Molecular and Cellular Biochemistry (2018) 449:63–72

https://doi.org/10.1007/s11010-018-3343-7

Low-grade neuroinflammation due to chronic sleep deprivation

results in anxiety and learning and memory impairments
Shaffi Manchanda1 · Harpal Singh1 · Taranjeet Kaur1 · Gurcharan Kaur1

Received: 28 December 2017 / Accepted: 7 March 2018 / Published online: 16 March 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Chronic sleep loss/fragmentation prevalent in the current 24/7 society is associated with irreversible consequences on health
and overall wellbeing. Various studies have well documented the ill effects of acute sleep loss on cognitive functions of
individuals; however, the underlying mechanism behind the chronic sleep loss is yet to be explored. The present study was
aimed to investigate whether chronic sleep deprivation (CSD) triggers anxiety-like behaviour and memory decline in male
Wistar rats. Rats were sleep deprived by placing them over slowly rotating drum (2 rpm) for 18 h (between 4 pm and 10 am)
followed by 6 h of recovery sleep for 21 consecutive days. Post CSD regimen, rats were subjected to behavioural tests such
as elevated plus maze (EPM), Novel Object Recognition (NOR) and Rotarod performance test and then sacrificed to remove
brain for further molecular studies. The study demonstrated that CSD rats showed anxiogenic behaviour along with recogni-
tion memory decline compared to control rats. CSD rats further showed elevated levels of inflammatory cytokines (TNFα,
IL-1β) along with activation of NFκB and AP1 transcription factors in hippocampus and piriform cortex (PC) regions of
brain. These observations were also accompanied by enhanced expression of GFAP and Iba1 in the two brain regions. The
data suggest that CSD triggered low-grade neuroinflammation which caused anxiogenic response and recognition memory
impairment. The study provides preliminary leads to further explore the role of astrocytes/microglial cells and inflammatory
cytokines in mediating these neurobehavioural consequences of chronic sleep loss and to develop effective interventions to
combat them.

Keywords  Chronic sleep deprivation · Neuroinflammation · Inflammatory mediators · Astrogliosis · Microgliosis

Introduction chronic periods of total sleep loss and sleep fragmentation

Chronic sleep loss, defined as sleep obtained ≤ 6-h/24-h
period, is prevalent in the modern societal setup due to pro-
fessional commitments and lifestyle factors. Serious conse-
quences of sleep loss documented so far include compro-
mised neurogenesis, cognitive functioning (poor vigilance
and performance, deficits in learning and memory), meta-
bolic and cardiovascular problems, immune disturbances
and blood–brain barrier disruption [1]. Both acute and
Shaffi Manchanda, Harpal Singh, Taranjeet Kaur have contributed
equally.
* Gurcharan Kaur
kgurcharan.neuro@yahoo.com
1
Medical Biotechnology Laboratory, Department
of Biotechnology, Guru Nanak Dev University, Amritsar,
Punjab, India
are associated with immune system dysfunction (cellular
and humoral) [2], but the mechanism behind this is not well
understood under different sleep loss conditions. In addition
to neurons, non-neuronal cells such as oligodendrocytes,
astrocytes and microglia also play critical role in synaptic
maintenance and homeostasis [3–5]. Recent studies provide
evidence that both chronic sleep loss and fragmentation can
activate these cell types and influence the behavioural and
physiological state [6–9]. Astrocytes demonstrate more
pronounced response to wakefulness than oligodendrocytes
with multifold activation in the genes during wake period
than sleep [8]. Impact of short-term sleep loss on learning
and memory impairment has now been firmly established
and demonstrated by various studies [10–14]. Acute sleep
loss has also been reported to lead to anxiety-like behaviour
and impairment in learning and memory as reported by our
lab [9, 13, 14] as well as others [15, 16]. Activation of sym-
pathetic nervous system and hypothalamic–pituitary–adrenal

