Melatonin regulates neuroinflammation ischemic stroke damage through interactions with microglia in reperfusion phase

Journal Pre-proofs Research report Melatonin Regulates Neuroinflammation Ischemic Stroke Damage through Interactions with Microglia in Reperfusion phase Fereshteh Azedi, Masoud Mehrpour, Saeed Talebi, Adib Zendedel, Somaieh Kazemnejad, Kazem Mousavizadeh, Cordian Beyer, Amir Hassan Zarnani, Mohammad Taghi Joghataei PII: DOI: Reference:

S0006-8993(19)30455-X https://doi.org/10.1016/j.brainres.2019.146401 BRES 146401

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Brain Research

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Please cite this article as: F. Azedi, M. Mehrpour, S. Talebi, A. Zendedel, S. Kazemnejad, K. Mousavizadeh, C. Beyer, A. Hassan Zarnani, M. Taghi Joghataei, Melatonin Regulates Neuroinflammation Ischemic Stroke Damage through Interactions with Microglia in Reperfusion phase, Brain Research (2019), doi: https://doi.org/10.1016/ j.brainres.2019.146401

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Effect of melatonin on microglia in reperfusion phase after ischemic stroke

Melatonin Regulates Neuroinflammation Ischemic Stroke Damage through Interactions with Microglia in Reperfusion phase Fereshteh Azedi1, Masoud Mehrpour2, Saeed Talebi3, Adib Zendedel4, Somaieh Kazemnejad5, Kazem Mousavizadeh6, Cordian Beyer4, Amir Hassan Zarnani7,8*, Mohammad Taghi Joghataei1,9* 1

Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences,

Tehran, Iran 2

Department of Neurology, Firoozgar Hospital, Iran University of Medical Sciences, Tehran, Iran

3

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

4

Institute of Neuroanatomy, RWTH Aachen University, Aachen, Germany

5

Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

6

Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical

Sciences, Tehran, Iran 7

Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

8

Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

9

Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran

*Corresponding Addresses: Mohammad Taghi Joghataei Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences P.O. Box: 14665-354 Tel: +98-21-86704698 Fax: +98-21-86704698 Email: [email protected], [email protected]

Amir-Hassan Zarnani Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran P.O. Box: 141556446 Tel: +98-21-88953132 Email: [email protected], [email protected]

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Abbreviations: ANOVA: Analysis of variance BBB: Blood brain barrier BDNF: Brain-derived neurotrophic factor CCL-5: Chemokine (C-C motif) ligand 5 CNS: Central nervous system CSFCS: Charcoal-stripped fetal calf serum DAB: Diaminobenzidine DMEM: Dulbecco’s modified Eagle medium EBST: Elevated body swing test ECA: External carotid artery GFAP: Glial fibrillary acidic protein H&E: Hematoxylin-eosin HIER: Heat-induced antigen retrieval HSPA1A: Heat shock 70 kDa protein 1 ICA: Internal carotid artery IL1B: Interleukin 1 beta iNOS: Inducible nitric oxide synthase

MAP2: Microtubule-associated protein 2 MCA: Middle cerebral artery MRI: Magnetic resonance imaging MT1: Melatonin receptor 1A MT2: Mtnr1b, Melatonin receptor 1B MT3: Melatonin receptor 1C OS: Oxidative stress PBS: Phosphate buffered saline PFA: Paraformaldehyde PPA: Pterygopalatine artery qrtPCR: Quantitative-real time PCR tMCAO: Transient middle cerebral artery occlusion TNFA: Tumor necrosis factor alpha TREM2: Triggering receptor expressed on myeloid cells 2 TTC: Triphenyltetrazolium chloride VEGF: Vascular endothelial growth factor

