Intravenous injection of apelin-13 improves sensory-motor balance deficits caused by cerebral ischemic reperfusion injury in male wistar rats via restoration of nitric oxide

Journal Pre-proof Intravenous injection of apelin-13 improves sensory-motor balance deficits caused by cerebral ischemic reperfusion injury in male wistar rats via restoration of nitric oxide Raheleh Gholamzadeh (Conceptualization) (Methodology) (Software) (Writing - original draft) (Investigation) (Formal analysis) (Resources), Nahid Aboutaleb (Supervision) (Visualization) (Validation) (Writing - review and editing) (Resources), Donia Nazarinia (Investigation)

PII:

S0891-0618(20)30155-1

DOI:

https://doi.org/10.1016/j.jchemneu.2020.101886

Reference:

CHENEU 101886

To appear in:

Journal of Chemical Neuroanatomy

Received Date:

29 August 2020

Revised Date:

10 November 2020

Accepted Date:

10 November 2020

Please cite this article as: Gholamzadeh R, Aboutaleb N, Nazarinia D, Intravenous injection of apelin-13 improves sensory-motor balance deficits caused by cerebral ischemic reperfusion injury in male wistar rats via restoration of nitric oxide, Journal of Chemical Neuroanatomy (2020), doi: https://doi.org/10.1016/j.jchemneu.2020.101886

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

1|Page

Intravenous injection of apelin-13 improves sensory-motor balance deficits caused by cerebral ischemic reperfusion injury in male wistar rats via restoration of nitric oxide

Raheleh Gholamzadeh 1,2, Nahid Aboutaleb 1,2*, Donia Nazarinia 3 Physiology Research Center, Iran University of Medical Sciences, Tehran, Iran

2

Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran

3

Department of Physiology, School of Paramedical Sciences, Dezful University of Medical Sciences,

of

1

ro

Dezful

*Corresponding author: Nahid Aboutaleb, Associate Professor of Physiology, Physiology Research

-p

Center, Iran University of Medical Sciences, Tehran, Iran. Department of Physiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran; Tel: 0982186704589, Email:

lP

re

[email protected]

ur na

Running title: IV injection effect of apelin-13 on stroke

Jo

Graphical abstract

re

-p

ro

of

2|Page

 

Abstract has

been reported

that apelin-13 possesses

neuroprotective

effects

against

cerebral

Jo

It

Cerebral ischemia/reperfusion injury can be resulted in neuronal death and sensorimotor deficits. Nitric oxide has dual role in neural loss after ischemic stroke. Apelin-13 decreases cerebral ischemia/reperfusion injury via enhancement of nitric oxide.

ur na



lP

Highlights

ischemia/reperfusion injury (IRI). Disabilities in sense, movement and balance are the major stroke complications which, result in a high rate of mortality. Here, effects of intravenous (IV) injection of apelin-13 on the severity of neural death, infarct volume, neurological defects and its association with nitric oxide (NO) were investigated. A rat model of cerebral IRI was created by middle cerebral artery occlusion (MCAO) for 60 min and restoration of blood flow for 23 h. Animals were randomly assigned into six groups: sham, ischemia (MCAO), vehicle (MCAO + PBS) and three treatment groups (MCAO + apelin-13 in 10, 20, 40 μg/kg doses, IV). All injections were carried out via tail vein

3|Page

injection 5 min before reperfusion. Neural loss and infarct volume were evaluated by Nissl and 2,3,5triphenyltetrazolium chloride (TTC) staining, respectively. Neurological defects were scored by standard modified criteria. Serum NO was measured by colorimetric method. Apelin-13 in doses of 20 and 40 µg/kg

significantly reduced neural death, infarct volume and disturbance of sensory-motor balance compared to control and vehicle groups (p<0.05). Serum NO levels reduced in MCAO groups compared to sham. Apelin-13 restored serum NO levels at 20 µg/kg dose (p<0.05). Our data showed beneficial effect of IV injection of apelin-13 on sensory-motor balance defects by reducing neural death and restoration of serum NO levels. The present study shows the validity of apelin-13 in treatment of

of

ischemic stroke in different administration methods.

ro

Key words: Apelin-13; Sensory-motor balance defects, Cerebral ischemia/reperfusion injury, Nitric

-p

oxide.

re

1. Introduction

Brain ischemia is one of the most important debilitating diseases around the world (Yang et al., 2014;

lP

Amani, Kazerooni, et al. 2019). It occurs through a brain vessel occlusion due to a thrombosis or embolism which cause neuronal death

(Khaksari et al., 2012; Amani, Mostafavi, et al. 2019).

Thrombolytic drugs can protect neural cells from ischemic injury via restoration of brain circulation.

ur na

Meanwhile, reperfusion may exacerbate oxidative stress and enhance neuronal death by increasing oxygen access, referred to as ischemic reperfusion injury (Duan et al., 2019; Puig, Brenna, and Magnus, 2018). Previous studies have shown that 50-65% of patients suffer from different transient or permanent disturbance in sensory-motor balance after cerebral ischemia (Chaegil, 2019). These conditions restrict patients’ activity and impose high costs for individual, family and community.

