-stem (Hasan, 1985).
1.7 AIMS Am OBJECTIVES
In the l i g h t of the ava i lab le l i t e r a t u r e , s t i l l there i s
a paucity of da ta on t h e r e s t r a i n t s t ress - induced a l t e r a t i o n s
in the b ra in l i p i d s of r a t . I t would, t he re fo re , be of great
i n t e r e s t to evaluate the l eve l s of brain l i p i d s and of t h e i r
p r o f i l e s following r e s t r a i n t s t r e s s to the r a t s .
This study i s confined to i nves t iga t e the changes in
t o t a l l i p i d s and l i p i d peroxidat ion in r e s t r a i n t s t r e s s -
induced aged r a t s . Since l i p i d s are e s s e n t i a l components of
23
all the cell membranea as well as of several crucial enzyme
systems including hormones, they are known to he involved in
numerous important biological processes including cellular
transport. Hence, any change in the levels of lipids might
result in a defective cellular metabolism. Therefore, the
present study was undertaken to determine the levels of total
lipids, phospholipids, cholesterol, gangliosides and lipid
peroxidation in cerebrum, cerebellum, brain stem and spinal
cord of normal as well as of stress-induced albino rats.
MATERIALS & METHODS
2 4
I I . MATERIAL AKD METHODS
2.1 ANIMALS
Healthy male albino rats (Charles Foster) of two differ
ent groups obtained from the animal colony of Central Drug
Research Institute, Lucknow were used in the present study.
Female rats were excluded from the study because of their
cyclic hormonal variations.
Group I: consisted of 24 rats, 6 months old, weighing
250 + 20 gm., and
Group II: consisted of 24 rats, 12 months old, weighing
450 + 20 gm.
They were fed on pellet diet (Hindustan Lever Ltd.,
India) ad libitam and had tree access to water. For the
biochemical estimation of lipid classes and lipid peroxida
tion, rats from the two age groups were equally divided into
two groups - control and experimental, comprising 6 rats in
each group.
2.2 RESTRAINT STRESS
The rats were individually taken out from their cages
and kept under stress in a specially designed cage (Fig. 4).
2.5 DISSECTIQIJ AND ISOLATION OF DIFFERENT PARTS OF BRAIJJ
Overnight fasted animals were sacrificed by decapitation.
Brains were rapidly removed, the blooa clots adhering to the
25
Pig. 4: Restraint cage lodging an al'bino rat,
immobilized for 24 hours.
26
b r a i n s were removed by washing in the normal s a l i n e . Then
the c e r e b r a l hemisphere , ce rebe l lum, b r a i n stem and s p i n a l
cords were r a p i d l y d i s s e c t e d out of t h e b r a i n s and weighed t o
the n e a r e s t m i l l i g r a m s on an e l e c t r i c a l ba l ance ( F i g . 5 & 6 ) .
2 .4 EXTRACTIOM OE LIPIDS FROM BRADi
D i f f e r e n t p a r t s of t h e b r a i n weighing 100-200 mg were
homogenized i n a g l a s s homogenizer wi th 6 ml of ch loroform-
methanol mix tu re (2 :1 v / v ) , s e p a r a t e l y , accord ing to the
method of Fo lch e t a l . (1951) and d e s c r i b e d by Is lam e t a l .
(1980) . Each homogenate was shaken p e r i o d i c a l l y for an hour
and f i l t e r e d laider vacuum th rough s i n t e r e d g l a s s funnel (G-4)
i n a g radua ted t e s t t u b e . The r e s i d u e of each t e s t tube was
aga in homogenized with 2 ml f r e s h chloroform-methanol mix tu re
and f i l t e r e d . The t e s t t u b e s were then r i n s e d wi th c h l o r o
form-methanol mix tu re and aga in f i l t e r e d . The f i n a l volume
of each e x t r a c t was made up to 10 ml wi th ch loroform-methanol
m i x t u r e . T h e r e a f t e r , 2 .5 ml of s a l i n e s o l u t i o n was added t o
the e x t r a c t i n each g radua ted t e s t t u b e . This was v i g o r o u s l y
shaken f o r t h e complete mixing and was p laced overn igh t i n a
r e f r i g e r a t o r a t 0-5 C. The next day t h e whole of the e x t r a c t
i n each t e s t tube s e p a r a t e d i n t o two d i s t i n c t l a y e r s . The
j u n c t i o n of t h e l a y e r s in each t e s t tube was marked and d e
s i r e d amount of the lower l a y e r of each t e s t tube was
c o l l e c t e d i n a s toppered t e s t tube w i th the he lp of a sy r inge
and s t o r ed a t 0-5°C u n t i l f i n a l u s e . The volume of lower
21
Fig . 5: Photograph showing do r sa l view of the ra t b r a i a .