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Abstract Even today, ischemic stroke is a major cause of death and disabilities because of its high incidence,

limited

treatments

and

poor

understanding

of

the

pathophysiology

of

ischemia/reperfusion, neuroinflammation and secondary injuries following ischemic stroke. The function of microglia as a part of the immune system of the brain following ischemic stroke can be destructive or protective. Recent surveys indicate that melatonin, a strong antioxidant agent, has receptors on microglial cells and can regulate them to protective form; yet, more findings are required for better understanding of this mechanism, particularly in the reperfusion phase. In this study, we initially aimed to evaluate the therapeutic efficacy of melatonin intra-arterially and to clarify the underlying mechanisms. After that by using an in vitro approach, we evaluated the protective effects of melatonin on microglial cells following the hypoxia condition. Our results proved that a single dose of melatonin at the beginning of reperfusion phase improved structural and behavioral outcomes. Melatonin increased NeuN and decreased GFAP, Iba1 and active caspase-3 at protein level. Furthermore, melatonin elevated BDNF, MAP2, HSPA1A and reduced VEGF at mRNA level. We also showed that melatonin receptor 1B highly expressed in microglial cells after 3 hours hypoxia. Besides, melatonin increased the ratio of TREM2/iNOS as a marker of the most protective form of microglia (M2). In summary, our data suggest that melatonin has the possibility to serve as targeting microglial action for preventing secondary injury of reperfusion phase after ischemic stroke. Keywords: Melatonin, Microglia, Neuroinflammation, Reperfusion phase, Ischemic stroke

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1. Introduction Ischemic stroke is one of the most severe types of neurological disorders which leads to longterm disability, high mortality and costs to the health care system (Feigin et al., 2014, Johnston et al., 2009). The majority (70–80%) of stroke cases are ischemic, while intracerebral hemorrhage accounts only for 10–20% of all stroke cases (Feigin et al., 2009). To date, there is still no effective treatment that increases the survival rate or improves the quality of life after ischemic and hemorrhagic stroke. Overproduction of oxidative stress (OS) and reactive oxygen species (ROS) following ischemic insult is known as a key factor in exacerbating brain damage (Dirnagl et al., 1999). OS, characterized by increased free radical damage, has been implicated to exacerbate stroke-induced pathophysiological and behavioral dysfunctions (Liu et al., 2006). Indeed, anti-ROS approaches such as applying free radical scavengers and antioxidants, have been extensively explored for the treatment of ischemic stroke and protect against cell death (Lee et al., 1999). Melatonin (N-acetyl-5-methoxy-tryptamine) is a neurohormone produced by the pineal gland and also in bone marrow (Reiter et al., 1999), which takes in a broad-spectrum antioxidant and potent free radical scavenger (Reiter et al., 2000). This hormone regulates the effect on the activities of enzymes involved in the generation of free radicals, and on microsomal membrane fluidity in situations of elevated oxidative stress. Moreover, its solubility in lipid and aqueous media, which allows it to cross morphophysiological barriers such as blood brain barrier (BBB) and enter subcellular compartments, permit melatonin to function as a highly effective inhibitor of oxidative damage. Three subtypes of melatonin receptors have been identified in mammals: melatonin receptor 1A (MT1), melatonin receptor 1B (MT2), and melatonin receptor 1C (MT3) of which MT1 and MT2 belong to the family of G protein-coupled, seven transmembrane

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receptors (von Gall et al., 2002). It is well established that the MT2 receptor has an important role in mediating the neuroprotective effects of melatonin following ischemia/reperfusion, as well as an association with a robust increase in neurogenesis (Lee et al., 2010, Chern et al., 2012). MT2 appeared to be strongly expressed by microglia, astrocytes and oligodendrocytes and recent findings showed that neuroprotective effect of melatonin is associated with a reduction of microglial activation and free radical scavenger production through MT2 receptor (Olivier et al., 2009). Microglia are the resident immune cells of the central nervous system (CNS) (Ransohoff and Brown, 2012, Ajami et al., 2007) which have a critical role to maintain the homeostasis of CNS (Neumann et al., 2006, Eyo and Dailey, 2013). After ischemic stroke, macrophages and microglia predominantly infiltrate the ischemic tissue in large numbers and make secondary injury in reperfusion phase. Microglia are be able to polarize to the classic pro-inflammatory type (M1-like) or alternative protective type (M2-like) by optimal condition (Ginhoux et al., 2010). M1-like microglia can produce pro-inflammatory cytokines, such as TNF-α, IL-1β, CCL5 and iNOS (Hartung and Finsen, 1998, Parada et al., 2013). In contrast, the protective M2-like microglia exerts their effect through the secretion of neuroprotective factors, such as TREM2 (Tocharus et al., 2010). It is proven that applying melatonin intravenously or intraperitoneally as a therapeutic agent can improve outcomes after ischemic stroke. Notably, it has shown that the different application routes of therapeutic agents have a clear various impacts on the final yield of the stroke treatment (Berkhemer et al., 2015, Mullen, 2015). Intravenous administration is one of the major ones; however, less diffuse distribution of most therapeutic agents in and around the infarct area compared to intra-parenchymal and intra-cerebroventricular is a main problem (Li et al., 2010,