Jo

Studies have shown that the rate of neuronal loss is directly correlated with the severity of neurological deficits (Faralli et al., 2013). The reduction of brain ischemia disabilities are of major concern in treatment, and therefore prevention of neural death can decrease these complications. It is well documented that reduction of blood flow to the brain activates the pathological process such as oxidative stress, excessive production of reactive oxygen species (ROS), mitochondrial dysfunction, inflammatory reactions, glutamate release and excitotoxicity. These factors cause the blood brain barrier (BBB) disruption and brain edema, which ultimately can result in brain neuronal death (Puig,

4|Page

Brenna, and Magnus, 2018). The damage to neurons reduces the brain synapses and subsequently gives rise to sensory and motor dysfunction (Faralli et al., 2013). Also, neuronal damage can reduce transmission of peripheral nerve information to the brain which, in turn lead to lack of balance (Chaegil, 2019). Nitric oxide (NO) as a vasodilator agent is one of the factors that increases after cerebral ischemia from different sources. Glutamate increases intracellular calcium via its receptors, which, in turn gives rise to NO production (Chen et al., 2017). According to the studies, NO has dual role in cerebral ischemia and neuronal death. Therefore, changes of NO during cerebral I/R and its

of

association with neuronal death and neurological deficits are fundamental issues that need much attention. (Castillo, Rama, and Dávalos, 2000; Li et al., 2018; Tanaka et al., 2018; Kawasaki et al.,

ro

2020).

Apelin-13 is an endogenous peptide that plays an important role in a large number of physiological

-p

processes. It is synthesized as a 77 amino acid pre-propeptide and converts to 13, 17 and 36 isoform by enzymatic breakdown. Apelin-13 is the most active form in the bloodstream (Khaksari et al., 2012;

re

O’Carroll et al., 2013; Yang et al., 2015). Its receptor is a G protein-coupled receptor (named APJ) that is similar to angiotensin II type1receptor ( AT1) but does not affect this receptor (Sun et al., 2011). Apelin-13 and the APJ receptor are expressed in various areas of the brain including neuronal

lP

cells, thalamus, hypothalamus, frontal cortex and vascular endothelial cells in both of rats and humans. Apelinergic system plays an important role in different physiological and pathophysiological activities

ur na

(Sun et al., 2011; Khaksari et al., 2012). Recently, the protective effects of apelin-13 on IRI has been demonstrated in several organs (Yang et al., 2015). Some studies showed protective effect of intravenous (IV) administration of apelin-13 on cardiac cells (Najafipour et al., 2012; Boal et al., 2016). Apelin-13 can diminish neuronal death by reducing cellular calcium and maintaining the mitochondrial membrane potential in epileptic cortical neurons (Esmaeili-Mahani, Sheibani, and

Jo

Najafipour, 2017). In brain ischemia, apelin-13 decreases neural death, infarct volume and improves neurological deficits through its anti-apoptotic and anti-inflammatory effects (Khaksari et al., 2012; Aboutaleb et al., 2014; Yang et al., 2014; Chen et al., 2015). A previous study has demonstrated that apelin-13 protects BBB by changing the expression of NO synthases enzymes (Chu et al., 2017). Previous reports have focused on administration of apelin-13 via intracerebroventricular (IVC) for treatment of cerebral I/R, which it is an invasive method and has no clinical application. Although Chen et al. showed the neuroprotective effects of intranasal administration of apelin-13 on brain ischemia, IV administration is the most effective method of drug delivery in clinic (Chen et al., 2015).

5|Page

Up to now, no study has been done on the effect of IV administration of apelin-13 on cerebral ischemia. So, in the present study, we aimed to investigate the effect of IV injection of apelin-13 on neuronal death, infarct volume, sensory-motor balance injury as well as its relationship with NO in cerebral IRI injury in rats. 2. Methods 2.1. Animals and experimental design

of

For the study design, 48 male Wistar rats (weighting 250-300 g) were obtained from Experimental and Comparative Studies Center of Iran University of Medical Sciences (IUMS). Animals were maintained

ro

under the standard conditions (12:12 light-dark cycle and controlled temperature of 23 ± 2° C), with free access to water and food. All the protocols and procedures were approved by the ethical

-p

committee of IUMS (ethical code: IR.IUMS.FMD.REC.1397.310).

According to Figure 1, rats were randomly divided into six groups (8 animals per group). 1) sham:

re

which only received surgical stress; 2) ischemia: The animals were subjected to middle cerebral artery occlusion (MCAO) for 60 min and reperfusion for 23 h to create the model; 3) vehicle: MCAO + IV

lP

administration of PBS via tail vein 5 min before reperfusion and three treatment groups: MCAO + IV administration of apelin-13 (cod: sc171835, Santa Cruz, USA) in 10, 20, 40 μg/kg doses from tail vein 5 min before reperfusion (Figure 1). In order to induce cerebral ischemia, the middle cerebral artery

ur na

(MCA) was closed for 60 min and then the blood flow restored for 23 h. The neuronal density, infarct volume, sensory- motor- balance deficits and NO serum level were evaluated in all the animals at 23 h

Jo

after reperfusion.

