International Journal of Biological & Medical ResearchThamaraiselvi K*, Mathangi DC a,Subhashini ASb. A R T I C L E I N F O A B S T R A C T Keywords: REM sleep deprivation Free radicals

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International Journal of Biological & Medical Research

Int J Biol Med Res. 2012; 3(2):1754-1759

Effect of increase in duration of REM sleep deprivation on lipid peroxidation* a bThamaraiselvi K , Mathangi DC ,Subhashini AS .

A R T I C L E I N F O A B S T R A C T

Keywords:

REM sleep deprivationFree radicalsLipid peroxidationMDA

Original article

1. Introduction

Sleep deprivation is becoming common in this fast moving world. Sleep deprivation has been

shown to have deleterious effect on the human body, giving rise to many diseases and disorders

and REM sleep deprivation is shown to increase free radicals and cause more harm as the

duration increases. The aim of this study is to assess the effect of increase in duration of REM

sleep deprivation on lipid peroxidation. REM sleep deprivation cause an increase of free radical

induced damage in blood. This can be shown by estimation of plasma malondialdehyde (MDA)

level. Wistar strain male albino rats were used to study the effect of REM sleep deprivation.

They were divided into two groups, control and REM sleep deprived. REM sleep deprivation

was obtained by inverted flowerpot technique. Heparinised blood sample was taken and

assessed for plasma MDA level. Body weight and food intake was monitored. The values

obtained were statistically analysed using one way analysis of variance (ANOVA). On

significant f test ratio, Tukey's multiple comparison tests were also performed. The level of

significance was kept at P<0.05. All the results obtained from the study are expressed as mean +

standard deviation (SD). The result showed an increase in plasma MDA level as the duration of

sleep deprivation increased, 48hrs (8.9* ± 2.87) and 72hrs (10.8* ± 2.4). There was only

minimal change in 24hrs (7.1 ± 0.76) and 96hrs (6.4 ± 1.6) when compared to the control

animals (5.4 ± 0.79) which were not significant. This increase in MDA level can be attributed to

increase in free radical formation and damage, as the duration of REM sleep deprivation

increases. So sleep is important in life, especially REM sleep.

In this fast moving world, with cost of living soaring sky high

people are finding it difficult to make both ends meet. To meet the

requirements money is needed and money is earned by sacrificing

the basic need of the body i.e. sleep. Sleep is one of the important

needs like oxygen, nutrition for survival. Sleep has been classified

into two stages, non rapid eye movement (NREM) and rapid eye

movement (REM) sleep. It has been shown that rapid eye

movement (REM) sleep is important as REM deprivation has shown

many deleterious and unwanted harmful effects. REM sleep might

participate in the genesis or regulation of at least the following

functions, dreaming experience, basic rest activity cycle,

motivational and adaptive behaviours, internal stimulation of the

immature brain, programming of the behavioural code, memory

consolidation, unlearning of trivial memory traces, amplifying of

Number of theories has been proposed on sleep like cerebral

anaemia, fatigue theory, theory of diminished sensory input and

chemical theories. Free radical flux theory of Reimund,[5] is a

recent addition. Free radical flux theory of sleep by Reimund, [5],

states that free radicals accumulate during wakefulness and are

removed during sleep. Removal of excess free radicals during sleep

is accomplished by decreased rate of free radicals and increased

efficiency of endogenous antioxidant mechanism. Thus sleep

essentially has antioxidative role.

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*Department of Physiology, SRM Medical College Hospital & Research Center, Kattangulathur, a Department of Physiology, Chettinadu Medical College Hospital, Chennai, b Department of Physiology, Sri Ramachandra Medical College Hospital & Research Institute, Chennai.

* Corresponding Author : Dr. K.ThamaraiSelvi

Associate Professor, Department of Physiology, SRM Medical College Hospital & Research Center, Kattankulathur, 603209, Kancheepuram District, Tamilnadu. E-mail: [email protected]

Copyright 2010 BioMedSciDirect Publications. All rights reserved.c

1755Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-1759

Paradoxical sleep deprivation was found to be followed by

deep destructive changes in mitochondrial membranes of the

brain and erythrocytes. At the same time the activity of the

antiradical defence system was abnormal changes in the ratio of

particular lipid components and impairing the functional activity

of biological membranes [6].

Experimental studies on sleep deprivation and the harmful

effects have been recognised for some time [7]. If the assumption

of Reimund, [5], ie., freeradicals produced during waking, are

removed during sleep were correct, one would predict that sleep

deprivation would be associated with an accumulation of free

radicals. It was suggested that free radicals are involved in the

pathogenesis of various human disease, psychological stress and

the normal ageing process [8]. In rats it was shown that prolonged

sleep deprivation leads to a syndrome characterized by increased

food intake, weight loss, increased energy expenditure,

progressive decline in body temperature which is invariably fatal

[9]. It was also proposed that the neural activity associated with

sleep deprivation for longer durations may damage brain cells and

eventually to cell death [10, 11]. Reimund, [5], showed that

increase in free radicals is associated with sleep deprivation.