V
28
CEREBELLUM
FIG 6. DISSECTION OF RAT BRAIN PARTS FOR BIOCHEMICAL STUDIES
'9
layer in each graduated t e s t tube was noted. I t was found
to be 7.0 + 0.1 ml. This ex t rac t was used for the es t imat ion
of t o t a l l i p i d s , phospholipids and c h o l e s t e r o l .
2.5 ESTIMATION OF TOTAL LIPIDS
Total l i p i d s were estimated according to the method of
Woodman and Pr ice (1972) as described below:
Standard Solution
A standard so lu t ion of 0.5 mg brain l i p i d s / m l in ch loro-
form-methanol mixture (2:1 v/v) was prepared by d i l u t i n g 1.0
ml of r e f r ige ra t ed stock solut ion (50 mg bra in I ip id /10 ml,
chloroform-methanol mixture) in a 10 ml stsuidard f l a sk and
made up the volume to 10 ml with chloroform-methanol mixture.
Colouring Reagent
Six gm KH2l*0. and 0.3 gm Vani l l in was dissolved by hea t
ing in double d i s t i l l e d water and made up the volume to 100
ml in a volvimetric f l a sk .
Brain Lipids
Lipids were i s o l a t e d from the ra t bra in by the technique
as described in the Ext rac t ion of Brain L ip ids . The ex t rac t
was dr ied by vacuum r o t a t o r y evaporator at 40^C and dried
l i p i d s were stored at 0-5°C.
Procedure
0.1 ml of brain extract and a set of standard in dupli-
30
0-60
100 600
Fig.7
200 300 400 500 Aig of Total Lipids
Calibration Curve for the Estimation of Total Lipids.
31
cate with 50 to 500 meg of brain lipids were taken to dryneee
in 18 X 150 mm Coming test tubes. 2.5 ml of concentrated
HoSO. were added to each test tube. Thereafter these test c 4
tubes were heated in boiling water bath for 20 minutes. Then
after cooling to room temperature, 5.0 ml colouring reagent
was added to each test tube. The absorption was read at 530
nm exactly after 10 minutes against a reagent blank. The
values of the standard curve were plotted by least square
method.
Calculation
Total lipids, phospholipids and cholesterol were cal
culated by the following formula:
C X V Lipids (mg/g fresh wt.) = •
Vt X Wt
where C = Concentration of lipids in Ug in 0.1 ml extract
(vol\ime taken for the estimation)
V = Total volume of the lower layer
Vt = Volume taken for the estimation
Vt = Fresh weight of the tissue in mg.
2.6 ESTIMATION OF PHOSPHATE
Phosphate was estimated by the well known method of
Fiske and Subba Row as described by Merinetti et al. (1962).
Reagent s
( i ) S t anda rd ; A s tandard of 0.01 mg P/ml was prepared by
32
d i l u t i n g 5 ml of r e f r ige ra t ed stock solut ion (0.43 gm
KH2PO./50O ml in double d i s t i l l e d water) in a volumetric
f lask and made up the volume to 100 ml with double d i s
t i l l e d water.
( i i ) Pe rch lo r ic acid 70?S.
( i i i ) Reducing Reagent; Reducing reagent was prepared by
d i s so lv ing 3.0 gm of a n a l y t i c a l grade sodium b i s u l p h i t e ,
0.6 gm sodium su lph i t e and 0,05 gm r e c r y s t a l l i s e d
l-amiB.o-2 naphthol-4-sulphonic acid (ANSA) in 25 ml of
double d i s t i l l e d water . A s l igh t yellow solut ion thus
obtained was stored in an amber coloured b o t t l e . The
colour i s s t ab l e for a week at room temperature.