Figure 1: Schematic diagram of the experimental groups

6|Page

2.2. Brain ischemia induction in rats Brain ischemia was induced by MCAO model according to the modified Longa et al. method (Longa et al., 1989; Aboutaleb et al., 2014). Briefly, the weighed animals were anesthetized with chloral hydrate (400mg/kg, ip, code: 102425, Merck, Germany) and placed in a supine position. Under cardiac monitoring and sterile condition, a 2 cm liner incision with a 0.3 cm distance from the sternum bone was made on the right side of the neck. Then, the common carotid artery (CCA), internal carotid artery

of

(ICA) and external carotid artery (ECA) were found and vagus nerve dissected from CCA. Following that, after the permanent ligation of CCA and ECA, a 3.0 silicone-coated nylon standard filament with

ro

a round tip (code: 403734PK5Re, doccol, USA) was moved through the CCA and the ICA to occlude the MCA. Insertion of 20-22 mm length of suture with mild resistance against further entrance

-p

confirmed that it was located in correct place. At 60 min after MCAO, the filament was removed and

2.3. Neural density assessment

re

blood flow was restored for 23 h.

lP

Nissl staining was used to evaluate motor cortical neuronal density (n=3, 3 sections from each brain) (Shamsaei et al., 2015). In summary, the animals received deep anesthesia at 23 h after reperfusion and were transcardially perfused with 200 ml of isotonic cool saline and 4% paraformaldehyde,

ur na

respectively. After the tissue fixation, 5μm-thick coronal sections were provided from paraffinembedded brains on gelatinized slides. Then, sections were deparafinated in 60°C and according to Nissl protocol were stained with 0.1% cresyl violet. Then, the images of the motor cortex were taken with a light microscope (x400, Labomed, USA) and transferred to the computer. The number of necrotic cells was counted by a technician blinded to the study. Neurons were counted in 0.160 mm2

Jo

area of motor cortex (distance to bragma:lateral = +1.1 mm, anterior = +2.2 mm) with the use of Image J software.

2.4. Measurement of infarct volume In order to determine the infarct volume (n=5), the animals were scarified with chloral hydrate (800 mg/kg) and were immediately decapitated at 23 h after reperfusion. The brain tissues were removed and placed in cool isotonic saline (4 ° C) for 10 min. Then, 8 coronal sections with 2 mm thickness

7|Page

were provided from the frontal to the temporal lobe by using a brain matrix. Tissue sections were immersed in 2% solution of TTC (Sigma, Germany) for 15 min at 37 ° C. Images were obtained from the sections with digital camera and were transferred to a computer. Then, they were analyzed with image J software and infarct volume was calculated according to the following formula. Corrected infarct volume (%) = Left hemisphere volume - (Right hemisphere volume - infracted volume)  100 (Khaksari et al., 2012).

of

2.5. Assessment of sensory-motor balance deficits According to table 1, animals (n=8) were evaluated for neurological deficits with a modified standard

ro

test 23 h after reperfusion (Chen et al., 2001). Assessment items included sensory, motor, balance, neurological reflexes and abnormal movement tests. Animals received 0 score against normal response

-p

to tests and 1 or higher scores in abnormal responses. Sensation was assessed by stimulating the organs with a sharp object. Movement was assessed by observing the position of the limb and the

re

ability to move on a flat surface. For balance assessment, rats were placed on center of a beam balance (60 cm height, 50 cm length and 2 cm wide) three times (each duration 1 min) with 1 min intervals and the last test was considered as the valid one. The animals were finally allocated a total score from 0-

lP

16, as not damaged (0) to severely injured (16).

ur na

Table 1. Sensory-motor balance deficits in animals at 23 h after reperfusion Table 1 Neurological score

score

Sensory tests Stimulation of the organs with a sharp object 0 Pulling back the limb 1 No response

1

Jo

Neurological deficits evaluation

Motor tests a) For performancing of this test animal was raised from tail 1 Flexion of fore limb 1 Flexion of hind limb 1 Rotate the head 10 degrees on the vertical axis within 30 seconds b) Observation of the animal walking on the ground 0 Normal walking 1 Disability for straight walking 2 Circling to the paralysis side of the body 3 Falling into paralysis side of the body

6

8|Page Beam balance tests 0 Standing and walking on beam balance with maintaining body posture 1 Grasps side of beam 2 Grips the beam and one limb falls down from the beam 3 Grips the beam and two limbs fall down from the beam, or rotate on beam in further than 60 seconds 4 Trying to keep the balance on the beam but falling in less than 40 seconds 5 Trying to keep the balance on the beam but falling in less than 20 seconds 6 Unable to maintain balance on the beam and falling in less than 20 seconds Reflexes tests a) Corneal reflex 0 blinking after mild cotton contact with eyes 1 No reflex b) Startle reflex 0 motor reaction after induced mild sound with clapping 1 No reflex Abnormal movement 1 Seizures