13
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64
axis after sleep disturbance [17] lead to the heightened
pro-inflammatory state [18]. Various clinical studies [19,
20] have also documented the occurrence of inflammation
caused due to sleep loss. Sleep disturbance has also been
reported to induce microglial activation in the mouse hip-
pocampus region along with elevated levels of IL-6 [21].
Literature reports on acute sleep deprivation regimen have
suggested the link between sleep loss and neuroinflammation
but whether chronic sleep loss causes impairments to same
extent as acute sleep loss is yet to be explored.
The present study was aimed to investigate whether
chronic sleep deprivation (CSD) of 18 h for 21 consecutive
days in male Wistar rats triggers anxiety-like behaviour and
memory decline accompanied by activation of inflammatory
mediators with reactive astrogliosis and microgliosis. Rats
were sleep deprived for 18 h (between 4 pm and 10 am) fol-
lowed by 6 h of recovery sleep for 21 consecutive days. We
have used rotating drum method to induce sleep deprivation
in rats. This method induces sleep fragmentation rather than
total sleep loss as animals may have microsleeps between the
time, rotating drum disc. Post CSD regimen, rats were first
subjected to behavioural tests such as elevated plus maze
(EPM) test to assess the anxiety levels, Novel Object Recog-
nition (NOR) test to elucidate learning and memory impair-
ments and Rotarod performance test for motor coordination
and then sacrificed to remove brain for further molecular
studies. To investigate the underlying mechanisms of chronic
sleep deprivation-associated neurobehavioural alterations,
we studied the expression of pro-inflammatory mediators,
viz. TNFα (tumour necrosis factor α), IL-1β (Interleukin-1β)
and IL-6 (Interleukin-6) and their downstream transcrip-
tional activation of NFκB (nuclear factor-κB) and AP1
(activator protein 1). NFκB occupies a vital position in the
inflammatory cascade, where it controls the production of
numerous pro-inflammatory mediators [22, 23], whereas
AP1 has been implicated in mediating cell survival by
inducing apoptosis under stressful conditions [24]. Occur-
rence of reactive astrogliosis and microgliosis under CSD
conditions was ascertained by studying the expression of
astroglial cell-specific marker GFAP (glial fibrillary acidic
protein) and microglial cell-specific marker Iba1 (Ionized
calcium-binding adapter molecule 1) from hippocampus and
piriform cortex (PC) regions of brain.
Materials and methods
Animals and sleep deprivation regimen
Wistar albino male rats in the age group of 4–8 months,
weighing 260–275 g were caged in the group of three under
controlled environmental conditions (constant temperature
of 25 ± 2 °C and constant dark/light cycle of 12:12 h) with ad
13
Molecular and Cellular Biochemistry (2018) 449:63–72
libitum food and water supply. For experimental study, ani-
mals were grouped into two groups—(I) control rats allowed
undisturbed sleep during light phase and (II) Chronic Sleep
Deprived (CSD) rats deprived of sleep for duration of 18 h
between 4 pm and 10 am for 21 days, Forced locomotion
method was used for sleep deprivation by placing animals
on slowly rotating drum with dimensions (40 cm diame-
ter × 30 cm height) operating at a speed of 2 rpm and were
allowed to sleep for 6-h duration between 10 am and 4 pm
for 21 consecutive days. All the animals were then subjected
to behavioural studies to test their memory and anxiety sta-
tus after the regimen. All animal experimental protocols
were approved by Institutional Animal Ethical Committee,
Guru Nanak Dev University, Amritsar, India (permission
number 226/CPCSEA-A/2014/37) and performed in accord-
ance with the guidelines of ‘Animal Care and Use’ laid down
by Institutional Animal Ethical Committee, Guru Nanak Dev
University.
Behavioural studies
Elevated plus maze (EPM) test
The animals (n = 8 per group) were subjected to EPM test
post-regimen on 22nd day starting from 10:00 am onwards,
which consisted of two opposing open (50 × 10 cm) and
closed arms (50 × 10 × 50 cm) extending from the central
platform (10 × 10 cm), crossing each other in the form of a
plus, elevated 50 cm above the floor. At the beginning of the
test, the individual rats were placed on the central platform
facing one of the closed arms and were allowed to freely
explore the apparatus for 5 min. Whole experiment was per-
formed in dim light to encourage rats to explore open arms.
The behaviour of each rat was monitored using a video cam-
era and their movements were registered and analysed. The
whole apparatus was thoroughly cleaned using 5% ethanol
before placing next rat. An entry into any arm was defined as
entry of all four limbs into that arm. A crossing was defined
as animals emerging from one arm and entering with all four
paws, to its opposite arm. Head dipping is defined as looking
down from the edge of an open arm or the central platform.
Videos were analysed by multiple observers blinded to the
experiment.
Novel object recognition (NOR) test
Rats (n = 8 per group) were subjected to NOR test for testing
their object recognition and working memory. The rats were
habituated to the empty box (100 × 50 × 50 cm) for 5 min