( iv) R e c r y s t a l l i s a t i o n of ANSA: 15.0 gm sodium metabisul-
p h i t e , 1,0 gm anhydrous sodium su lph i t e and 1,5 gm crude
ANSA were dissolved in 100 ml double d i s t i l l e d water by
hea t ing . Hot so lu t ion was f i l t e r e d through the f i l t e r
paper. In f i l t r a t e 1.0 ml concentrated HCl was added
and s t i r r e d . P r e c i p i t a t e s are formed which were f i l
tered with suct ion pump and washed with about 30 ml
double d i s t i l l e d water and f i n a l l y with alcohol t i l l
washing i s co lou r l e s s . This pur i f ied ANSA was dried in
oven at 100 C for 1 hour with l e a s t poss ib le exposure
to l i g h t and was t rans fe r red to amber coloured b o t t l e .
Procedure
0.2 ml of brain samples in duplicate were placed in
33
18 I 150 mm Coming test tubes. All the solvent "Has removed
by evaporating on a boiling water bath. 1.0 ml of reagent
grade perchloric acid (70^) was added to each of the samples.
Then the ssunples were digested by heating on a heater for 30
minutes or till the samples were clear. After the digestion,
the samples were cooled to room temperature and 1.5 ml of
ammonium molybdate, 0.2 ml reducing reagent and 7.0 ml double
distilled water were added to each of the samples. Thereafter,
the whole mixture was mixed thoroughly in each test tube. The
tubes were heated for 7 minutes in a boiling water bath. The
colour intensity was read at 700 nm after 30 minutes against
a blank which was prepeared by taking 1.0 ml perchloric acid
and treating it in the same way as samples except digestion.
Preparation of Calibration Curve
A calibration curve was prepared by taking 1.0 meg to
8,0 meg (1.0 meg, 2.0 meg, 3.0 meg, 7.0 meg, and
8.0 meg) of phosphorus in different test tubes and adding
1.0 ml of perchloric acid to each of them and adopting the
same procedure as in the ease of samples except digestion.
The absorption is a linear function of the phosphorus con
tent. The values of these standards were plotted by the least
square method.
Calculation
The amount of vinknown samples can be calculated by
direct proportion with the absorbance obtained for the stan-
34
0-30
60
Fig.8
2 0 3 0 4 0 5 0 ALg of Phosphorous
Calibration Curve for the Estimation of Phosphorus.
35
d a r d . The amoxmt of phosphol ip id i s c a l c u l a t e d by m u l t i p l y i n g
wi th a f a c t o r of 25 .
2 .7 ESTIMATION OF CHOLESTEROL
C h o l e s t e r o l was determined by Lieberman-Burchard r e a c t i o n
as de sc r ibed by Bloor e t a l . (1922) .
Reagent s
(i) Stock Stemdard; A stock standard cholesterol solution
was prepared by dissolving 100 mg AnalR grade choles
terol (made in Germany) in chloroform in a volumetric
flask (10 ml). After the dissolution of the choles
terol, the volxime was made upto the mark, i.e., 10 ml
with chloroform. This gives 10 mg/ml cholesterol solu
tion. It was kept in refrigerator.
(ii) Working Standstrd; Working standard cholesterol solution
was prepared by taking 1.0 ml refrigerated stock stan
dard cholesterol solution in a 10.0 ml volumetric flask
and making the volume with A.R. grade chloroform. This
gives 1.0 mg/ml cholesterol solution.
(iii) Colour Reagent; 50.0 ml analytical grade acetic anhyd
ride was placed in a deep freeze for one hour and 5.0 ml
A.R. grade concentrated sulphuric acid was added drop-
wise with stirring. It was allowed to stand for 10
minutes before use. This reagent must be prepared fresh
each time it is used.
36
Procedure
0.5 ml of brain eamplee vere taken in 15 x 125 nm screw
capped test tubes. To this 4,5 ml analytical grade chloro
form and 1.0 ml colour reagent were added. The mixture was
mixed thoroughly and the test tubes were recapped. There
after the tubes were kept in dark for 50 minutes at 25°C.
The colour intensity was read at 660 nm in a photoelectric
colorimeter against reagent blsink which was prepared by taking
5.0 ml chloroform and 1.0 ml colour regent, and treating it
in the same way as samples.
Calibration of Standard Curve
A standard curve was calibrated by taking 0.1, 0.2, 0.3
0.7 and 0.8 ml of working standgurd cholesterol solu
tion in different screw capped test tubes, making the voliune
up to 5.0 ml in each test tube with chloroform and adding
1.0 ml colour reagent to each test tube and treating them as
samples. The absorption is a linear fimction of the choles
terol content. The values of these standards were plotted
by the least square method.