6

Total score

16

ro

of

2

1

-p

Animals received a score of 1if they were unable to show normal response or did not have a nervous reflex

lP

2.6. Measurement of NO serum level

re

1-6 mild injury, 7-12 moderate injury, 13-16 severe injury

In order to evaluate serum level of NO, blood samples were taken from the animals (n=7) at 23 h after reperfusion and immediately centrifuged (4000 rmp, 4°C) for 10 min. All serums were collected and

ur na

stored in -20°C prior to analysis. The NO level was measured according to the NO assay kit protocol (Cib Biotech company, Tehran, Iran), by Greiss calorimetric method (Castillo, Rama, and Dávalos, 2000). Since NO is an invisible gas, the basis of this method is measurement of its stable metabolites, which are nitrite and nitrate. Briefly, the samples were thawed at room temperature and loaded in the wells. Next, sulfanilic acid was added to samples. Serum nitrite (NO2-) reacted with sulfanilic acid and

Jo

produced diazonium ion. Then, this ion is paired with a combination of 1-naphthyl ethylene diamine, which was added to all wells and produced a pink combination of azo derivatives. Finally, absorbance was read in 570 nm using ELAISA reader and NO concentrations were calculated using the standard curve. 2.7. Statistical analysis

9|Page

The collected data were analyzed using SPSS software (version 16.0) through one-way analysis of variances (ANOVA) and Tukey's post hoc test. The results were expressed based on the comparison of the mean ± SEM values, and then p<0.05 was considered statistically significant. 3. Results 3.1. Apelin-13 prevents reduction of neural density following cerebral I/R injury For evaluation of the neural loss, dead cells were counted in motor cortex of Nissl-stained brain tissue

of

sections. Neural loss was markedly enhanced in MCAO groups in comparison with the sham group. No significant difference was observed between ischemia (57.92 ± 2.11) and vehicle (51.7 ± 7.9)

ro

groups. Apelin-13 treatment groups in doses of 20 μg (36 ± 3.6) and 40 μg (22.66 ± 2.51) significantly diminished neural death compared to ischemia and vehicle groups. Also, there was a significant

Jo

ur na

lP

re

-p

difference in neural death between doses of 10 μg and 40 μg (p=001) (Figure 2).

10 | P a g e

B

of

A

-p

ro

C

70 50

***

*** !!,$

40

$$$ ###

30 20

ur na

10 0

*** $$$ ###

re

***

lP

Neural loss (%)

60

Sham Ischemia Vehicle Apelin 10

Apelin Apelin 20 40

Jo

Figure 2. Comparison of neural loss between experimental groups (n=3). A: Area of the neuron counting in the motor cortex B: Nissl-stained images of brain motor cortical neurons with 100X and 400X magnification. Red arrows indicate necrotic cells. C: IV administration of apelin-13 reduced neural loss after IRI (***P<0.0001 vs Sham, $P<0.05 & $$$ P<0.0001 vs Ischemia, ###P<0.0001vs Vehicle, !!P<0.01 vs dose 40μg).

3.2. Apelin-13 decreases the brain infarct volume following cerebral I/R injury At 23 h after MCAO, all tissues were stained with TTC to evaluate brain infarct volume. The whole brain tissues in the sham group were turned as red, which is a sign of the healthy tissue. All MCAO groups showed extensive tissue damage than the sham group. There were no significant differences in

11 | P a g e

infarct volume between ischemia (36.62 ± 9.04) and vehicle (36.17 ± 16.20) groups. Apelin-13 treatment groups showed a decrease in infarct volume compared to vehicle and ischemia groups, which was significant in 20 μg (P=0.011) and 40 μg (P=0.004) groups. There was no significant difference in stroke volume between the treatment groups (Figure 3).

re

-p

ro

of

A

***

lP

50 45 40 35 30 25 20 15 10 5 0

***

Jo

ur na

Infarct volume(%)

B

**

$ #

$$ ##

12 | P a g e

Figure 3: Comparison of total infarct volume between experimental groups (n=5). A) Brain tissue sctions stained with TTC, red area:safe tissue, wihte area: injury tissue . B: IV administration of apelin-13 reduced infarct volume after IRI ( ** P<0.01 & *** P<0.0001 vs Sham, $P<0.05 & $$P<0.01 vs Ischemia, #P<0.05 & ##P<0.01 vs Vehicle).

3.3. Apelin-13 improves the sensory- motor- balance deficits following cerebral I/R injury The severity of neurological damage was assessed at 23 h after reperfusion using a complete 16criteria sensory-motor and balance test. No abnormalities were observed in the sham group. All MCAO groups showed marked sensory-motor balance deficits in comparison with sham. In the case of

of

ischemia (10.75 ± 1.48) and vehicle (10.87 ± 1.88) groups, the severity of the injury was almost the same. Apelin-13 treatment groups in doses of 20 μg (P=0.000) and 40 μg (P=0.000) showed a

ro

significant decrease in severity of neurological deficits compared to vehicle and ischemia groups. Also, in comparison between the treatment groups, dose of 10 μg (p=0.027) showed a significant

re

-p

difference from 20 μg (Figure 4).

