Cardioprotective effects of acute sleep deprivation on ischemia reperfusion injury

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Autonomic Neuroscience: Basic and Clinical 230 (2021) 102761
Available online 4 December 2020
1566-0702/© 2020 Elsevier B.V. All rights reserved.
Cardioprotective effects of acute sleep deprivation on ischemia/ 
reperfusion injury 
Zohreh Edalatyzadeh a,1, Marjan Aghajani a,c,1,2, Alireza Imani a,b,*, Mahdieh Faghihi a, 
Khosro Sadeghniiat-Haghighi b, Sahar Askari a,1, Samira Choopani a,3 
a Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 
b Occupational Sleep Research Center, Tehran University of Medical Sciences, Tehran, Iran 
c Department of Physiology, School of Medicine, Shahed University, Tehran, Iran 
A R T I C L E I N F O 
Keywords: 
Sleep deprivation 
Sympathectomy 
Ischemic preconditioning 
Epinephrine 
Oxidative stress 
A B S T R A C T 
Objectives: Modulation of sympathetic activity during acute sleep deprivation can produce various effects on body 
functions. We studied the effects of acute sleep deprivation before ischemia/reperfusion on myocardial injury in 
isolated rat hearts, and the role of sympathetic nervous system that may mediate these sleep deprivation induced 
effects. 
Methods: The animals were randomized into four groups (n = 11 per group): Ischemia- Reperfusion group (IR), 
Acute sleep deprivation group (SD), Control group for sleep deprivation (CON-SD) and Sympathectomy + ASD 
group (SYM-SD). In SD group, sleep deprivation paradigm was used 24 h prior to induction of ischemia/ 
reperfusion. In SYM-SD group, the animals were chemically sympathectomized using 6-hydroxydopamine, 24 h 
before sleep deprivation. Then, the hearts of animals were perfused using Langendorff setup and were subjected 
to 30 min regional ischemia followed by 60 min of reperfusion. Throughout the experiment, the hearts were 
allowed to beat spontaneously and left ventricular developed pressure (LVDP) and rate pressure product (RPP) 
were recorded. At the end of study, infarct size and percentage of the area at risk were determined. 
Results: We found that SD increased LVDP and RPP, while reducing the myocardial infarct size. Moreover, 
sympathectomy reversed SD induced reduction in infarct size and showed no differences as compared to IR. 
Conclusion: This study shows cardioprotective effects of acute sleep deprivation, which can be abolished by 
chemical sympathectomy in isolated hearts of rats. 
1. Introduction 
Nowadays, altered patterns of sleep can be seen frequently due to the 
changes of lifestyle. The health repercussions of sleep deprivation (SD) 
have lured attention of a significant body of researchers. Chronic sleep 
deprivation (CSD) has detrimental effects on cardiovascular system, and 
a prominent role of sympathetic activation has been proposed as 
possible trigger of these effects. Extended hours of wakefulness increases 
heart rate and blood pressure, thereby contributing to elevated risk of 
cardiovascular diseases (Perry et al., 2011). Interestingly, acute sleep 
deprivation (ASD) attenuates central and peripheral inflammatory re-
sponses and may provide protection against cerebral ischemia and 
neuronal cell death. ASD is associated with a shift in sympatho-vagal 
balance towards sympathetic predominance and decreased para-
sympathetic cardiovascular modulation (Weil et al., 2009). In addition, 
sympathetic hyperactivity involves adrenergic overdrive mediated by α1 
adrenergic receptors (Kuo et al., 2012). 
Ischemic heart diseases are the leading cause of death worldwide. 
Obstruction of coronary blood flow results in a wide array of structural 
and functional changes within myocardium that may lead to irreversible 
injury (Ruiz-Meana and García-Dorado, 2009). Rapid restoration of 
blood flow is the most effect plan against ischemic injury. Paradoxically, 
reperfusion and re‑oxygenation of anoxic tissues potentiate an addi-
tional injury (‘reperfusion injury’) characterized by a complex cascade 
* Corresponding at: Department of Physiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. 
E-mail address: aimani@tums.ac.ir (A. Imani). 