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Fischer et al., 2009). Among these, the intra-arterial (IA) route takes in a high potential for clinical translation, particularly for clinical application of endovascular therapy in the treatment of ischemic stroke, however, there are numerable studies applying intra-arterial delivery of therapeutic agents such as melatonin (Cloft et al., 2009). Briefly, some comprehensive studies about the effects of melatonin on microglial cells, intraarterially during the reperfusion phase is essential. Indeed, investigations on the role of melatonin and MT2 receptor in microglia after ischemic stroke and alteration in reperfusion phase can improve the understanding of the pathophysiology of ischemic stroke and secondary injury. According to the above studies, we proposed to investigate if intra-arterial administration of melatonin after transient middle cerebral artery occlusion (tMCAO) has a more protective effect on stroke outcome? Further by using an in vitro approach, we were going to study the protective effects of melatonin on microglial cells following hypoxia condition. 2. Results 2.1.

In vivo study

2.1.1. Validation of tMCAO model and MRI scanning MRI scanning (T2-weighted image) was performed on tMCAO rats after 24h from surgery to set the best time of tMCAO. The brain edema volume was 177.1±16.34 (Figure.3B: a). Infarct areas in the MCA territory could be remarked also in H&E and Nissl staining (Figure.3B: b-c). 2.1.2. Effect of melatonin on TTC stain and infarction volume TTC staining of tMCAO brain sections showed reproducible and readily detectable lesions in the areas that were supplied by the MCA. The lesions were present in the striatum and the overlying cortex. Melatonin treatment reduced the infarct volume significantly (p<0.05) as compared to tMCAO+vehicle group (Figure 4.A-B). 6

2.1.3. Effect of melatonin on neurological output In order to further evaluate the therapeutic potential of melatonin in stroke, we next examined the efficacy of intra-arterial delivery of melatonin in an established in vivo rat stroke model. Intraarterial injection of melatonin after experimentally induced ischemic stroke in adult rats significantly reduced behavioral abnormalities compared to vehicle-injected rats. ANOVA revealed significant treatment effects in all 2 behavioral tests (Cylinder test, F=586.6, P<0.0001, figure 4.C, EBST, F=42.09, P<0.0001, figure 4.D), with post hoc Bonferroni’s multiple comparison test showing that melatonin ameliorated these motor and neurological impairments. 2.1.4. Effect of melatonin on neural genes in tMCAO brain tissue Evaluation of gene expression in the cortex on days 1 and 7 showed that the gene expression of BDNF in melatonin group was higher than vehicle group on day 1 and this difference was significant (P<0.001). Similarly, this trend could be observed on day 7. Moreover, melatonin upregulated MAP2 and HSPA1A genes on day 7 (P<0.001). The analysis of gene expression of GFAP demonstrated that this gene expressed in melatonin group more than vehicle group on days 1 and 7 (P<0.001). In contrast, VEGF had lower expression in melatonin compared to vehicle group on days 1 and 7 (P<0.001) (Fig.5a-b). When we evaluated the gene expression in the striatum on days 1 and 7, results showed that BDNF and MAP2 expressed higher in melatonin group on day 1 compared to vehicle group (P<0.001). HSPA1A gene in melatonin group decreased significantly on day 1 compared to vehicle group (P<0.001). GFAP had higher expression in melatonin group compared to vehicle group both on days 1 and 7 (P<0.001) (Fig.5c-d). 2.1.5. Effect of melatonin on brain damage after tMCAO