***

12 8 4 2 0

***

*** !

10 6

lP

14

ur na

Neurological defects score

16

Apelin 10

*** $$$ ###

Apelin 20

Apelin 40

Jo

Sham Ischemia Vehicle

** $$$ ###

Figure 4. Comparison of neurological deficits in experimental groups (n=8). IV administration of apelin- 13 improved neurological deficits after IRI (**P<0.01 & ***P<0.0001 vs Sham, $$$P<0.0001 vs Ischemia, ###P<0.0001 vs Vehicle, ! P<0.05 vs dose 20μg).

3.4. Apelin-13 prevents reduction of NO serum level following cerebral I/R injury

13 | P a g e

Serum NO level was measured at 23 h after reperfusion by the Griss method. In all MCAO groups, serum NO level was markedly decreased compared to sham. There were no significant differences in serum NO levels between ischemic (2.75 ± 0.28) and vehicle (2.82 ± 0.34) groups. Apelin-13 treatment increased serum NO level, which was significant with the 20µg dose (p=0.003) compared to ischemia and vehicle groups. Also, there were no significant differences between the treatment groups

of

5

$$ ##

***

***

-p

3

ro

* !

4

1 0 Sham Ischemia Vehicle Apelin 10

re

2

Apelin Apelin 20 40

ur na

lP

Serum nitric oxide level(μmol/dl)

(Figure 5).

Figure 5) Comparison of serum NO level between experimental groups (n=7). IV administration of apelin-13 increases serum NO level. (*P<0.05 & ***P<0.0001 vs Sham, $$ P<0.01 vs Ischemia, ##P<0.01 vs Vehicle).

4. Discussion

Jo

One of the major problems after cerebral I/R is sensory-motor and balance deficits, the severity of which is related to the rate of neuronal death and the extent of infarct volume (Faralli et al., 2013; Chaegil, 2019). Also, the amount of release of NO, a brain circulation regulator after cerebral I/R, plays an important role in severity of neuronal death and its outcomes (Castillo, Rama, and Dávalos, 2000; Chen et al., 2017; Kawasaki et al., 2020). Despite the availability of the numerous ways (oral, subcutaneous, IV) for the drug administration in stroke patients, especially in acute phase, the most effective method is IV injection. Our study for the first time showed that IV administration of apelin-

14 | P a g e

13 at 5 min before reperfusion could markedly improve neurological defects after IRI by reducing neuronal death and infarct volume through prevention of NO depletion in male Wistar rats. In accordance with previous studies (Khaksari et al., 2012; Aboutaleb et al., 2014; Yang et al., 2014; Chu et al., 2017; Duan et al., 2019), our results showed that IRI drastically increased neuronal death, and this change was accompanied by an increase in infarct volume and sensory-motor balance defects. Khaksari et al. showed that establishment of reperfusion after cerebral ischemia resulted in extensive damage to brain tissue with significant edema (Khaksari et al., 2012). Aboutaleb et al. demonstrated

of

MCAO for 60 min induced apoptosis in brain cortical neurons and caused severe neurological defects (Aboutaleb et al., 2014). The metabolism of neuronal cells can shift to anaerobic when blockade of the

ro

blood flow to the brain reduce access of the oxygen and glucose to cells. Subsequently, activation of various pathological processes, including activation of glutamate receptors, intracellular calcium

-p

overload, mitochondrial dysfunction, ROS formation, and secretion of neuroinflammatory factors result in damage to neural cells and subsequently necrotic and apoptotic cell death in the ischemic

re

core and penumbra, respectively (Puig, Brenna, and Magnus, 2018). Neural death reduces brain synaptic communications and disrupts the comprehensive function of the central nervous system neurovascular unit (Faralli et al., 2013; Puig, Brenna, and Magnus, 2018). Therefore, it reduces the

lP

transmission of information between different parts of the brain, as well as from the environment to the brain, which, in turn results in various disorders in sensation, movement and balance (Chaegil,

ur na

2019). In agreement with our findings, previous studies have shown that the higher rate of neuronal death is associated with the greater infarct volume and neurological defects (Faralli et al., 2013; Yang et al., 2014; Chen et al., 2015; Huang et al., 2016) . In present study, IV injection of apelin-13 notably reduced neuronal death and infarct volume following IRI in a dose dependent manner, and was associated with considerable improvements in

Jo

sensory-motor and balance deficits. IV injection of apelin-13 in dose of 10 µg/kg did not affect the examined parameters but doses of 20 and 40 µg/kg showed completely favorable effects in reducing IRI. Based on our findings, apelin-13 in dose of 20 µg/kg is suggested as minimum IV effective dose on brain IRI. This result was in accordance with the other studies. Khaksari et al. showed that IVC injections of apelin-13 in three different doses decreased brain infarct volume, neurological deficits and brain edema with the best observable effects in higher dose rates (Khaksari et al., 2012). Also, another study demonstrated that IVC injections of apelin-13 could improve neurological defects