1 These authors contributed equally to this work. 
2 Department of physiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 
3 Department of physiology, School of Medicine, Isfahan University of Medical Sciences, Tehran, Iran. 
Contents lists available at ScienceDirect 
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journal homepage: www.elsevier.com/locate/autneu 
https://doi.org/10.1016/j.autneu.2020.102761 
Received 7 September 2020; Received in revised form 8 November 2020; Accepted 2 December 2020 
mailto:aimani@tums.ac.ir
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Autonomic Neuroscience: Basic and Clinical 230 (2021) 102761
2
of inflammation, oxidative stress, apoptosis and necrosis. However, 
many advances have been made in order to diminish these additional 
lethal effects which brought about by reperfusion. Among these in-
terventions, cardiac preconditioning (i.e., applying of mechanical or 
pharmacological strategies prior to sustained lethal myocardial ischemic 
event) has revealed promising results (Imani et al., 2008). Activation of 
sympathetic nervous system is one of the major factors determining the 
extent of ischemic/reperfusion damage (Perry et al., 2011), such that, in 
long-term, it exerts deleterious effects on heart, yet, short-term activa-
tion can confer significant cardio-protection (Imani et al., 2008; Naderi 
et al., 2010). 
Keeping these findings in view, the current study was designed to 
study the effects of ASD before ischemia/reperfusion on myocardial 
injury in isolated rat hearts, and the role of sympathetic nervous system 
in mediating these SD induced effects. 
2. Methods 
2.1. Animals 
The experimental protocols followed in this study were conformed to 
Guidelines for the Care and Use of Laboratory Animals published by the 
US National Institute of Health (NIH Publication no. 85–23, revised 
2011) and were further approved by the institutional ethical committee 
at Tehran University of Medical Sciences (Tehran, Iran). This study was 
performed in 2016 (April to December) in the Faculty of Medicine, 
Teharn University of Medical Sciences. Forthy-four male Wistar rats 
weighing 250–300 g which were housed in an air-conditioned colony 
room mainatianed at 21–23 ◦C and 12 h light-dark cycle. 
The animals were randomly divided into four groups (n = 11 per 
group): 
1. Ischemia-Reperfusion group (IR): Isolated hearts underwent 30 min 
of regional ischemia followed by 60 min reperfusion using Langen-
dorff setup. 
2. Acute sleep deprivation group (SD): Multiple platform method was 
applied to induce ASD for 24 h. Following SD, isolated hearts un-
derwent to 30 min of ischemia and then 60 min of reperfusion using 
Langendorff setup. 
3. Control group for sleep deprivation (CON-SD): As compared to ASD 
group, these rats were placed on wider platforms, so they could 
position themselves to sleep with minimal or no water immersions. 
Following this control protocol for SD, isolated hearts underwent 30 
min of ischemia and then 60 min of reperfusion using Langendorff 
setup. 
4. Sympathectomy+ ASD group (SYM-SD): Twenty-four hours after 
chemical sympathectomy, rats were subjected to ASD for 24 h. 
Following this, isolated hearts underwent 30 min of ischemia and 60 
min of reperfusion using Langendorff setup. It should be noted that in 
our previous study we had a control group of sympathectomy which 
underwent I/R one day after sympathectomy and our results 
regarding the infarct size and hemodynamic parameters showed no 
significant change in this group compared to I/R group (Rakhshan 
et al., 2015). So,in the current study we did not consider another 
sympathectomy control group. 
2.2. Preparation of isolated hearts 
The animals were anesthetized with Sodium Pentobarbital (60 mg/ 
kg intraperitoneally (IP); Sigma, Munich, Germany). Blood samples 
were collected and centrifuged (2000 rpm for 15 min at 4 ◦C) to obtain 
serum. Then, the hearts were isolated and retrogradely perfused at a 
constant hydrostatic pressure (80 mmHg) according to the Langendorff 
technique. Briefly, the aorta of excised heart was cannulated and 
perfused with modified Krebs-Henseleit bicarbonate buffer containing 
(in mmol/L): NaHCO3 25; KCl 4.7; NaCl 118.5; MgSO4 1.2; KH2PO4 1.2; 
glucose 11; CaCl2 2.5, gassed with 95% O2 5% CO2 (pH 7.35–7.45 at 
37 ◦C). A saline-filled latex balloon was introduced into left ventricle 
and was inflated to give a preload of 8–10 mmHg. The balloon was 
connected to a pressure transducer (Harvard, March-Hugstetten, Ger-
many) which allowed real time measurement of intraventricular pres-
sures. A surgical needle (5–0 silk suture) was passed under the origin of 
left anterior descending coronary (LAD) artery, and the ends of suture 
were passed through a pipette tip to form a snare. Local ischemia was 
induced by tightening the snare and reperfusion was performed by 
releasing the ends of suture. Ischemia was confirmed by ST elevation 
and increase in R- wave amplitude in ECG. The perfusion apparatus was 
water-jacketed to maintain a constant perfusion temperature at 37 ◦C. 