over 2 days at the start of dark phase. The activities of the
rats were recorded using a video camera. This was followed
by the familiarization phase during which rats were placed in
the box with two similar sample objects without having any
Molecular and Cellular Biochemistry (2018) 449:63–72 65
specific odour and were allowed to explore both the objects
for 5 min up to 3 days. The objects were cleaned thoroughly
with 70% ethanol to ensure the absence of olfactory cues.
On the test phase, the least preferred sample object was
replaced by a novel object to check the recognition memory
of the rats. Between each phase, there was a gap of 24 h. The
exploration of object was defined by sniffing, licking, chew-
ing by rats or by moving vibrissae while directing the nose
towards and less than 1 cm from the object. The number of
episodes and time spent in exploration of each object by each
animal was recoded. The preference index between the two
objects was calculated as
Preference index = Time spent in exploring new object∕Total time in exploration.
Grooming and rearing
We further analysed grooming behaviour in these animals in
the open field box during NOR test to correlate the effect of
stress and anxiety caused by extended wakefulness on mem-
ory and cognition. The grooming behaviour in rats is defined
as the duration of time spent by the animal in licking various
body parts including paw licking, head washing, body fur
grooming with hind paws, leg and genital scratching. Any
grooming bout of duration more than 5 s was recorded as
actual grooming bout. Total time spent in grooming activity
and latency of grooming i.e. grooming bouts was recorded
using video recording and averaged for each group. Rearing
is a frequent behavioural act defined as standing up on the
hind limbs with or without keeping the forelimbs on the
wall. It is a measure of environmental novelty used to meas-
ure learning and memory. Number of rearing episodes were
recorded for each rat for every 5-min session during NOR.
Rotarod performance test
Rotarod apparatus is an automatic motor-driven tread-
mill (Rotamex-5; Columbus Instruments) consisting of a
7.0 × 9.5 cm spindle diameter with a fall height of 44.5 cm
from the centre. The Rotarod performance test was per-
formed on each animal (n = 8 per group) at a fixed speed of
10 rpm for duration of 300 s. The number of falls and the
time spent on the rotating rod was recorded as a measure of
running performance for each trial and averaged for each
group.
Western blotting
Rats (n = 4 for each group) were anesthetized using thio-
pentone injection (1 unit per 10 g) and sacrificed to dissect
out their brain. PC and hippocampus regions were dissected
out and lysed in lysing buffer using tissue homogenizer and
centrifuged at 5000 rpm for 10 min at 4 ̊C. Supernatant was
collected after centrifugation and used for protein estima-
tion by Bradford method. 50 µg of protein from each sample
was mixed with 6X sample buffer. Protein samples were
resolved in 7–10% SDS-PAGE followed by transfer onto a
0.45 µm pore size PVDF membrane (Hybond-P from Amer-
sham Biosciences UK Limited) using the semi-dry Novablot
system (Amersham Biosciences UK Limited) at 90 mA for
90 min. Transferred membranes were then blocked with
5% skimmed milk in 0.1% TBS-Tween 20 for 2 h. Further,
membranes were probed with mouse monoclonal anti-Iba1
(1:1000) (EMD Millipore, Catalogue number MABN92),
anti-GFAP (1:5000) (Sigma-Aldrich, Catalogue number
G3893), anti-AP1 (1:2000) (Sigma-Aldrich, Catalogue num-
ber A5968), anti-TNFα (1:1000) (Sigma-Aldrich, Catalogue
number SAB1404480), anti-IL-1β (1:1000) (Sigma-Aldrich,
Catalogue number SRP8033), anti-IL-6 (1:1000) (Sigma-
Aldrich, Catalogue number SAB5300276) and anti- rabbit
NFκB (1:2000) (Cell Signaling Technology, Catalogue num-
ber 8242) for overnight at 4 °C. This was followed by three
washings with 0.1% TBS-Triton-X 100 of 10 min each and
incubation with HRP-labelled anti-mouse IgG (1:2500) or
anti-rabbit IgG (1:2500) secondary antibodies (Genei) for
2 h at 25 °C. Membranes were again washed 0.1% TBS-
Triton-X 100 thrice for 10 min each. Immunoreactive bands
were detected by Amersham ECL Plus Western blot detec-
tion system (GE Healthcare Life Sciences UK Limited)
using Image Quant LAS 4000 (GE Healthcare Life Sciences
UK Limited). α-tubulin has been used as an endogenous
control for normalizing the expression of the proteins probed
on the membrane. Change in expression of protein of interest
was taken as the average of integrated density values (IDV)
obtained from at least three independent experiments.
Statistical analysis
Values were expressed as mean ± SEM of the values
obtained from at least three independent experiments.
The Sigma Stat for Windows (version 3.5) was used to
analyse the results by Student’s t test and one-way ANOVA
(Holm–Sidak post hoc method), in order to determine
the significance of the mean values. Two-way ANOVA
was performed on EPM test data and NOR test data to
determine the level of significance for the parameters
studied within the group and between the groups. One-
way ANOVA was used to analyse the data of rotarod
performance test and Western blotting. Values with p
value ≤ 0.05 were considered as significant.
13