15 | P a g e

caused by IRI in a dose dependent manner (Aboutaleb et al., 2014). Yang et al. reported that apelin-13 could reduce apoptosis through activation of phosphatidylinositol 3-kinase /protein kinase B (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Also, another

study demonstrated that apelin-13 reduced infarct volume and neurological deficit by regulation of balance between apoptotic markers (Bax , Bcl2 and caspase-3) (Yang et al., 2014). Huang et al. found that post-ischemic IVC injection of apelin-13 enhanced survival of neuronal and vascular endothelial cells as well as decreased sensory-motor and balance deficits through overexpression of vascular

of

endothelial growth factor (VEGF) and protecting integrity of neurovascular unit (Huang et al., 2016). Our study indicated reduction of the serum NO levels in MCAO group at 23 h after reperfusion in

ro

comparison with sham group. Some studies explained that the serum NO levels increased after brain ischemia compare to healthy group (Chen et al., 2017; Tsai et al., 2007). But, in agreement with our

-p

study, Serrano-Ponz et al. illustrated a significant reduction of the serum NO level at 24 h after brain ischemia compared to healthy individuals (Serrano‑Ponz et al., 2016). Also, other research showed

re

significant elevated levels of serum NO at 1 h after I/R and restoration to basal level at 24 h after reperfusion (Hussein, Omayma, and Elwakil, 2012). The reason for these different results is probably

lP

related to the various methods and time of the NO measurement and also different sources of NO release. Three isoform enzymes produce NO after reperfusion including endothelial NO synthase (eNOS), neural NO synthase (nNOS) and inducible NO synthase (iNOS) (Chen et al., 2017). The

ur na

balance between eNOS, nNOS and iNOS activity determines the NO serum and tissue concentrations (Serrano‑Ponz et al., 2016; Chen et al., 2017). The elevated Serum levels of NO in the early hours of ischemia and reperfusion is more likely related to neural and inducible sources. This NO increases ROS production and secretion of pro-inflammatory cytokines via mitochondria impairment which causes more ischemia and reperfusion insult (Chen et al., 2017; Puig, Brenna, and Magnus, 2018). In

Jo

contrast, increasing NO level at 24 h after reperfusion probably has an endothelial origin that reduces brain IRI by increasing cerebral blood flow (Serrano‑Ponz et al., 2016). In addition, agents that increase NO by eNOS origin or reduce iNOS and nNOS, showed protective effects on brain ischemia (Kawasaki et al., 2020; Khan et al., 2009; Li et al., 2018; Khan et al., 2005). In keeping with these previous studies, our findings showed that the diminution in serum NO level at 23 h after reperfusion was associated with a massive neuronal death, extension of infarct volume and neurological deficits while IV administration of apelin-13 reversed these effects by restoration of NO serum levels.

16 | P a g e

Similarly, Kawasaki et al. proved NO enhancement and nNOS expression decrement after cerebral I/R protected neural cells (Kawasaki et al., 2020). One study illustrated that the administration of a slow donor NO protected brain from IRI by iNOS inhibition and increased cerebral blood flow (Khan et al., 2005). Chu et al. documented that apelin-13 protected the brain from IRI via regulation of iNOS and eNOS activity (Chu et al., 2017). In addition, apelin-13 intranasal delivery showed neuroprotective effects against IRI by increasing cerebral blood flow (Chen et al., 2015). According to the results obtained from this work as well as the reported data by the reviewed documents, it could be

of

said that IV injection of apelin-13 may act as a NO donor and reduces neuronal death and improves sensory-motor equilibrium defects through restoration of brain blood flow. However, further studies

ro

are needed to confirm this hypothesis.

The present study for the first time showed IV injection of apelin-13 at a lower dose than IVC and

-p

intranasal injection could significantly reduce the brain IRI. Similarity, Najafipour et al. indicated IV administration of apelin-13 could improve cardiomyopathy caused by hypertension induced by renal

re

ischemia (Najafipour et al., 2012). Also, IV injection of apelin-13 protected cardiac cells from IRI by inhibiting apoptosis (Boal et al., 2016). Thus, apelin-13 IV injections may open new avenue to treat cerebral I/R injury. Although IVC administration of the drug is more effective than IV, it is an

lP

invasive procedure limited by the bony skull and may increase brain tissue damage. Intranasal drug delivery is a non-invasive method without BBB limitation, but would require a particular drug

ur na

formulation due to the mucosillary clearance and low permeability of nasal epithelial cells (Mittal et al., 2014). In confirmation of this, Chen et al. prescribed hyaluronidase in to the nostril of all animals before apelin-13 administration to increase tissue permeability, which it may cause damage to nasal mucosa (Chen et al., 2015). In addition, intranasal delivery of drugs requires the correct position of the head and there is a possibility of drug entry into the trachea and nasopharynx, which leads to repeated

Jo

dosing and increased complications. Also, direct drug delivery into the brain increases the risk of neurotoxicity (Mittal et al., 2014), as studies showed that highly potent peptides can affect the brain without the requirement to cross the BBB (Banks, 2015). Therefore, IV administration of apelin-13, especially in the acute phase of stroke, seems to be a good method in drug delivery for treatment of stroke. 5. Conclusion

17 | P a g e

Our study demonstrated association between neural death, infarct volume, neurological deficits and serum NO level. IV injection of apelin-13 could markedly ameliorate neural death, sensory-motor balance defects, and infarct volume via restoration of serum NO level. Our study showed that IV administration of apelin-13 may contribute to obtain a pathway one step closer to clinical use, but understanding its mechanisms and side effects need to further investigations. Author contributions

of

Raheleh Gholamzadeh: Conceptualization, Methodology, Software, Writing- Original draft preparation, Investigation, Formal analysis, Resources. Nahid Aboutaleb: Supervision, financial

ro

supporting, Visualization, Validation, Writing- Reviewing and Editing, Resources. Donia Nazarinia:

Conflict of interest

re

All authors affirm that have no conflict of interest.