Hearts were allowed to beat spontaneously throughout the experiments. 
The hemodynamic parameters including left ventricular developed 
pressure (LVDP; mmHg) and rate pressure product (RPP) were recorded 
by an investigator who was blinded to the identity of the groups. LVDP 
was defined as the peak systolic pressure minus the end diastolic pres-
sure, and RPP calculated by multiplying LVDP and heart rate (HR). Rats 
with ventricular fibrillation (VF) for more than 5 min were removed 
from the study. 
2.3. Determination of infarct size and area at risk 
After completion of the reperfusion period, the left coronary artery 
was re-occluded, and Evans Blue dye (1.5 ml 0.5% Evans blue in distilled 
water; Sigma, Munich, Germany) was infused via aorta to differentiate 
ischemic area at risk (AAR; unstained) and from non-ischemic area 
(stained blue). Hearts were frozen overnight and then sliced into 2-mm 
transverse sections from apex to base. The slices were incubated in 1% 
triphenyl tetrazolium chloride (TTC (Sigma, Munich, Germany) in 0.1 M 
phosphate buffer, pH 7.4, 37 ◦C) for 20 min followed by tissue fixation 
(10% phosphate-buffered formalin) for 24 h. TTC reacts with the viable 
tissue, producing a red formazan derivative, which is distinct from the 
white necrotic area. Sections were scanned by an investigator who was 
blinded to the identity of them to determine non-ischemic area, AAR and 
infarct size (IS) by calculating pixels occupied by each area using Adobe 
Photoshop software (Adobe Systems Seattle, WA). Area at risk was 
expressed as a percentage of left ventricular (LV) volume for each heart. 
The infarct size was determined by using computer-aided planimetry 
and expressed as a percentage of area at risk (%IS/AAR) (Naderi et al., 
2010; Imani et al., 2011). 
2.4. Sleep deprivation paradigm- modified multiple platform method 
For inducing ASD, rats were placed in an acrylic tank (125 cm × 44 
cm × 44 cm) containing 8 circular platforms, 6.5 cm in diameter and 10 
cm in height. The tank was filled with water (20 ◦C) up to 1 cm below 
platform level. The animals were capable of moving and jumping inside 
the tank, from one platform to another. When rats lapsed into REM- 
sleep, because of muscular atonia they abruptly fell into water. In 
addition, wider platforms (14 cm in diameter and 10 cm in height) were 
used for controls; thus, allowing them to acquire sleep without falling 
into the water. The rats were allowed to move around freely inside the 
tanks from one platform to another. Food and water were made avail-
able through a grid placed on top of the water tank (Aghajani et al., 
2017; Ma et al., 2014; Parsa et al., 2017) 
2.5. Chemical sympathectomy 
Chemical Sympathectomy was achieved by injecting 6-hydroxydop-
amine (100 mg/kg; Sigma-Munich, Germany) diluted in NaCl 9% and 
Ascorbic Acid 1% (Daroupakhsh Co. Iran), subcutaneously (Martinelli 
et al., 2002). 
Z. Edalatyzadeh et al. 
Autonomic Neuroscience: Basic and Clinical 230 (2021) 102761
3
2.6. Measurement of serum pro-oxidant-antioxidant balance (PAB) and 
epinephrine 
PAB assay employs Tetramethylbenzidine 3, 3′, 5.5′ (TMB) and its 
cations as the indicators of oxidation-reduction. For determining PAB, 
two different reactions were performed; an enzymatic reaction where 
the chromogen TMB was oxidized to a colored cation by peroxides and a 
chemical reaction where the cation of TMB was reduced to a colorless 
compound by antioxidants. Next, photometric absorbance was 
compared with the absorbances given by a series of standard solutions 
(Faramarzi et al., 2012). PAB values were expressed in arbitrary HK 
units based on percentage of H2O2 in the standard solution. A low PAB 
value is indicative of high antioxidant concentration, while a high PAB 
value represents low antioxidant concentration. 