66 Molecular and Cellular Biochemistry (2018) 449:63–72

(a) (b)
300 Open Arm 7 Open Arm
Closed Arm #
Closed Arm

No. of entries in open and closed

#
Time spent in open and closed

250 Central Open Area # 6

#
5
200
arm (seconds)

arm
150
3
100 $
$ 2
50 1
*
0 0
Control CSD Control CSD

(c) 5 Open Arm (d)

Closed Arm 7
#
No. of crossings in open and

4 6

No. of head dips 5

closed arm

3
4

2 *# 3

2
1 *
1

0 0
Control CSD Control CSD

(e) (f)
2.5 350
300
2
250
Time spent (sec.)
No. of Falls

1.5 200
150
1
100
0.5
50

0 0
Control CSD Control CSD

Results impairment and elicited inflammatory response and caused

reactive gliosis in hippocampus and PC regions of brain.
The current data suggest that CSD for 21  days in male During EPM test, although both control and CSD animals
Wistar rats induced anxiety-like behaviour and memory spent significant duration of time in closed arm (p ≤ 0.001)

13
 Molecular and Cellular Biochemistry (2018) 449:63–72 67
◂Fig. 1  Chronic sleep loss resulted in anxiety-like behaviour and poor
grip strength: a histogram represents average time spent by the rats in
open and closed arms of EPM apparatus. CSD animals spent maxi-
mum time in the closed arm than open arm and central open area as
compared to control rats. The exploratory activity of animals of each
group is show by the number of entries (b) and number of crossings
(c) in open and closed arms. CSD rats showed complete avoidance of
the open arm compared to control rats. d Histogram represents the
number of head dips by the rats of each group. e, f Histograms repre-
senting number of falls and time spent by rats on rotarod. Values are
expressed as  mean ± SEM, *p ≤ 0.05 control versus CSD, #p ≤ 0.05
open versus closed arm and, $p ≤ 0.05 closed versus central open area.
Holm–Sidak method after one-way and two-way ANOVA. (Color fig-
ure online)
but CSD rats completely prohibited themselves from enter-
ing the open arm and spent no time at all in open arm unlike
control rats (p ≤ 0.05) (Fig. 1a). These observations are in
line with the number of entries and crossings in an arm,
where CSD rats entered closed arm with more number of
entries and crossings than open arm (p ≤ 0.001) as compared
to control rats (Fig. 1b, c). However, both control and CSD
rats demonstrated no significant difference for the time spent
in central open area (Fig. 1a). Further the number of head
dips shown by CSD rats was significantly lesser (p ≤ 0.01)
than the control rats (Fig. 1d). These observations suggest
that CSD induced anxiety-like behaviour in male rats. We
also evaluated motor activity of these rats and found that
CSD rats after 21 days of chronic sleep deprivation had
33.33% more number of falls (Fig. 1e). There was no differ-
ence in the time spent by the CSD rats on rotating rod from
that of control rats (Fig. 1f).
We further analysed recognition memory response of rats
during NOR test and found that CSD rats had lesser pref-
erence index score (15% lesser) for the novel object than
control rats (Fig. 2c) and spent 25% lesser time in exploring
the novel object as compared to control rats (Fig. 2b). In
parallel, time spent by CSD rats in exploring the old object
was higher (52.83%) as compared to control rats (Fig. 2b).
However, there was marginal difference between the two
groups for the number of episodes towards both the objects
(Fig. 2a) and the rearing activity of CSD rats was also 75%
higher as compared to control rats (Fig. 2d). Furthermore,