-p

Investigation.

Funding

lP

This research is result of a PhD student thesis in physiology, which is supported economically by Iran University of Medical Sciences (grant number: 97-3-4-12866).

ur na

All authors confirm all below items:

1. Animals were maintained under the standard conditions (12:12 light- dark cycle and controlled temperature of 22–24° C ), with freely water and food access. All animal work and procedures were approved by the ethical committee of IUMS

Jo

(ethical cod: IR.IUMS.FMD.REC.1397.310) accordance with National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).

2. All authors affirm that have no conflict of interest.

18 | P a g e

3. This manuscript is result of a PhD student thesis in physiology, which is supported economically by Iran University of Medical Sciences (grant number: 97-3-4-12866) and we have not received financial supporting from any

ro

of

organization.

Acknowledgment

Sciences, for her kindly scientific collaboration.

re

References

-p

We would like to thank Mr. Karimi, M.Sc of Razi Drug Research Center of Iran University of Medical

Jo

ur na

lP

Aboutaleb, N., Kalalianmoghaddam, H., Eftekhari, S., Shahbazi, A., Abbaspour, H., Khaksari, M., 2014. Apelin13 inhibits apoptosis of cortical neurons following brain ischemic reperfusion injury in a transient model of focal cerebral ischemia. Int J Pept Res Ther 20, 127-132. Amani, Hamed, Hanif Kazerooni, Hossein Hassanpoor, Abolfazl Akbarzadeh, and Hamidreza PazokiToroudi. 2019. 'Tailoring synthetic polymeric biomaterials towards nerve tissue engineering: a review', Artificial cells, nanomedicine, and biotechnology, 47: 3524-39. Amani, Hamed, Ebrahim Mostafavi, Mahmoud Reza Alebouyeh, Hamidreza Arzaghi, Abolfazl Akbarzadeh, Hamidreza Pazoki-Toroudi, and Thomas J Webster. 2019. 'Would Colloidal Gold Nanocarriers Present An Effective Diagnosis Or Treatment For Ischemic Stroke?', International Journal of Nanomedicine, 14: 8013 Banks, W. A,. 2015. Peptides and the blood–brain barrier. Peptides 72, 16-19. Boal, F., Timotin, A., Roumegoux, J., Alfarano, C., Calise, D., Anesia, R., Parini, A., Valet, P., Tronchere, H., Kunduzova, O,. 2016. Apelin‐13 administration protects against ischaemia/reperfusion‐mediated apoptosis through the FoxO1 pathway in high‐fat diet‐induced obesity. Br J Pharmacol 173, 18501863. Castillo, J., Rama, R., Dávalos, A., 2000. Nitric oxide–related brain damage in acute ischemic stroke. Stroke 31, 852-857. Chaegil, L., 2019. Multi-Sensorimotor Training Improves Proprioception and Balance in Subacute Stroke Patients: A Randomized Controlled Pilot Trial. Front Neurol 10, 157. Chen, D., Lee, J., Gu, X., Wei, L., Yu, S.P,. 2015. Intranasal delivery of apelin-13 is neuroprotective and promotes angiogenesis after ischemic stroke in mice. ASN neuro 7, 1759091415605114. Chen, J., Li, Y., Wang, L., Zhang, Z., Lu, D., Lu, M., Chopp, M., 2001. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 32, 1005-1011.