Serum levels of epinephrine were measured using a commercially 
avialable ELISA kit (R&D, Minneapolis, MN, USA). 
All measurements were performed by an investigator who was 
blinded to the identity of the groups. 
2.7. Statistical analysis 
Statistical comparison of means between groups for homodynamic 
parameters was performed by two-way ANOVA, followed by Tukey test. 
We used one-way ANOVA followed by Tukey test in order to compare 
infarct size as well as serum levels of PAB and epinephrine between 
groups. All statistical analyses were performed using SPSS software 
(Version 20, SPSS Inc., Chicago, IL, USA) and p < 0.05 was considered 
statistically significant. 
3. Results 
3.1. Homodynamic and ECG parameters 
The ECG changes that occurred in our experimental groups were 
similar to those we had seen previously (Edalatyzadeh et al., 2016). 
Accordingly, we reported that experience of 24 h’ SD prior IS decreased 
the number of ventricular tachycardia (VT) episodes during ischemia 
period, and chemical sympathectomy put down the beneficial effects of 
acute SD on preconditioning response. Moreover, there was no signifi-
cant effect of SD on the incidence of VT upon reperfusion in all experi-
mental groups. Also, statistical analysis of VF incidence data indicated 
that there was no occurrence of VF in SYM-SD group. Totally, the hearts 
of three animals in IS and CON-SD groups (3/11) and five animals in 
SYM group (5/11) were excluded from the study due to occurrence of VF 
for more than 5 min upon ischemia period. 
Table 1 indicates cardiac functional parameters including LVDP and 
RPP for IR, SD, CON-SD and SYM-SD groups. In CON-SD and SYM-SD 
groups, LVDP at baseline was significantly reduced as compared to IR 
and SD groups. Moreover, in SYM-SD group, LVDP was also decreased as 
compared to CON-SD group. After induction of ischemia and subsequent 
reperfusion, LVDP was increased in SD group and reduced in SYM-SD 
group, as compared to IR group. Furthermore, ischemia and reperfu-
sion markedly decreased LVDP in CON-SD and SYM-SD groups as 
compared to SD group (p < 0.001), but there was no difference between 
CON-SD and IR groups. At baseline and reperfusion, sympathectomy 
reduced LVDP when compared to CON-SD group (p < 0.001). Repeated 
measurement of ANOVA for LVDP showed significant differences (p <
0.05) between baseline, ischemia and reperfusion period in IR, SD and 
CON-SD groups. In addition, duringischemia and reperfusion period, 
RPP in SD animals was increased when compared to IR, CON-SD and 
SYM-SD animals (p < 0.05). 
3.2. Area at risk and infarct size 
As shown in Fig. 1, no significant differences in the ratio of AAR to 
total LV area were found between experimental groups. The ratio of IS to 
AAR in SD group was significantly reduced as compared to IR group (p 
< 0.001). Moreover, this ratio was significantly increased in SYM-SD 
group comparing IR, SD and CON-SD groups. No significant differ-
ences were found in AAR to total LV area ratio between IR and CON-SD. 
3.3. Serum levels of epinephrine 
There was a significant statistical (p = 0.003) difference in serum 
levels of epinephrine (Fig. 2) between IR and SD groups. However, 
epinephrine levels were significantly reduced in SYM-SD group as 
compared to the SD group (p < 0.001). 
3.4. Pro-oxidant/antioxidant balance 
As shown in Fig. 3, there were no significant differences in PAB (HK 
unit) between experimental groups. 
4. Discussion 
There exists a complex and bidirectional relationship between sleep 
and cardiovascular system. Sleep disorders may increase the risk of 
cardiovascular diseases, while on the other hand, cardiovascular dis-
eases can result in altered sleep patterns (Tobaldini et al., 2014). The 
current study demonstrates the effect of ASD before myocardial infarc-
tion, on hemodynamic and histological parameters in isolated rat hearts. 