we analysed grooming behaviour of rats and found that CSD
rats had more grooming bouts (percentage increase was
62.5%) compared to control rats (Fig. 2e). However, they
spent 20% lesser time in grooming themselves as compared
to control rats (Fig. 2f).
CSD and neuroinflammation
Chronic sleep deprivation resulted in upregulation in the
expression of astrocytic cell-specific protein GFAP (Fig. 3a
upper panel, b, c) and microglial cell specific marker Iba1
(Fig. 3a second panel, b, c) in hippocampus and PC regions
of brain, which is indicative of astrogliosis and microgliosis
under CSD conditions. Further activation of NFκB (which
is implicated in sleep regulation) was observed in CSD rats
in hippocampus (p ≤ 0.05) and PC regions of brain as com-
pared to control rats (Fig. 3a third panel, b, c). This was also
accompanied by activation of AP1 (dimer of c-Jun and c-fos)
in CSD rats in both the brain regions (p ≤ 0.05) (Fig. 3a
fourth panel, b, c). We further evaluated the inflammatory
molecules expression such as TNFα, IL-1β and IL-6 using
Western blotting. CSD rats showed enhanced expression of
TNFα in both the brain regions (p ≤ 0.05) (Fig. 4a upper
panel, b, c) as well as IL-6 in hippocampus region (p ≤ 0.05)
(Fig. 4a second panel, b). However, a marginal decrease in
the expression of IL-6 was observed in PC region which was
not statistically significant (Fig. 4a second panel, c). CSD
rats showed no change in the expression of IL-1β in both the
brain regions studied (Fig. 4a third panel, b, c).
Discussion
Chronic sleep deprivation for 21  days was observed to
induce anxiety-like behaviour and recognition memory
impairment in young adult male Wistar rats. Animals were
subjected to EPM test to ascertain their anxiety levels. CSD
group rats showed minimum number of entries and time
spent in open arm as compared to closed arm, which indi-
cates their higher anxiety levels triggered due to chronic
sleep loss. Complete avoidance for the open arm demon-
strated by CSD rats as evident form the number of crossings
in open arm further supports their anxiogenic response after
CSD. CSD rats also showed fear-like response as evident
from the marked reduction in the number of head dips as
compared to control rats. Various sleep deprivation para-
digms have shown time-dependent feature of anxiety-like
behaviour triggered due to SD as evaluated by EPM [15, 25,
26]. A recent study by Yin et al. [27] has shown that mice
exhibited anxiety-like behaviour following 3 days of inter-
mittent and paradoxical SD with more pronounced increase
when deprived for 7 days of SD paradigm. Sleep deprivation
and anxiety are further known to affect the motor perfor-
mance of animals. More number of falls from the rotating
rod indicated the impaired motor coordination of CSD rats.
However, we did not find any difference in the length of time
spent by CSD rats on rotating rod from that of control rats.
The current data suggest that in CSD regimen of 18 h of
sleep deprivation, although the animals were daily allowed
undisturbed sleep for 6 h, but there was sleep debt due to
curtailment of daily sleep dose, which may have led to anxi-
ety and low-grade inflammatory response.
Since anxiety is further associated with poor performance,
alertness and cognitive dysfunction, therefore we analysed
the recognition memory and learning response of the rats to
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68 Molecular and Cellular Biochemistry (2018) 449:63–72