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The H&E and Nissl staining revealed prominent structural brain damage in the ischemic regions with increasing intensity in the vehicle group (Fig. 6 a-b). Representative magnified images of the cortex in ipsilateral and contralateral regions of the ischemic core showed interstitial edema and pyknotic nuclei that were absent in shams. H&E and Nissl staining of tMCAO rats treated with melatonin clearly demonstrated the lower structural damage in the ischemic brain regions (Fig. 6) 2.1.6. Effects of melatonin on expression of neural markers at protein level The extent of neuronal injury could be identified by an absence as well as an attenuation of NeuN immunoreactivity. After 1 day of tMCAO, lack of NeuN could be observed in the ipsilateral but not in the contralateral part. The major cell loss was found on day 7. After melatonin treatment, absence of NeuN in the infarct areas, decreased after 1 and 7 days of tMCAO, mainly after 7 days. In fact, melatonin highly increased NeuN in the ipsilateral part (Fig.7a). Evaluation of Iba-1 showed massive infiltration of microglia on day 1 and day 7, however, the Iba-1 expression was decreased in the tMCAO group with melatonin treatment highly on day 7 (Fig.7b). We could find attenuated GFAP immunoractivity in the ipsilateral part of tMCAO-vehicle group, mainly on day 7. Moreover, GFAP expression after melatonin treatment declined both on days 1 and 7 (Fig.8c). 2.1.7. Apoptosis after melatonin treatment The role of apoptosis in cerebral ischemia has been regarded as a basic pathogenesis. To evaluate whether the neuroprotective effect on apoptosis in the pathogenesis of cerebral ischemia is due to melatonin, the effect of melatonin on the apoptosis protein active caspase-3 was examined. As active caspase-3 is a marker of apoptosis pathway, it was measured by immunohistochemistry method to evaluate the activity of apoptosis. As shown in Figure 7, active caspase-3 was more 8

down regulated through treatment of melatonin, suggesting that melatonin retarded the activity of apoptosis (Fig. 7d). 2.2.

In vitro study

2.2.1. Expression of melatonin receptor 1B (Mtnr1b) in BV-2 cells Because the effect of melatonin in stroke treatment is recognized to be mediated through specific G-protein coupled receptor Mtnr1b mainly, we asked the question whether this receptor was expressed in BV-2 cells and how can its expression change during hypoxia. We tested this using qrtPCR. Mtnr1b appeared to be expressed in hypoxic BV-2 cells. Moreover, Mtnr1b gene during hypoxic stimulation expressed highly after 3 h (15.16±0.56) compared to 1 h hypoxia (0.63±0.007) (p<0.001) and 2 h hypoxia (5.38±0.028) (P<0.0001) (Figure 8.a). 2.2.2. Cytotoxic effects of melatonin on BV-2 cells We examined the vulnerability of BV-2 cells against 24 h exposure to melatonin with 1 µM, 5 µM, 10 µM, 30 µM, 100 µM and 300 µM concentrations to understand that these conditions can be toxic to the cells or not. The melatonin showed cytotoxic effects in a dose-dependent manner on BV-2 cells when compared with the control cells (Figure 8.b). The increasing concentrations of the melatonin induced cytotoxicity (P<0.05). The highest dose (300 μM) of melatonin administered to the BV-2 cells showed more significant reduction (30%–40%) in viability (P<0.05). The IC50 value of the melatonin was shown to be 14.77 μM (R2=0.542) (Figure 8.c). Deference between morphology and number of melatonin-treated BV-2 cells and vehicle-treated BV-2 cells during 3h hypoxia stimulation and the reperfusion phase (3h, 6h and 24h) could be observed in Figure 8d. Although, the loss of cell volume or cell shrinkage in vehicle-treated BV2 could be seen in all steps, melatonin-treated BV-2 cells showed apparently normal structure. This experimental setting was further chosen for all other experiments, since we aimed at 9