Calculation
The values of the unknown samples can be directly cal
culated by the proportion with the absorbance obtained for
the standards.
37
0-45
0-40 -
E c
ig 0-30 A
0-25 -
200 300 ^00 500 /Ug of Cho les te ro l
Fig. 9 Calibration Curve for the Estimation of Cholesterol.
38
2.8 ESTIMATION OF GANGLIOSIDES
Gangllosides were estimated according to the method of
Pollet et al. (1978) as described belov.
Reagents
( i ) Standard Solut ion: A stock standard vas prepared by
d i s so lv ing 100 mg N-acetylneuraminic acid (Sigma Chemi
c a l s Co., U.S.A.) in double d i s t i l l e d water and making
the finsil volume to 100 ml. This gives 1.0 mg N-ace ty l
neuraminic acid/ml. Working standard vas prepared by
taking 1.0 ml of stock standard d i l u t i n g i t to 10 ml in
a volumetric f lask with double d i s t i l l e d water. This
i s 100 Ug N-acetylneuraminic acid/ml .
( i i ) Resorcinol Reagent: This reagent was prepared by mixing
10.0 ml of y^ r e so rc ino l so lu t ion in double d i s t i l l e d
water, 80.0 ml of concentrated hydrochloric acid , 0.25
ml of 0.1 M copper sulphate and water (double d i s t i l l e d )
upto 100.0 ml.
Procedure
To 2.0 ml of the upper layer of the l i p i d ex t rac t was
added 2.0 ml of r e so rc ino l reagent in a t e s t tube . I t was
heated in a bo i l i ng water bath for 30 minutes. After cooling
5.0 ml of a mixture of bu ty l ace ta te and n-butanol (85:15,
v/v) was added to each tube . The tubes were shaken thoroughly
and kept for 15 minutes to separate the organic phase. About
39
0.30-
0.28
E c o OQ IT)
no
'in c
Q
u • « - '
a o
004-
100 200 300 ^00 500
/ U g of G a n g l i o s i d e
600
Fig.fO Calibration Curve for the Estimation
of Ganglioside
40
3-4 ml of organic phase was taken and absorbance was measured
at 580 nm against reagent blank in a photoelectric colori
meter.
A standard curve with different concentrations of N-ace-
tylneuraminic acid (5 to 30 meg) having 2.0 ml final volume
of water was prepared by treating in the same way as samples.
Calculation
C X V Gangliosides (mg/g fresh weight) = Vt X Wt
where C = Concentration in tig in 2,0 ml extract
V = Total volume of the upper layer
Vt = Voliime taken for the estimation
Wt = Fresh weight of the tissue in mg.
2.9 DETERMINATION OF RATE OF LIPID PEROXIDATION
The amount of malonaldialdehyde formed/30 minutes during
lipid peroxidation was estimated according to the method of
Utely et al. (1967) as described below.
Reagents
( i ) 0.13 M Potassium Chloride; 2.2368 gm of KCl was d i sso l
ved in double d i s t i l l e d water and the volume was made
200.0 ml.
( i l ) ^0^ (w/v) Tr ich loroace t ic Acid: 10.0 gm of TCA was
41
dissolved in double d i s t i l l e d water and the volume was
made upto 100 ml in a volumetric f lask with double d i s
t i l l e d water.
( i i i ) 0.67?^ 2-Thiobarbi tur ic Acid (TBA) : This was prepared
by d i s so lv ing 0.67 gm of TBA in double d i s t i l l e d water
(25-50 ml) . Two p e l l e t s of NaOH were added to i t and
the f i n a l volume was made upto 100 ml with double d i s
t i l l e d water in a vol\imetric f l ask . The pH of the
so lu t ion was adjusted to 7.2 with HCl (1 .0 N).
Procedure
Different parts of the brain were homogenized (10^ w/v)
in chilled 0.15M potassium chloride. One ml of each homoge-
nate was taken in 25 ml conical flasks and incubated at 37 +
1°C in a metabolic shaker (120 strokes/min., amplitude 1 cm)
for 5 hours. After incubation 1.0 ml of 10?S TCA was added to
it for the precipitation of protein. Thereafter, 1.0 ml of
each reaction mixture was pipetted in centrifuge tubes and
centrifuged at 3»000 rpm for 10 minutes. One ml of the clear
supernatant was mixed with 1.0 ml 0.679 TBA and 1.0 ml double
distilled water. The tubes were placed in a boiling water
bath for 10 minutes, cooled and the absorbance of the coloured
solution was read at 535 nm in a photoelectric colorimeter.