We observed that ASD confers cardioprotection against MI by increasing 
LVDP and RPP and by limiting infarct size. 
Ischemia- reperfusion injury is accompanied with the damage of 
myocardial infarction which leads to interruption of the blood supply to 
tissues. Today some strategies are used to limit the consequences of 
ischemia and myocardial infarction, one of which is ischemic pre-
conditioning (Kloner, 2009). The role of sympathetic nervous system in 
early preconditioning has been extensively studied. Pharmacological 
activation of α1-adrenoceptors has been shown to mimic early pre-
conditioning (Imani et al., 2008; Naderi et al., 2010; Salvi, 2001; Gomez 
et al., 2008) and its blockade can abolish preconditioning induced 
Table 1 
Cardiac functional parameters: left ventricular diastolic pressure (LVDP) and rate pressure product (RPP). 
Groups LVDP RPP 
Baseline Ischemia Reperfusion Baseline Ischemia Reperfusion 
IR 55.87 ± 3.96 47.33 ± 1.47 61.67 ± 1.78 31,089 ± 1915 8974 ± 1789 10,572 ± 1680 
SD 93.07 ± 1.31 77.87 ± 2.25a 85.46 ± 1.63a 27,197 ± 1060 21,257 ± 1466a 18,893 ± 2085a 
CON-SD 60.28 ± 7.04a,b 40.15 ± 3.34b 59.7 ± 1.87b 14,516 ± 1736 7987 ± 1436b 10,152 ± 2050 
SYM-SD 38.46 ± 1.43a,b,c 35.38 ± 1.17a,b 38.28 ± 2.27a,b,c 10,064 ± 893 6349 ± 937 b 8485 ± 1425b 
Data were presented as mean ± standard error of mean (SEM); ischemia/Reperfusion (IR), Acute sleep deprivation group (SD), Control group for sleep deprivation 
(CON-SD), sympathectomy group (SYM-SD). 
a p < 0.05 versus IR group. 
b p < 0.05 versus SD group. 
c p < 0.05 versus SD-CON group. 
Z. Edalatyzadeh et al. 
Autonomic Neuroscience: Basic and Clinical 230 (2021) 102761
4
cardioprotection (Piascik and Perez, 2001). Based on controlled chro-
nobiological studies, it is reported that sleep is more important for 
sympathetic regulation of the heart, whereas, parasympathetic nervous 
system activity is mostly influenced by circadian system (Mullington 
et al., 2009). Besids, it seems the effects of ASD and/or periods of sleep 
loss is not comparable of chronic sleep deprivation (CSD) and/or pro-
longed sleep loss; i.e. not only may CSD put detrimental effects on 
daytime alertness, but also on different organs including cardiovascular 
Fig. 1. Box and whisker plots of the ratio of area at risk to total left ventricular area and ratio of infarct size to area at risk. The mean value is represented by the cross 
sign. Ischemia/Reperfusion (IR), Acute sleep deprivation group (SD), Control group for sleep deprivation (CON-SD) and sympathectomy group (SYM-SD); and Area 
At Risk to total Left Ventricular Area ratio (AAR/LV), Infarct Size to Area At Risk ratio (IS/AAR); *: p < 0.05 versus IR group, ̂ : p < 0.05 versus SD group, &: p < 0.05 
versus SD-CON group. 
Fig. 2. Box and whisker plots of the changes in serum level of epinephrine (pg/ 
ml) in Ischemia/Reperfusion (IR), Acute sleep deprivation group (SD) and 
sympathectomy group (SYM-SD); The mean value is represented by the cross 
sign. *: p < 0.05 versus IR group, ^: p < 0.05 versus SD group, &: p < 0.05 
versus SD-CON group. 
Fig. 3. Box and whisker plots of the serum Pro-oxidant/Antioxidant Balance in 
Ischemia/Reperfusion (IR), Acute sleep deprivation group (SD), Control group 
for sleep deprivation (CON-SD) and sympathectomy group (SYM-SD). The mean 
value is represented by the cross sign. 