19 | P a g e

Jo

ur na

lP

re

-p

ro

of

Chen, Z-q., Mou, R-t., Feng, D-x., Wang, Z., Chen, G., 2017. The role of nitric oxide in stroke. Med Gas Res 7, 194. Chu, H., Yang, X., Huang, C., Gao, Z., Tang, Y., Dong, Q., 2017. Apelin-13 protects against ischemic blood-brain barrier damage through the effects of aquaporin-4. Cerebrovasc Dis 44, 10-25. Duan, J., Cui, J., Yang, Z., Guo, C., Cao, J., Xi, M., Weng, Y., Yin, Y., Wang, Y., Wei, G., 2019. Neuroprotective effect of Apelin 13 on ischemic stroke by activating AMPK/GSK-3β/Nrf2 signaling. J. Neuroinflammation 16, 24. Esmaeili-Mahani, S., Sheibani, V., Najafipour, H., 2017. Apelin-13 protects rat primary cortical glia-neuron coculture against pentylenetetrazole-induced toxicity. Biomed Pharmacother 87, 661-668. Faralli, A., Bigoni, M., Mauro, A., Rossi, F., Carulli, D., 2013. Noninvasive strategies to promote functional recovery after stroke. J Neural Transplant Plast, 2013. Huang, C., Dai, C., Gong, K., Zuo, H., Chu, H., 2016. Apelin-13 protects neurovascular unit against ischemic injuries through the effects of vascular endothelial growth factor. Neuropeptides 60, 67-74. Hussein, S.A., Omayma, A.R., Elwakil, A., 2012. Biochemical Abnormalities in Brain Tissues during. J. Med. Sci 12, 121-130. Kawasaki, H., Ito, Y., Kitabayashi, C., Tanaka, A., Nishioka, R., Yamazato, M., Ishizawa, K., Nagai, T., Hirayama, M., Takahashi, K., 2020. Effects of Edaravone on Nitric Oxide, Hydroxyl Radicals and Neuronal Nitric Oxide Synthase During Cerebral Ischemia and Reperfusion in Mice. J Stroke Cerebrovasc Dis 29, 104531. Khaksari, M., Aboutaleb, N., Nasirinezhad, F., Vakili, A., Madjd, Z., 2012. Apelin-13 protects the brain against ischemic reperfusion injury and cerebral edema in a transient model of focal cerebral ischemia. J Mol Neurosci 48, 201-208. Khan, M., Im, Y-B., Shunmugavel, A., Gilg, A.G., Dhindsa, R.K., Singh, A.K., Singh, I., 2009. Administration of Snitrosoglutathione after traumatic brain injury protects the neurovascular unit and reduces secondary injury in a rat model of controlled cortical impact. J Neuroinflammation 6, 32. Khan, M., Sekhon, B., Giri, S., Jatana, M., Gilg, A.G., Ayasolla, K., Elango, C., Singh, A.K., Singh, I., 2005. SNitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke. J Cereb Blood Flow Metab 25, 177-192. Li, S., Wang, Y., Jiang, Z., Huai, Y., Liao, J.K., Lynch, K.A., Zafonte, R., Wood, L.J., Wang, Q.M., 2018. Impaired cognitive performance in endothelial nitric oxide synthase knock-out mice after ischemic stroke, a pilot study. Am J Phys Med Rehabil 97, 492. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins ,R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. stroke 20, 84-91. Mittal, D., Asgar A., Shadab, Md., Sanjula, B., Jasjeet, K.S., Javed, A., 2014. Insights into direct nose to brain delivery: current status and future perspective. Drug deliv 21, 75-86. Najafipour, H., Soltani.H, A., Nekooian, A.A., Esmaeili-Mahani, S., 2012. Apelin receptor expression in ischemic and non-ischemic kidneys and cardiovascular responses to apelin in chronic two‐kidney–one‐clip hypertension in rats. Regul Pept 178, 43-50. O’Carroll, A-M., Lolait, S.J., Harris, L.E., Pope, G.R., 2013. The apelin receptor APJ: journey from an orphan to a multifaceted regulator of homeostasis. J Endocrinol 219, R13-35. Puig, B., Brenna, S., Magnus, T., 2018. 'Molecular communication of a dying neuron in stroke. Int J Mol Sci 19, 2834. Serrano‑P, M., Rodrigo‑G, C., Siles, E., Martínez‑L, E., Ochoa‑C, L., Martínez, A., 2016. Temporal profiles of blood pressure, circulating nitric oxide, and adrenomedullin as predictors of clinical outcome in acute ischemic stroke patients. Mol Med Rep 13, 3724-3734. Shamsaei, N., Khaksari, M., Erfani, S., Rajabi, H., Aboutaleb, N., 2015. Exercise preconditioning exhibits neuroprotective effects on hippocampal CA1 neuronal damage after cerebral ischemia. Neural Regen Res 10, 1245.

20 | P a g e

Jo

ur na

lP

re

-p

ro

of

Sun, X., Iida, S., Yoshikawa, A., Senbonmatsu, R., Imanaka, K., Maruyama, K., Nishimura, S., Inagami, T., Senbonmatsu, T., 2011. Non-activated APJ suppresses the angiotensin II type 1 receptor, whereas apelin-activated APJ acts conversely. Hypertens Res 34, 701-706. Tanaka, A., Ito, Y., Kawasaki, H., Kitabayashi, C., Nishioka, R., Yamazato, M., Ishizawa, K., Nagai, T., Hirayama, M., Takahashi, K., 2018. Effects of memantine on nitric oxide production and hydroxyl radical metabolism during cerebral ischemia and reperfusion in mice. J Stroke Cerebrovasc Dis 27, 1609-1615. Tsai, S-K., Hung, L-M., Fu, Y-T., Cheng, H., Nien, M-W., Liu, H-Y., Zhang, F.B-Y., Huang, S-S., 2007. Resveratrol neuroprotective effects during focal cerebral ischemia injury via nitric oxide mechanism in rats. J Vasc Surg 46, 346-353. Yang, Y., Lv, S-Y., Lyu, S-K., Wu, D., Chen, Q., 2015. The protective effect of apelin on ischemia/reperfusion injury. Peptides 63, 43-46. Yang, Y., Zhang, X., Cui, H., Zhang, C., Zhu, C., Li, L., 2014. Apelin-13 protects the brain against ischemia/reperfusion injury through activating PI3K/Akt and ERK1/2 signaling pathways. Neurosci Lett 568, 44-49.