The rate of lipid peroxidation was expressed as nanomoles of
malonaldialdehyde formed per 30 minutes using extinction co
efficient 1.56x10^ as described by Utely et al. (1967).
42
Calculation
Lipid peroxidation was calculated by using the follow
ing formula:
O.D. X 30 X 1000 X 1000 x 1000 x 3 x 2 X =
1.56 X 100000 X 1000 x 180
O.D. X 10
1.56
where X = nanomoles of malonaidialdehyde formed/30 minutes;
O.D, = change of optical density at zero hour and after
3 hours incubation of the same samples.
2.10 STATISTICAL ANALYSIS
The da ta were analysed using S tuden t ' s ' t ' t e s t . Signi
f icant d i f fe rences between means of experimental and cont ro l
groups were ca lcula ted and P values were obtained. P values
l e s s than 0.05 were considered s i g n i f i c a n t .
2.11 ANALYSIS OF VARIANCE (AUOVA)
ANOVA was calculated by using computer "VAX-11/TSO".
The object of Ai OVA was to break up the total variation into
components due to each of the two factors.
(i) Variation within regions,
(ii) Variation due to following 24 hours restraint stress.
They were compared by "F" test.
OBSERVATIONS
43
I I I . OBSERVATIONS
3.1 PHYSICAL SIGNS
After continuous restraint stress for a period of
24 hours, the 6 months as well as 12 months old rats lost
their weights, becsime dull euad lethsurgic. Most of the
animals were quite irritable in comparison to the con
trols (Fig. 11a & 11b).
Autopsy of the stomach revealed production of ulcers
in the gastric mucosa as an important feature in the
stressed rata (Fig. 12a & 12b).
3.2 EFFECT OF RESTRAINT STRESS ON REGIONAL
BRAIN LIPID LEVELS IN AGED RATS
la 6 months old stressed rats, the contents of total
lipids were significantly increased in the cerebrum (P<, 0 .05)
and the cerebellum (P<f0.05), but in the brain stem, increase
was non-significant (9.70?^), however, in the spinal cord the
total lipid levels showed decrease by 6.07^ only, whereas,
in the 12 months old stressed rats, the contents of total
lipids were significantly elevated in the cerebellum
(P<^ 0.001), however, the contents of total lipids were
significantly decreased in the cerebr\im (P<^ 0.05), the
brain stem (P<^0.001) and non-significantly in the spinal
cord (1.54^) (Tables 1 4 2).
Table 3 shows that in 6 months old stressed rats, the
44
Fig. 11: (a) Control anima.'
^^) Efrect of res t ra in t s t ress on the animal.
»
Fig . 12: Gas t r ic mucosal surface of r a t stomach (a) I A cont ro l animals.
F i g . Gast r ic mucosal surface of r a t stomach (b) l a s t ressed ones, mul t ip le u l c e r s
(arrow).
46
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48
l e v e l s of phospholipid were s ign i f i can t ly increased in the
cerebrum ( P < 0 . 0 1 ) , the cerebellum (P<^ 0.05) and the bra in
stem (P<^ 0 .001) , but l eve l s were decreased n o n - s i g n i f i
cant ly in the sp ina l cord ( -14.65^) . On the other hand,
the phospholipid l eve l s in 12 months old s t ressed r a t s showed
s ign i f i can t decrement in a l l the regions of CNS , the ce re
brum (P<Co .05) , the cerebellum ( P < ^ 0 . 0 0 1 ) , the brain stem
(P<^0.01) and the sp ina l cord (P<^0.01) (Table 4 ) .
The concentrat ion of cho les te ro l in 6 months old
s t ressed r a t s (Table 5) was found to be s i g n i f i c a n t l y in
creased in the cerebellum ( P < ^ 0 . 0 5 ) , but non-s ign i f ican t
increase in t he cerebrum (3.58^) as well as in the bra in
stem (6.84?^) while sp ina l cord showed a decrease by 5.25^
only. However, in the 12 months old s t ressed r a t s s i g n i
f ican t deple t ion in the concentrat ion of cho le s t e ro l was
observed in the cerebrum (P<r0 .001) and the bra in stem
(P<^ 0 ,05 ) , but in the cerebellum nonsignif icant e levat ion
was d i s c e r n i b l e . However, in the sp ina l cord decrease was
by 7.58?S only (Table 6 ) .