imitating a physiological situation after hypoxia where microglial cells survive and have the ability to initiate their inflammatory and/or protective function. This part was close to what we did at in vivo. 2.2.3. Effect of melatonin on inflammation-related genes in BV-2 cells In the present study to investigate whether melatonin regulates the hypoxia-induced proinflammatory cytokines, the expression of different inflammatory genes was analyzed by q-PCR. In fact, we examined basal expression levels of key pro-inflammatory genes typically associated with the M1 phenotype (IL1B, TNFA, iNOS and CCL-5) and the M2 phenotype (TREM2). Notably, the gene expression assessments revealed IL1B and TNFA were expressed in variable manners at various time points. By raising the reperfusion time, IL1B expression increased as well and a typical difference in IL1B expression between melatonin and vehicle groups was found 6 hours after reperfusion (P<0.05). Indeed, IL1B expression in melatonin group was less than that of the vehicle group (Figure 9.a). In contrast to IL1B, TNFA had a higher level after 3 hours of reperfusion, both in melatonin and vehicle treatments. Although the application of melatonin slightly inhibited the TNFA rise, it was not significant (Figure 9.b). Moreover, the expression analysis of CCL5 demonstrated considerable difference between groups treated with melatonin at 6 hours and 24 hours post reperfusion (P<0.01). In the vehicle group, the highest level of CCL5 expression was observed at 24 hours, post reperfusion with a significant difference in comparison to other assessed reperfusion times (3h and 6h, P<0.01) (Figure 9.e). TREM2 like TNFA had a downward trend from the 3rd hour to the 24th hour of reperfusion. TREM2 expression had significant differences between hours 3 and 24 of reperfusion after melatonin treatment (P<0.01). TREM2 expression in the melatonin group after hour 3 reperfusion had significant difference with hour 6 (P<0.001) and hour 24 (P<0.0001) (Figure 10

9.c). Finally, we evaluated the TREM2/iNOS ratio as a marker of the most protective form of Microglia (M2). The TREM2/iNOS ratio was at its peak at hour 3 after reperfusion compared to hour 6 (P<0.001) and hour 24 (P<0.0001). Melatonin increased this ratio to a much larger extent compared to the vehicle group (p<0.01) (Figure 9.f). 3. Discussion Melatonin is one of the strongest anti-ROS agents. Recent findings showed that effective modulations or interventions on microglial cells in reperfusion phase, particularly for preventing secondary injury could be incredibly impressive in stroke outcomes. In this study, at first we attempted to show that single dose of melatonin intra-arterially in the first of reperfusion phase could increase structural and behavioral outcomes in tMCAO rats. We evaluated the effect of melatonin in acute and subacute phases of ischemic stroke. Long term effects of melatonin on structural and behavioral outcomes are demonstrated by previous studies (Letechipia-Vallejo et al., 2007, Rennie et al., 2008); however, it is well established that the positive effect of melatonin in ischemic stroke is mainly related to its neuroprotective action in the acute stage of ischemic stroke compared to the chronic phase (Lin and Lee, 2009). Notably, we could insert the tip of intra-arterial catheter in the ICA after the PPA branch and increase the efficacy of melatonin delivery into the ischemic brain. It is desirable to temporarily withdraw the blood circulation of ICA into the PPA when therapeutics agents are delivered into the ICA (Chen et al., 2008, Guo et al., 2013). Injection of melatonin directly to the ICA hasn’t been performed in any survey yet, however, Kilic et al indicated that injection of melatonin through tail artery at the first of ischemic phase and also reperfusion phase reduced infarct volume and significantly improved neurologic deficit scores in pinealectomized rats subjected to tMCAO (Kilic et al., 1999). Similar to previous studies, we couldn’t observe apparent 11