(a) (b)
6 Old New 20 # Old New
18
5 16

Time spent (sec.)

14
No. of episodes

4
12
3 10
8
2 6
4
1
2
0 0
Control CSD Control CSD

(c) (d)

0.9 12
0.8
10
0.7
Preference Index

0.6 8
Rearings

0.5
6
0.4
0.3 4
0.2
2
0.1
0 0
Control CSD Control CSD

(e) (f)

6 80
70
5
60
Grooming time (sec.)
Grooming bouts

4
50
3 40
30
2
20
1
10
0 0
Control CSD Control CSD

Fig. 2  Chronic sleep loss caused learning and recognition memory
deficits: histograms represent several parameters analysed dur-
ing NOR test a number of episodes, b time spent in exploration of
objects, c preference index for the novel object, d number of rearings.
CSD rats spent relatively lesser time in exploring the novel object
with lower preference score for the novel object compared to control
rats. e, f Histograms representing grooming bouts and time spent in
grooming by the two groups. CSD rats had more number of grooming
bouts (p = 0.07) with lesser time spent in grooming (p = 0.45) as com-
pared to control rats. Values are expressed as mean ± SEM, #p ≤ 0.05
old versus new. Holm–Sidak method after one-way and two-way
ANOVA. (Color figure online)

13
Molecular and Cellular Biochemistry (2018) 449:63–72 69

(a)
Hippocampus Piriform Cortex
Control CSD Control CSD
GFAP 50 kDa

Iba1 17 kDa

NFKB 65 kDa

AP1 39 kDa

α-tubulin 52 kDa

(b) (c)
180 * Control 160 Control
CSD
160 140 CSD
140 * Relative expression in PC region *
120
in hippocampus region

*
Relave expression

120
100
*
100
80
80
60
60
40
40
20 20

0 0
GFAP Iba1 NFκB AP-1 GFAP Iba1 NFκB AP-1

Fig. 3  Chronic sleep loss activated astrocytes and microglial cells
and inflammatory pathway: a representative western blot images of
GFAP, Iba1, NFκB and AP1. b, c Quantitative densitometric analysis
of the markers in the hippocampus and piriform cortex regions of rat
brain among the two groups (n = 4 for each group). The expression
of all the markers were upregulated in response to chronic sleep loss
in CSD rats. Values are expressed as mean ± SEM, *p ≤ 0.05 control
versus CSD, Holm–Sidak method after one-way ANOVA. (Color fig-
ure online)