The concentrat ion of gangl ios ides has been shown in
Tables 7 and 8. The l e v e l of gangl ios ides showed s i g n i
f icant deple t ion in the bra in stem (P<^0,001) and in the
cerebrum and the cerebellum non-s ign i f ican t deple t ion
(10.36% and 11.66%, respec t ive ly) was observed in the
49
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55
6 months old streesed rate. However, the levels of gang-
liosides in the spinal cord were found significantly ele
vated (P <J).01). In the 12 months old stressed albino
rats, the levels of gangliosides showed significant eleva
tion in the cerebellxim (P<^0,01), however, in the cerebrum
and the brain stem significant depletion was observed
(P <J[).001). In the spinal cord, these levels remained
virtually \inaltered (only 2.66^ decrease).
3.3 EFFECT OF RESTRAINT STRESS ON
THE RATE OF LIPID PEROXIDATION
After the restraint stress for 24 hours, alterations
in the rate of lipid peroxidation in different regions of
the central nervous system of aged albino rats (6 months
and 12 months) have been presented in the Tables 9 and 10.
In 6 months old stressed albino rats the rate of lipid per
oxidation showed significant depletion in the cerebrum
(P<^0.01), the cerebellum (P<]0.01) and the brain stem
(P<^0.05). However, in the spinal cord elevation was
only by 4.88^. In 12 months old stressed rats the rate of
lipid peroxidation showed significant increase in the cere
brum (P<^0.01), the cerebellum (P<^0.001), the brain stem
(P<^0.CC1) and the spinal cord (P<^0.01).
56
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58
3.4 AIJALYSIS OF VARIAJ CE (AJ^OVA)
A perusal of tables (11-20) reveals that levels of
total lipids, cholesterol, ganglioside and rate of lipid
peroxidation are significant between different regions of
brain of 6 months old as well as that of 12 months old rats.
However, the levels of phospholipid between different regions
of brain show non-significant trend in both the age groups.
On the other hand, levels of phospholipid and rate of lipid
peroxidation in different regions of brain of stressed rats
of both the age groups, i.e., 6 months and 12 months old are
significant, while the levels of cholesterol show different
trend (significant in 12 months old rats and non-significant
in 6 months old rats). However, the levels of total lipids
and gangliosides in different regions of brain of 6 months
old rats as well as that of 12 months old rats are non-signi
ficant after restraint stress for 24 hours.
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DISCUSSION
69
IV. DISCUSSlOI^
In the present study, following restraint stress, the
levels of total lipids, phospholipids, cholesterol, anglio-
sides and the rate of lipid peroxidation were detennined in
different regions of the brain and the spinal cord of the
albino rats aged six and twelve months. On going through
the available literature, no related references of any pre
vious study dealing with this subject could be obtained.
It is well known that the brain is a very complex and
heterogeneous organ. In some way, it is more a collection
of disparate organs than of a single entity. This hetero
geneity is of great importance in the evaluation and inter
pretation of the biochemical findings (Hertz, 1969).
Lipids are essential components of the central nervous
system and are, therefore, highly enriched in the nervous
system of the vertebrates (Ordy and Kaack, 1975). Myelin
sheath and neuropil of gray matter account for much of the
total lipid in brain tissue (Robins, 1956), making upto 65"
of the dry weight of the white matter and 35-40^ of gray
matter (Brante, 1949). The lipids in the nervous system
form an important part of neurochemical investigations. It
is well known that distinct regional differences occur in
lipid content and turnover in cell types and various pathways
and centres of the brain (Ordy and Kaack, 1975). These pro-
70
cesses are regulated by the appropriate enzymic activities.
According to Veils et al. (1967), lipid content in
creases in developing rat brain. In the present investiga
tion total lipids showed elevation in the cerebrum, cerebellum
and the brain stem in six months old rats. However, total
lipid levels were decreased in all regions except the cere
bellum of the 12 months old rats after 24 hours restraint
stress. Conflicting reports have been appearing in litera
ture regarding the relationship between stressors and total
lipids. Saxena and Tyagi (1981) reported increased lipid
deposition in various parts of the brain during X-irradiation.