correlations between the extent of the infarct area and behavior. Previous surveys indicated that some treatment strategies such as cell therapy in ischemic stroke could be less effective in improving structural outcome, but more effective in improving functional outcome (Lees et al., 2012). Likewise, we were able to observe the effect of melatonin on astrocyte by decreasing GFAP and on neurons by increasing NeuN at protein level especially on day 7. Furthermore, we indicated that melatonin decreased active caspase-3. Previous findings demonstrated that intraperitoneal injection of melatonin reduced disseminates neuronal injury after mild focal ischemia in mice via inhibition of caspase-3 (KilicU et al., 2004). Interestingly, it was noted that expression of Iba1 (as a marker of microglial cells) in tMCAO+vehicle rats increased highly in ipsilateral part on day 1 and day 7. Melatonin decreased Iba1 expression in tMCAO+melatonin rats, both on day 1 and 7. Similarly, Ito et al showed that Iba1+ microglia was apparent 3.5 to 12 hours after reperfusion in a transient ischemia model. Also, over 24 to 48 hours, Iba1+ cells appeared throughout the core of infarct area. Furthermore, heavily Iba1 immunoreactive cells rapidly appeared at 3.5 hours after reperfusion and immunoreactivity further increased and peaked on day 7 (Ito et al., 2001). Moreover, previous findings demonstrated that melatonin attenuated ischemic damage by reducing the infiltration of microglia (Chung SY, 2003, Liang et al., 2014) especially through MT2 receptor (Lee et al., 2010). For further investigations, we evaluated the gene expression of GFAP, VEGF, BDNF, MAP2 and HSPA1A which were involved in stroke recovery. Our findings showed that melatonin increased BDNF, MAP2 and HSPA1A. The role of BDNF and MAP2 in brain repair after ischemic stroke was reported by several studies (Imbesi et al., 2008, Watson et al., 2016, Lee et al., 2012). HSPA1A, a member of the 70 kDa class of molecular chaperones or heat shock 12

proteins (Hsps) regulates both apoptotic and necrotic cell death and when overexpressed is associated with reduced oxidative stress (Hoehn et al., 2001, Giffard et al., 2004). Previous studies showed that overexpressing of HSPA1A gene protects from cerebral ischemia (Hoehn et al., 2001, Giffard et al., 2008). We demonstrated that melatonin increased HSPA1A in the cortex after ischemic stroke. Evaluation of GFAP gene indicated that after melatonin treatment GFAP at mRNA level increased in contradiction to the protein level. It is well established that occasionally, protein expression does not show accordance with gene expression on a continuous fashion due to some post-transcriptional changes in the level of proteins (Schwanhäusser et al., 2011, Maier et al., 2009). Besides, the discordance between levels of GFAP mRNA and GFAP protein has been reported in previous surveys (Galbavy et al., 2015, Latrémolière et al., 2008). Although the reasons for GFAP gene elevation are not clear, the beneficial role of GFAP in restoration of brain injuries has been corroborated by some studies (Yang and Wang, 2015, Triolo et al., 2006). It assumed that post-injury-GFAP induction and associated reactive gliosis might in fact promote neuroregeneration (Yang and Wang, 2015). Moreover, GFAP might contribute to form macrocomplexes to initiate mitogenic and differentiating signals for efficient nerve regeneration (Triolo et al., 2006). Therefore, it seems that more findings on the role of GFAP during stroke recovery are required. We also evaluated the gene expression of VEGF. During ischemia, VEGF contributes to the disruption of the BBB. VEGF increases the permeability of blood vessels and leads to vasogenic edema (Pichiule et al., 1999) while inhibition of VEGF reduces BBB permeability (Zhang et al., 2000). Based on our result, melatonin decreased the gene expression of VEGF. Similarly,