According to Zepelin and Rechtschaffen, [12], sleep seems to limit

metabolic requirements. Therefore sleep deprivation could

enhance the metabolic rate and in turn increase oxidative stress.

But Almeida et al., [13], showed that there is no oxidative stress

following paradoxical sleep deprivation in rats. Cirelli,[14], has

shown that there is no evidence of brain cell degeneration after a

long –term sleep deprivation. No change was detected in TBAR

substances [15] .Gopalakrishnan et al showed that there was no

evidence of oxidative damage at the lipid or the peripheral tissues

[16].Neurons are more prone to oxidative stress relative to other

cell types [17].

As the oxidative products of free radicals in the body may be

present in the blood, so an estimation of these products in the

blood will show the level of free radical induced damage in the

body and the importance of REM sleep.

Reports of free radical level and free radical induced cell

damage following sleep deprivation are scanty and also

contradictory. Aim of the study is to study the effects of REM sleep

deprivation on free radical induced damage in the blood in Wistar

strain rats and to elucidate the effect of duration of REM sleep

deprivation on these damages.

Wistar strain male albino rats weighing between 150- 180

gms, were used as the experimental animal for this study. Ethical

clearance was obtained from the Ethical Committee through the

institution before the study.

The animals were divided into three groups of Control, REM

sleep deprived and REM sleep control.

Group I – Control animals (n=6): These animals were used for

evaluating the baseline values for the various parameters to be

assessed.

2. Materials and method

2.1 Food intake and body weight

2.2 Estimation of plasma MDA level for lipid peroxidation [19]

2.3 Statistical Analysis

Group II – REM sleep deprived animals: These animals were used

to study the effect of REM sleep deprivation on lipid peroxidation

in the blood.

Group III – REM sleep control animals : These animals were used to

assess whether the REM sleep deprivation or any other effect like

isolation, immobilisation involved in the REM sleep deprivation

procedure.

The groups II and III were further subdivided into four groups

based on the duration of study as (a) 24 hrs. (b) 48 hrs. (c) 72 hrs.

(d) 96 hrs. With six animals in each group.

The animals were provided with food and water ad libitium, with

12 hrs light and 12 hrs dark cycle at ambient room temperature (24

- 28°C).

A ] REM sleep deprivation

An ingenous method of Jovet et al.,[18], was used for REM sleep

deprivation. Here the animals were placed on small platform

(diameter 6.5 cms) surrounded by a pool of water, where the

platform is small enough with reference to the body size of the

animal. This technique is based on the loss of skeletal muscle tone

typical of REM sleep is accompanied by the animal falling into

water.

B] REM sleep control

The REM sleep control animals were also placed in the similar

condition but with the platform size increased (diameter15 cms)

which allowed the relaxed position of REM sleep. This was done to

rule out the possibilities of non- specific effects like isolation.

The body weight of the animals was estimated before and

during the REM sleep deprivation and amount of food intake was

also measured during the period of REM sleep deprivation. This

was done to see whether there was any change in the body weight

and amount of food due to REM sleep deprivation.

The animals were sacrificed with mild ether anaesthesia at the

stipulated time as per the subgrouping (ie.24, 48, 72 and 96 hours).

Heparinised blood samples were collected from jugular vein.

The heparinised blood was centrifuged at 3000 rpm for half an

hour and plasma was obtained. Malondialdehyde reacts with

thiobarbituric acid to generate a coloured complex which was read

in spectrophotometer at an absorbance of 532nm.

The values obtained were statistically analysed using one way

analysis of variance (ANOVA). On significant f test ratio, Tukey's

multiple comparison tests was also performed. The level of

significance was kept at P<0.05.

All the results obtained from the study are expressed as mean +

standard deviation (SD).

1756

The animals subjected to REM sleep deprivation showed an increase in food intake in all the subgroups, 24hrs (57 + 19), 48hrs (57 +26) ,

72 hrs (73 + 25) and 96hrs (70 ±27), as compared to control (17 ±2.4)

b) Body weight (gms) (Table -1, Figure-II)

Body weight monitored showed a steady decline in all subgrouping of REM sleep deprivation inspite of being hyperphagic. i.e.,24hrs (166

+ 19), 48hrs (160±+18) , 72 hrs (157 ± 19) and 96hrs (147 ± 17), as compared to control (180 ±18)

c) Lipid peroxidation ( moles MDA /ml) Table -2, Figure-III)

MDA level estimated in the animals showed an increase following REM sleep deprivation for 48 hrs (8.9 ± 2.87) and 72 hrs (10.8 ± 2.4).