Lahiri et al. (1981) observed decrease in total lipids, phos
pholipids and cholesterol contents during beat stress. Simi
larly, the present study revealed that the levels of total
lipids were decreased in the cerebrum, brain stem and the
spinal cord after the restraint stress. White et al. (1978)
have shown that lipids of various tissues are known to be in
a dynamic steady state and there is continuous replacement of
existing molecules by new ones.
Individual lipid classes, particularly membrane phospho
lipids are undergoing constant turnover at different rates
with respect to the structure of the lipid and localization
in different cells and membranes (Porcellati, 1983). Phospho
lipids play an important role in aging studies because they
are an integral part of the membrane lipid layer and changes
71
in composition may result in alteration of the membrane pro
tein activities (S\in and Faudin, 1984). Present study shows
that after restraint stress, in six months old rats, the
levels of phospholipids were increased in the cerebrum, cere
bellum and the brain stem. However, phospholipid levels were
decreased in the cerebrum, cerebellum, brain stem and the
spinal cord of 12 months old rats. A report by Benjamins and
McKhann (1981) showed that the contents of phospholipids
moderately increased (409C-50 ) during development. On the
other hand, Lahiri et al. (1981) reported that the levels of
phospholipids were decreased after heat stress. Norton and
Poduslo (1973) and Horrocks (1968) have also shown decrease
in phospholipid levels in aging processes. The results of the
present study showed that the levels of phospholipids decreased
in 12 months old rats after restraint stress. The present
findings are well in accordance with the above mentioned
reports.
The findings in the present study show that the concen
tration of cholesterol in six months old rats was increased
in the cerebrum, cerebellum and the brain stem, which is in
accordance with the findings of Rouser and Yamamoto (1968,
1969). In 12 months old rats, the level of cholesterol was
decreased in the cerebinim, brain stem and the spinal cord
which is in agreement with Lahiri et al. (1981).
Gangliosides are found in major quantities in the brain
(Ramsay and Nicholas, 1972). They are involved in nerve
impulse conduction since they act as receptor sites for neuro
toxin (Van Heyninger, 1959; North et al., 1961). It has been
demonstrated that the distribution of gangliosides resembles
that of -aminobutyric acid (Lowden and Wolfe, 1964). Results
in the present investigation shov that the concentration of
gangliosides was decreased in various regions of the brain,
and increased in the spinal cord in six months old rats. How
ever, in 12 months old rats, the concentration was decreased
in cerebrum, brain stem and spinal cord, but increased in
cerebellum after restraint stress. Recent reports have shown
that depletion of gangliosides concentration during ageing
is probably related to degeneration of the gangliosides rich
components such as neuronal membrane (Rahmann, 1980; Segler
et al., 1983). Some investigators are of the view that the
degradation of brain gangliosides may be a cause of sequential
removal of mono-saccharides and N-acetylneuraminic acid by
gangliosidases and neuroaminidases (Ledeen et al., 1973; 1977).
The brain membranes are also highly sensitive to peroxi-
dative reactions which are known to occur intracellularly
(Hollcher and Griffin, 1969). Since generation of free radi
cal reaction and lipid peroxidation are on-going processes
occurring throughout the life time of the neurons, damage to
the neuronal membrane is expected to occur with increasing
age.
!'''>
A direct reaction of oxygen and lipid to form free
radical intermediates and to produce semi-stable peroxides
occurs resulting in lipid peroxidation. Apparently, the
brain horaogenate contains the iinsaturated fatty acids and
catalysis necessary for peroxidation in the architecture of
the cell itself, being available for reaction with molecular
oxygen to undergo lipid peroxidation. Subcellular organelles
and biomembrane are the major sites of lipid peroxidation
damage (Tappel, 1970). The peroxidative changes triggered by
free radicals in brain fatty acids and phospholipids may be
of importance in the development of brain cell damage. Highly
reactive molecules with an unpaired electron in an outer orbi
tal, which are known as free radicals, are being formed cons
tantly in various reactions essential for aerobic life. The
best studied effect of free radical attack is that causing
lipid peroxidation, i.e., oxidation of a-methylene bridges of
unsaturated fatty acids, resulting in the formation of lipid
peroxides and hydroperoxides, leading finally to fragmenta
tion of lipids. Mitochondrial swelling and decrease in oxi
dative phosphorylation (Utsumi et al., 1965), and change in
endoplasmic reticulum (Bidlack and Tappel, 1974) have all
been described as biochemical distortions following lipid
peroxidation. Previous studies suggest that radiation hazards
(Inouye et al., 1979) or influence of various environmental
pollutants (Mudd and Freeman, 1977) are closely related to
lipid peroxidation. It was reported that tissues most euscep-
74
ttble to lipid peroxidation appear to be those with low
mitotic rate such as brain (Barber and Wilbur, 1959). Accord
ing to Kartha and Krishnamurthy (1978), the brain showed a
considerably high degree of peroxidation among the various
organs. This might be explained on the basis that the brain
has the largest amount of lipid of all body organs. Recently,
Tayyaba and Hasan (1985) showed that administration of organo-
phoephate metasystox causes dose-dependent increment of lipid
peroxidation in all the regions of central nervous system.