a novel environment. Reduced preference of CSD rats for
the novel object as evident from the preference index score
(Fig. 2c) and lesser time spent by CSD rats in exploration of
novel object as compared to control rats (Fig. 2b) indicated
their impaired recognition memory and learning response
to the novel environment. Recent study has revealed that
paradoxical SD for 3 consecutive days did not cause work-
ing memory impairment as shown on Y-maze task, whereas
7 days of paradoxical SD significantly impaired working
memory indicating that SD-induced impairment is degree
dependent [27]. These findings may suggest that chronically
reducing daily quota of sleep also impairs the learning and
memory function along with anxiety.
Self-grooming is an innate self-care behaviour for the
cleaning of skin and fur, adopted under various stressful
and non-stressful conditions [28–31]. Rats perform dif-
ferent types of grooming behaviour depending on envi-
ronmental conditions [32]. Under non-stressful conditions
such as home cage, rats show spontaneous self-grooming
pattern characterized by a typical cephalocaudal sequence
[28, 29]. Under anxiogenic conditions such as the open-
field test (OFT) or the EPM, rats show interrupted and dis-
organized grooming pattern that depends on their anxiety
levels [30, 31, 33]. In the present study, we have analysed
self-grooming behaviour of rats under non-stressful condi-
tions of environmental novelity (NOR). Thus, reduced time
spent by CSD rats in self-grooming supported their anxi-
ogenic behaviour triggered due to sleep debt and chronic
sleep fragmentation, whereas control rats showed normal
grooming behaviour attained as a result of adequate sleep.

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70 Molecular and Cellular Biochemistry (2018) 449:63–72

(a) Hippocampus Piriform Cortex mice to water-based environment may be due to long-term
Control CSD Control CSD utilization of flower pot method of sleep deprivation.
TNF-α 17 kDa
Chronic sleep deprivation caused
IL-1β 17 kDa neuroinflammation by activating inflammatory
mediators, astrocytes and microglial cells
IL-6 21 kDa

α-tubulin
Sleep deprivation, sleep curtailment and sleep fragmentation
52 kDa
are known to cause low-grade inflammation and blood–brain
(b) barrier disruption mediated by inflammatory molecules,
140
*
astrocytes and microglia. For instance, chronic sleep depri-
Control CSD
120 * vation for period of 72 h elevated the levels of TNFα, IL-6
and IL-1β in hippocampus and basal forebrain of rats [34].
Relative expression in
hippocampus region

100 Similarly, in the present study, chronic sleep deprivation also

80 resulted in activation of pro-inflammatory cytokines in rats
as evident from the marked increase in the expression of
60
TNFα in both the brain regions and IL-6 in hippocampus
40 region (Fig. 4a–c). A recent study has shown that intermit-
20 tent or paradoxical sleep for 3 and 7 days repetitively for
3 months caused sustained increase in expression of TNFα
0
TNFα IL-1β IL-6 without any significant change in IL-1β and IL-6 compared
to control mice [27]. Further, we evaluated the expression
(c) of NFκB and AP1 which are known to be involved in sleep
140
regulation [35, 36]. NFκB activation converges in cellular
Control CSD
* pathways considered as hallmark of inflammation [37]. Acti-
Relative expression in PC region