Z. Edalatyzadeh et al. 
Autonomic Neuroscience: Basic and Clinical 230 (2021) 102761
5
system due to inflammatory and oxidative stress (Aghajani et al., 2017; 
Philip et al., 2012). Despite the exact mechanisms of ASD on remote 
preconditioning are not still clear, clinical trials have shown that it 
would be as a powerful approach to eliminate I/R injury due to its anti- 
inflammatory effects (Koch et al., 2014; Cam et al., 2013; Brager et al., 
2016). Although Kato et al. (2000) have reported unaltered levels of 
serum catecholamines following sleep loss, there are some studies which 
have shown inadequate sleep and sleep disturbances often co-exist with 
sympathetic hyperactivity (Kuo et al., 2012; Dimitrov et al., 2009; Jeddi 
et al., 2016). Accordingly, in the current study with the hypothesis that 
SD may trigger adrenergic overdrive, we showed that serum levels of 
epinephrine in animals which experienced SD before IR was more than 
IR subjects. Moreover, to determine the relationship between SD and 
cardiovascular dysfunction, some of the experimental animals were 
sympathectomized using 6-hydroxydopamine. We found lower levels of 
epinephrine in the serum of SYM-SD animals as compared to SD animals 
and this finding reveals that the procedure of sypathectomy before MI- 
induction was more effective to decrease epinephrin’s levels in SYM- 
SD animals. Early observations in our laboratory had indicated that 
there is not any difference between control group of sympathectomy and 
I/R group regarding hemodynamic parameters and preconditioning 
response. With this in mind, it seems 6-hydroxy-dopamine does not have 
any off-target effects on cardiac preconditioning (Rakhshan et al., 2015). 
In further support of this view, Edward O Weselcouch et al. with the aim 
to test whether endogenous catecholamines are involved in cardiac 
preconditioning, used either reserpine or 6-hydroxydopamine. They 
found that catecholamine depletion with either reserpine or 6-hydroxy-
dopamine did not affect pre-ischemic coronary flow or cardiac function 
(Weselcouch et al., 1995). However, as regards 6-hydroxydopamine 
may have some effects on cardiovascular parameters on its own; i.e. 
any prolonged hypotension and bradycardia pre-I/R would have some 
preconditioning effects (Zoccal et al., 2007; Breese and Traylor, 1970; 
Archer et al., 1986), it would also be more convincing to consider an 
alternative method of sympathectomy in our future studies to control for 
any off-target effects of 6-hydroxydopamine. 
The instrumental models that are used for induction of SD, result in 
altered indices of stress (Ma et al., 2014; Machado et al., 2006). The SD 
paradigm used in our study -modified multiple platform method- has 
beenreported to augment adrenocorticotropic and corticosterone re-
sponses (data is not shown). Short-term increase in glucocorticoids en-
hances immunity and is acccompanied with anti-inflammatory effects, 
whereas a chronic increase in corticostrone causes immunosuppression 
(Hirotsu et al., 2015; Sebastiano et al., 2017). It is said that cortico-
strone/cortisol levels rise steadily during ASD and reach to their peak 
levels after few days; i.e. corticosterone concentrations may increase and 
reach to a moderate level 24 h after one night of SD (Dimitrov et al., 
2009; Leenaars et al., 2011; Suchecki et al., 2002). Therefore, the rise in 
LVDP and RPP and decrease in infarct size observed in SD animals, can 
be attributed to the stress effects of tiled water tank used for SD 
(Aghajani et al., 2017). These findings are in agreement with our pre-
vious work in which we demonstrated the infarct-sparing effect of acute 
forced swimming stress on isolated rat hearts (Moghimian et al., 2012). 
Moreover, there were no differences in hemodynamics and infarct size 
between IR and CON-SD group, which further intensifies the influence of 
stress caused by SD protocol. In further support of this results, we pre-
viously reported that acute SD can cause early preconditioning against 
IS-induced ventricular arrhythmia through activation of sympathetic 
nervous system; especially by the decrease in the incidence of VF 
(Edalatyzadeh et al., 2016). 