Hasan and All (1981) observed that the increased rate of lipid
peroxidation is associated with increased deposition of lipo-
fuscin pigment in brain cells after organophoephate adminis
tration.
In the present study the rate of lipid peroxidation was
decreased in six months old rats after restraint stress,
whereas in 12 months old rats, it was increased in all the
regions of CNS. In aging process free radical peroxides are
formed leading to disruption of membrane process occurring
throughout the life time. Furthermore, elevation of lipid
peroxides has been observed under the effect of a number of
chemical agents, oxygen toxicity and irradiation (Sharma and
Krishnamurthy, 1980).
Interestingly, the present investigation revealed that
the levels of total lipids were increased but lipid peroxi
dation was decreased in the different brain regions of the
7 5
six months old rats following restraint stress. On the con
trary, total lipid contents were decreased and lipid peroxi
dation was enhanced in the different "brain regions of the
12 months old rats.
From this report, it is apparent that increased aging
has certainly affected the regulation of lipids and their
metabolites. Also, the restraint stress has enhanced the
lipid peroxidation, leading to membrane damage.
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7 6
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LIST OF PUBLICATION AND PRBSENTATIONS
PUBLICATION
1. Gupta A and Haeaa M. A g e - r e l a t e d changes i n l i p i d p r o f i l e s and l i p i d p e r o x i d a t i o n in t h e GNS fo l lowing r e s t r a i n t s t r e s s . Ind J Med Res . ( i n P r e s s ) .
PRESENTATIONS
1. Grupta A and Hasaa M. R e s t r a i n t s t r e s s - i m d u c e d a l t e r a t i o n s
i n CHS l i p i d p r o f i l e s of t h e ag ing r a t s . P r e s e n t e d a t
Thi rd N a t i o n a l Conference on Aging, -vrith a s p e c i a l s e s s i o n
on B r a i n Aging, he ld a t School of L i f e S c i e n c e s , U n i v e r
s i t y of Hyderabad, Hyderabad on Nov. 26 -28 , 1986.
2 . Gupta A, Hasan M and Haider S S. A l t e r a t i o n s i n l i p i d
p r o f i l e s and l i p i d p e r o x i d a t i o n i n v a r i o u s r e g i o n s of t h e
b r a i n of aging r a t s fo l l owing r e s t r a i n t s t r e s s . P r e s e n t e d
a t XXIV N a t i o n a l Conference of Anatomical Soc ie ty of I n d i a
he ld a t J . N . Medica l C o l l e g e , A.M.U., A l i g a r h on Dec.
28-50 , 1986.
5 . Hasan M, Gupta A and Chandra S V. Trace e lements in d i s
c r e t e r e g i o n s of t h e r a t CNS: Age r e l a t e d and r e s t r a i n t
s t r e s s - i n d u c e d changes . P r e s e n t e d a t I n t e r n a t i o n a l sympo
sium on Metabolism of m i n e r a l s and t r a c e e lements i n human
d i s e a s e s h e l d a t A l i g a r h , New D e l h i and S r i Nagar (Kashmir)
from 18 t o 30 S e p t . , 1987.
4 . Hasan M, Gupta A and Chandra S V. S i g n i f i c a n t a g e - r e l a t e d
e l e v a t i o n of aluminium i n d i s c r e t e r e g i o n s of t h e r a t CHS:
D e p l e t i o n by r e s t r a i n t s t r e s s . P r e s e n t e d a t t he 36 th
N a t i o n a l Conference of Anatomical S o c i e t y of I n d i a he ld on
28-30 D e c , 1987 a t P t . J.N.M. Medica l CoUege , Ra ipu r .