But there was only minimum change in 24hrs (7.1 ± 0.760 and 96 hrs (6.4 ± 1.6) when compared to the control animals (5.4 ± 0.790 which

were not significant.

To rule out the possible non-specific effects attributed to the technique of REM sleep deprivation like isolation, immobility, all the results

were compared with sleep control animals.

3. Results

I.REM sleep Deprivation

a) Food Intake (gms) (Table -1, Figure-I)

Table -1. Effect of REM sleep deprivation on food intake & body weight changes

Food intake (gms)

Body weight (gms)

REM deprived

REM control

REM deprived

REM control

n=6 in each group, values given as mean ± SD, *P<0.05

17 2.4

17 3

180 18

168 8

±

±

±

±

57* 19

37* 9

166 19

159 12

±

±

±

±

57* 26

56* 11

160 18

153 13

±

±

±

±

73* 25

71* 71

57 19

153 12

±

±

±

±

70* 25

72* 61

47* 17

150 12

±

±

±

±

6.531

54.252

2.25

2.02

Control 24hrs 48hrs 72hrs 96hrs 'F' test ratio

Table -2. Effect of REM sleep deprivation on lipid peroxidation

Figure I Effect of REM sleep deprivation on food intake. Figure II Effect of REM sleep deprivation on body weight

LPO (n moles MDA / ml plasma


5.4+ 0.79 7.1+ 0.76 8.9*+ 2.87 10.8*+ 2.4 6.4+ 1.6 7.745

Control 24hrs 48hrs 72hrs 96hrs 'F' test ratio

Thamaraiselvi et.al / Int J Biol Med Res. 2012; 3(2):1754-1759

1757

II- REM sleep control

a) Food intake (gms) table-1, Figure-I)

The food intake estimated showed an increase in all the

subgroups of 24hrs (37 ± 9), 48hrs (56 ± 11), 72hrs (71 ± 7) and 96

hrs (72 ± 6) compared to control animals.

b) Body weight (gms) (table-2. Figure-II)

Body weight monitored showed only minimal changes in all the

subgroups of 24hrs (159 ± 12), 48hrs (153 ±13) , 72hrs (153 ± 12),

and 96 hrs (150 ± 12) compared to control animals (168 +8)

c) Lipid peroxidation (n moles MDA/ml) (Table -3 Figure-III )

REM sleep control showed minimum changes in MDA level in all

subgroups of 24hrs (4.75 ± 1), 48hrs (6.319 ± 2.24), 72hrs (5.29

± 1.5) and 96hrs (4.888 ± 1.1) when compared to the control

animals (5.4 ± 0.79 ) which were also not significant.

The above table shows comparison between control, REM sleep

deprived and REM sleep control on lipid peroxidation values of 24

hrs duration.


deprived and REM sleep control, on lipid peroxidation values of 48

hrs duration.


deprived and REM sleep control, on lipid peroxidation values of 72

hrs duration.




Table 3

Table 4

Table 5

LPO (n moles MDA / ml plasma)

LPO(n moles MDA / ml plasma)


5.4 0.79±

5.4 0.79±

5.4 0.79±

7.1 0.76±

8.9* 2.87±

10.8* 2.4±

4.75 1.0±

6.319 2.24±

5.29 1.5±

12.077

4.398

20.428

24 hrs

48 hrs

72 hrs

Control

Control

Control

REM deprived

REM deprived

REM deprived

REM control

REM control

REM control

'F' test ratio

'F' test ratio

'F' test ratio


deprived and REM sleep control, on lipid peroxidation values of

96hrs duration.

Free radicals are very reactive molecules or fraction of

molecules, which contain one or more unpaired electrons; they

exist independently and are very unstable. The free radical often

reacts non-specifically with any other molecules, which are usually

paired and forms another free radical. Oxidative stress refers to a

disturbance in the pro and antioxidant balance in favour of the pro

oxidant [20]. Oxidative stress ensues when free radicals evade or

overwhelm the antioxidant protective mechanisms of cells and

tissues. It induces cytotoxic effects that are mediated by

perturbation of intracellular free calcium and thiol homeostasis. An

early response to oxidative stress is depletion of cellular soluble

and protein bound thiols. Therefore it causes decreased plasma

membrane Ca2+ ATPase activity, plasma membrane blebbing,

decreased mitochondrial ability to retain Ca2+, decreased Ca

sequestration capacity into endoplasmic reticulum and alters

microsomal Ca homeostasis. Free radical damage includes lipid

peroxidation of membrane, mitochondrial damage, lesions in DNA

and cross linking of proteins [21]


Table 6

Figure III Effect of REM sleep deprivation on lipid peroxidation


5.4 0.79± 6.4 1.6± 4.888 1.1± 2.551

96 hrs Control REM deprived REM control 'F' test ratio

4. Discussion


In todays world free radicals are held culprit to various

diseases that affect human from infection to ageing and cancer. Co

morbidities linked to disturbed or impaired sleep such as

cardiovascular disease, diabetes, arthritis are increased.[ 22,23]

increased lipid peroxidation was seen in diabetes.[ 24 ]

This effect is enhanced by increase in amount of sources that

produce free radicals like pollution, smoking etc. the effect of free

radicals on our body are due to the damage caused to cellular

elements, and lipid peroxidation is one of them.