120 vation of NFκB and its subsequent translocation to nucleus

100
80
60
40
20
0 due to extended wakefulness [36, 38, 39]. Enhanced expres-
TNFα
Fig. 4  Chronic sleep loss ameliorated the base levels of inflamma-
tory cytokines: a representative western blot images of inflamma-
tory cytokines TNFα, IL-1β and IL-6. b, c Quantitative densitomet-
ric analysis of the markers in the hippocampus and piriform cortex
regions of rat brain among the two groups (n = 4 for each group).
Values are expressed as mean ± SEM, *p ≤ 0.05 control versus CSD,
Holm–Sidak method after one-way ANOVA. (Color figure online)
Further, the rearing activity of CSD rats for the open area
was higher compared to control rats which could be due
to their increased familiarity to the open area which was
similar to the area used in rotating drum method for SD.
This is in line with a recent study by Yin et al. [27] which
has revealed that intermittent paradoxical sleep deprived
mice took lesser time in reaching the hidden/visible plat-
form during Morris water maze test compared to control
mice. The study indicated concluded that familiarity of SD
may promote the transcriptional activation of inflammation-
related genes such as TNFα, IL-1β [1, 17]. Although NFκB
showed marginal increase in its expression in the two brain
regions but consistent change may suggest the role of this
transcription factor in mediating the inflammatory response
to CSD. Several studies based on SD have shown induction
of AP1 and c-fos (immediate early gene) in young adult rats
IL-1β IL-6
sion of AP1 in the current study may be the outcome of
increased sleep debt in CSD rats as long-term lack of sleep
disrupts the homeostatic balance of sleep–wake cycle lead-
ing to cognitive impairments.
Astrocyte/microglial activation along with release of
inflammatory cytokines is considered to be the main source
of neuroinflammation among other factors [40]. Various ani-
mal studies from our lab and others have shown that both
acute and chronic periods of sleep loss induce astroglial and
microglial activation [12, 14, 27]. In a recent study by Hur-
tado-Alvarado et al. [41], it was reported that 20 h of sleep
deprivation for 10 consecutive days induced overexpression
of markers of reactive astroglia (GFAP) and microglia (Iba1)
and disrupts the blood–brain barrier via adenosine-mediated
signalling. Similarly, in our recent lab studies of 12 h of
acute sleep deprivation in rats, we observed reactive gliosis
in astrocytic and microglial population along with secretion
of pro-inflammatory mediators such as TNFα, IL-6 [12, 14].
Activation of inflammatory cytokines along with profound

13
Molecular and Cellular Biochemistry (2018) 449:63–72 71
astrogliosis and microgliosis (Fig. 3a–c) and anxiety-like
behaviour (Fig. 1a–d) triggered due to chronic sleep depriva-
tion is further indicative of neuroinflammation.
Conclusion
The current data suggest that inflammatory mediators
increase during chronic sleep deprivation and trigger the
activation of glial and microglial cells in discrete brain
regions. Upregulation in the protein expression of inflamma-
tory cytokines, transcription factors as well as astroglial and
microglial cells activation may explain the higher anxiety
levels and learning and memory impairment associated with
chronic sleep deprivation and may prove to be the potential
targets to develop effective therapeutic interventions to over-
come these conditions.
Acknowledgements  This work was supported by Department of Sci-
ence and Technology (DST), Government of India (GOI) project Grant
[Grant No. C/639/(IFD)/2015–2016] to Gurcharan Kaur. Shaffi Man-
chanda is thankful to Council of Scientific and Industrial Research
(CSIR), GOI for fellowship grant during the entire course of study.
Harpal Singh and Taranjeet Kaur are thankful to University Grants
Commission (UGC), GOI and its scheme CPEPA (Centre with Poten-
tial for Excellence in Particular Area) for fellowship grant. Infrastruc-
ture provided by University Grants Commission (UGC), India under
University with Potential for Excellence (UPE) and CPEPA schemes
and Department of Biotechnology (DBT), India under DISC facility is
highly acknowledged. The funding source had no role in study design;
collection, analysis and interpretation of data; in writing of report; and
in decision to submit the article for publication.
Compliance with ethical standards  severe phenotype of the disorder. Sleep Med Rev 17:241–254
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical approval  All applicable international, national and/or institu-
tional guidelines for the care and use of animals were followed. All
procedures performed in studies involving animals were in accordance
with the ethical standards of the institution or practice at which the
studies were conducted.
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