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previous studies showed that the gene expression of VEGF was reduced by melatonin treatment (Song et al., 2014). In this study, we tried to find out that if melatonin could modulate microglial cells in vivo, how does it occur in an in vitro experiment and what are the main results by focusing on the role of microglial cells in the reperfusion phase? Therefore, after the in vivo study, we used an in vitro model (hypoxia-stimulated BV-2 cells) for in vitro investigations. According to our results, Mtnr1b (melatonin receptor 1B, MT2), which has important roles in stroke treatment was expressed by a mouse microglial cell line (BV-2). Furthermore, the gene expression of Mtnr1b increased significantly during hypoxia in a time-dependent manner. There isn’t any report about alteration of Mtnr1b gene expression in microglial cells after hypoxia; however, very few studies showed changes in expression of this gene in other circumstances. For instance, it was reported that chronic hypoxia in the rat carotid body led to upregulation of Mtnr1b (Tjong et al., 2006). For more findings, we evaluated the effect of melatonin on pro-inflammatory cytokines. The effects of melatonin on cytokines have been demonstrated by many studies (Tocharus et al., 2010, Lee et al., 2007); however, it is the first time that the effects of melatonin on inflammation-related genes in hypoxia-stimulated BV-2 cells have been reported. According to our research, after 3 hours of hypoxia, melatonin inhibited IL-1β expression during the reperfusion phase significantly, after 6 hours of reperfusion; however, no significant changes in TNFA were observed. A recent study suggested that melatonin inhibited IL-1β and TNFA expression in microglia effectively (Ding et al., 2014). Besides, according to Park et al, melatonin effectively suppressed the upregulation of IL-1β and also TNFA at mRNA level in manganese and/or lipopolysaccharide-stimulated BV2 cells. Based on Park et al, melatonin pretreatment significantly attenuated the expressions of iNOS and nitric oxide in BV2 microglial 14

cells (Park and Chun, 2017). Similarly, the upregulation of iNOS after 24 hours of reperfusion could be seen that were not rather significant. These different findings took place because we used hypoxic stimulation for activating BV-2 cells instead of manganese and lipopolysaccharide. Likewise, different melatonin concentration, different melatonin pre-treatment time and performing evaluations in ischemic or reperfusion phase could yield different results. In our study, we determined the ratio of TREM2/iNOS too. Interestingly, recent findings showed that the putative switch between M1/M2 phenotype of hypoxic BV-2 cells could be estimated by evaluation of the ratio between TREM2 and iNOS. Therefore, the high ratio of TREM2/iNOS is as a marker of the most protective form of microglia (M2). Based on our results, melatonin profoundly increased this ratio after 3 hours of reperfusion. 4. Conclusion To effectively summarize, scrutinizing the microglia modulation by melatonin in the reperfusion phase of ischemic stroke brings about broader insights on the mechanisms of secondary injury and brain repair and helps us to discover new strategies for ischemic stroke therapy. In this study, we indicated that the neuroprotective potential of melatonin intra-arterially was mediated by microglial action through melatonin receptor 1B. Indeed, melatonin modulates pro-inflammatory cytokines in microglial cells in the reperfusion phase and eventually elevates protective form of microglial cells. Nevertheless, more studies seem to be required for getting more information about mechanisms, monitoring changes during the reperfusion phase. 5. Experimental procedures 5.1.

In vivo

5.1.1. Animal preparation All experiments were conducted according to the institutional guidelines with the protocol 15

approved by the Committee for the Use of Live Animals in Teaching and Research, the Iran University of Medical Sciences and health ministry. Adult male Wistar rats, weighting between 270 and 315 g, were purchased from the Animal Laboratory Unit, Iran University of Medical Sciences. The rats were maintained under a stable lighting condition (12 hours of light beginning at 6 AM) provided with unlimited source of food and water for a minimum of 4 days before experimentation. 5.1.2. Experimental design To investigate the effects of melatonin in an experimental model of focal cerebral ischemia, we used the rat tMCAO model as described by Longa et al (Longa et al., 1989). The following experimental groups were designed: Sham group which received all the surgical procedures except tMCAO and melatonin injection; tMCAO vehicle group which received intra-arterially saline with 5% ethanol solution; tMCAO treatment group which received intra-arterially melatonin, i.e., ischemia was induced for 35 min followed by reperfusion. Melatonin was purchased from Sigma (Sigma, USA) and dissolved in saline with 5% ethanol (Merck, Germany) and given in a dose of 4 mg/kg intra-arterially after the onset of reperfusion. The optimal dose of melatonin treatment regimen used in this experiment was supported from previous studies showing that this amount provided the maximal protective effects in the treatment of different types of disease and metal toxicity. After the completion of the reperfusion period, the animals were assessed for neurobehavioral activity and then sacrificed at day 1 and day 7 after tMCAO. The brains were taken out for biochemical estimations. A schematic illustration of the treatment set-up is given in Fig. 1. 5.1.3. Transient middle cerebral artery occlusion (tMCAO)