Reimund [5], hypothesised that free radicals which

accumulate during wakefulness are removed during sleep.

Removal of excess free radicals during sleep is accomplished by

decreased rate of formation of free radicals and increased

efficiency of endogenous antioxidant mechanisms. Thus sleep has

an antioxidative role.

Studies have been done on the effect of REM sleep deprivation

on lipid peroxidation, but these studies are scanty and also

contradictory, so the aim of our study is to know the effect of REM

sleep deprivation on the lipid peroxidation in the blood of Wistar

strain rats.

To deprive REM sleep in rats the technique developed by

Jouvet et al., [15], was followed where the animals are placed on a

small platform surrounded by water. The advantages of this

technique are that several animals can be deprived

s i m u l t a n e o u s ly, w i t h o u t l a b o r i o u s m o n i t o r i n g o f

electrophysiological characteristics of sleep.

Coenen and Van Luijtelaar [25], have shown that there was not

much difference in the various REM sleep deprivation protocols

like classical platform, pendulum, multiple platform. Both stress

index, (Selyes classical indices, adrenal hypertrophy, thymus

atrophy, body weight loss and stomach ulceration) as well as EEG

recording did not show significant differences between the

various REM sleep deprivation procedures [26].

But this technique was heavily criticised for its several stress

factors like restriction of movement, isolation, recurrent wetting,

emotional distress which are inherent to the procedure [27]. This

technique is at present accepted as a valid method if comparisons

were made with animals on large platform allowing the relaxed

position of REM sleep and without danger of falling into water

[2,29]. Hence a REM sleep control was also included in this study.

The body weight and food intake were monitored in this study

and it was found that there was a steady decline in the body weight

as the duration of REM sleep deprivation increases, though there

was a steady increase in food intake. This loss of weight despite

h y p e r p h a g i a w a s a l s o s h o w n b y o t h e r w o r k s

[25,29,30,31,32].Study by Kushida et al.,[31], also showed that

REM sleep deprivation caused weight loss in spite of increased

food intake, which they attributed to increased energy

expenditure with mean levels reaching more than twice base line

values. Though increased food intake was also observed in REM

sleep control animals it was not accompanied by decreased body

weight is peculiar to REM sleep deprivation as also shown [32].

4.Conclusion

5.References

The 24hrs and 96hrs REM sleep deprived animals of the

present study showed no change in the MDA level. This shows that

both short term sleep deprivation and long term sleep deprivation

do not produce symptoms of oxidative stress. This in agreement

with the reports of Cerelli et al.[14] ( REM sleep deprivation of 8hrs

) and Almedia et al.[13] ( REM sleep deprivation of 96hrs

respectively. 48hrs and 72hrs of REM sleep deprived animals

showed an increase in the level of MDA and REM sleep control

animals showed minimal changes. This is in correlation with

studies which showed that 96 hrs REM sleep deprivation caused

severe motor weakness, lesion in various organs and systems and

eventually death in the animals [9]. This was attributed to

accumulation of free radicals. Halliwell and Gutteridge [8], also

showed an increase in the MDA level in REM sleep deprived

animals. According to Zeppellin, and Rechtschaffen [12] sleep

seems to limit metabolic requirements. Therefore sleep

deprivation could enhance the metabolic rate and in turn increase

oxidative stress.

From the results obtained by this study and in comparison

with other studies we can infer that REM sleep has an antioxidative

effect and deprivation leads increase in free radical generation and

free radical induced cell damage. This study supports Reimunds

hypothesis of antioxidative function for sleep. In spite of the

number of studies conducted and various contradictory results

that were put forward, the problem to arrive at a definite

conclusion is still an open question. So the future to such study lies

in devising an easy technique of REM sleep deprivation without the

non specific effects, study the behavioural changes due to REM

sleep deprivation, and study the antioxidant defense mechanism.

Study the effect of reabsorptive sleep, to see the return to baseline

value. Free radicals, the cause of many diseases, formed during

awake state are removed during sleep and the effect is more as the

duration of sleep deprivation increases. So sleep is very important

for a person to lead a normal healthy lifes.

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International Journal of Biological & Medical ResearchThamaraiselvi K*, Mathangi DC a,Subhashini ASb. A R T I C L E I N F O A B S T R A C T Keywords: REM sleep deprivation Free radicals