Effects of stress may be of two main types: local and remote. It has 
been documented that acute stress evokes several responses that may 
prevent the development of myocardial IR injury. The interaction be-
tween sympathetic nervous system, neuroendocrine system and car-
diovascular system by this acute stress response may ultimately result in 
the release of cardioprotective substances to limit infarct zone (Moghi-
mian et al., 2012). According to one study by Jeddi et al. it is indicated 
that hearts from SD had increased infarct size, lower basal cardiac 
function and less tolerance to IR injury due to elevated levels of CK-MB 
and nitric oxide (NO) (Jeddi et al., 2016). This contrasting results can be 
related to SD duration; it means that we used an ASD paradigm of 24 h 
not a CSD paradigm which last 96 h and caused detrimental effects on 
the heart. In addition, in another study we observed an increase in CK- 
MB levels just in I/R animals not in subjects which experienced SD 
before I/R inury (data is not published). After IR injury permeability of 
cardiomyocytes is incresed and causes CK-MB to leak from damaged 
heart to the blood stream and lead to increased infarct size (Mishra et al., 
2011). In this context it seems moderate increase in the levels of corti-
costrone secondary to SD have prevented higher release of CK-MB and 
subsequently further damage of cardiomyocytes. Moreover, low range of 
corticostrone may have an anti-inflammatory effect on myocytes and 
limit infarct development (Flower et al., 2015). Furthermore, in the 
current study we reported that sympathectomy may abolish SD induced 
favorable effects, suggesting the role of activation of sympathetic ner-
vous system in conferring stress generated cardioprotection. Moreover, 
studies have shown a positive correlation between infarct size and car-
diac arrhythmias and mortality following MI (Zaman and Kovoor, 
2014), which was evidenced in our study by smaller infarct size and 
improved cardiac function in SD animals than in IR group. In our study, 
the extent of the ischemic zone (AAR) was not different among the 
experimental groups which indicates that all animals underwent similar 
extent of ischemia and the results achieved are merely the effects of SD 
or sympathectomy. 
ROS production can be increased due to overproducction of NO. This 
oxidative stress plays a vital role in initiating and promoting IR induced 
myocardial injuries (Aghajani et al., 2017; Logue et al., 2005). However, 
on the other hand, it is reported that generation of reactive oxygen 
species (ROS) is necessary to create cardioprotection due to pre-
conditioning. In this regard, a study from our laboratory has reported 
that inhibition of ROS generation abolishes noradrenaline induced 
preconditioning effect (Imani et al., 2011). In this regard it seems low 
increase of NO production (due to sympathetic overdrive) (Imani et al., 
2011) by specific signaling pathways pevents further ROS production 
(Jeddi et al., 2016), and eventually limits infarct size (Imani et al., 
2011). Despite these all explanations, in the current study we did not 
evaluate NO levels to connect it with the obtaining results of PAB con-
centrion. An increase in antioxidative activity after short term SD was 
reported in the literature (Ramanathan and Siegel, 2011; Ramanathan 
et al., 2010), suggesting that compensatory mechanisms of ASD protect 
the heart from oxidative stress. However, it was reported that oxidative 
response could not necessarily be the result of SD (Villafuerte et al., 
2015); so this notion describes why oxidative stress in the current study 
did not have any role in creating the acute sleep deprivation-induced 
preconditioning effect and it seems further studies are needed to know 
SD induced preconditioning effects of oxiative stress markers. 
5. Conclusion 
This study showed ASD induces early preconditioning and confers 
cardioprotection. In addition, sympathectomy abolishes this protective 
effect, indicating the role of sympathetic nervous system activation in 
inducing SD induced cardioprotection. 
Acknowledgements 
This research did not receive any specific grant from funding 
agencies in the public, commercial, or not-for-profit sectors. 
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	Cardioprotective effects of acute sleep deprivation on ischemia/reperfusion injury
	1 Introduction
	2 Methods
	2.1 Animals
	2.2 Preparation of isolated hearts
	2.3 Determination of infarct size and area at risk
	2.4 Sleep deprivation paradigm- modified multiple platform method
	2.5 Chemical sympathectomy
	2.6 Measurement of serum pro-oxidant-antioxidant balance (PAB) and epinephrine
	2.7 Statistical analysis
	3 Results
	3.1 Homodynamic and ECG parameters
	3.2 Area at risk and infarct size
	3.3 Serum levels of epinephrine
	3.4 Pro-oxidant/antioxidant balance
	4 Discussion
	5 Conclusion
	Acknowledgements
	References