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Rats were anesthetized with ketamine (80 mg/kg, Rotexmedica, Germany) /xylazine (7 mg/kg, Interchemie, Holland) intraperitoneally. Body temperature was maintained at 37±0.5°C using a heating pad. This parameter was monitored continuously during all procedures. Animal surgery was performed as previously described (Niizuma et al., 2009). Briefly, a 4-0 microfilament (Doccol, USA) with round tip and silicon coating was introduced from the right external carotid artery (ECA) into the internal carotid artery (ICA) and reached the circle of Willis to occlude the origin of the middle cerebral artery (MCA). Reperfusion was performed by withdrawing the microfilament after 35 min tMCAO. 5.1.4. Magnetic resonance imaging (MRI) MRI scanning was performed in the national brain mapping laboratory, Iran, with a 3.0 Tesla prisma MRI scanner (Siemens, Germany). MRI scanner with1H rat coil and holder was done in rats with tMCAO after 24 h. T2-weighted MR images were acquired with the TR=2300 ms and TE=106 ms. Brain edema volume was assessed from T2-weighted images by summing up the edema area measured from all slices using ImageJ 1.52n. 5.1.5. Insertion of intra-arterial catheter in ICA for melatonin administration After 35min ischemia and removing the microfilament, an arterial catheter (16.1×45mm) was inserted into the ICA through the ECA. The catheter was finally inserted in the ICA after the pterygopalatine artery (PPA) branch for increasing the efficacy of melatonin injection to the ischemic brain. Afterwards that, a single dose of melatonin/vehicle was injected slowly during 2 min via the arterial catheter. 5.1.6. Neurological score and motor assessment scale

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Behavioral assessment was performed by using semi-quantitative analyses of motor asymmetry (elevated body swing test, EBST) and motor coordination (cylinder test) at days 1 and 7 after melatonin treatment. The EBST was used to test the asymmetrical motor behavior. Rats were maintained by the root of the tail and raised 10 cm above the testing surface. The initial direction of swing, determined as the turning of the upper body by >10° to either side, was recorded in 20 trials in each rat, performed over 5 min. The number of turns in each (left or right) direction was recorded for each rat. The cylinder test was used to measure the degree of forepaw asymmetry. Rats were put in a transparent cylinder (diameter: 20 cm, height: 30 cm) for 3 min with the number of forepaw contacts to the cylinder wall counted. The score of the cylinder test in this study was calculated as a contralateral bias: [(the number of contacts with the contralateral limb) – (the number of contacts with the ipsilateral limb) / (the number of total contacts) × 100]. 5.1.7. Triphenyltetrazolium chloride (TTC) staining and quantification of infarct volume The rats were anesthetized 1 or 7 days after the MCA occlusion with Ketamine/Xylazine and decapitated. Their brains were removed and coronally sectioned at 2mm interval by a brain matrix. The brain slices were incubated for 20 min in a 2% solution of TTC (Sigma, USA) at 37°C, fixed with 4% paraformaldehyde (PFA) (Merck, Germany) and photographed using a Nikon Eclipse 55i (Nikon, Germany). The infarct volume was quantified using ImageJ 1.52n by summing the infarct areas in all sections and multiplying by the slice thickness (2 mm). 5.1.8. RNA isolation and semi-quantitative real-Time PCR The total RNA was extracted from brain tissue, using Trizol (Biocompare, USA). The gene expression studies were performed with tissues corresponding to the epicenter of injury which was determined by TTC staining (Kramer et al., 2010). RNA concentration was measured using a NanoDrop 1000 device (PeqLab, Germany). Complementary DNA of 1 μg of total rat brain 18

RNA was synthesized using the Takara (Japan). Semi quantitative-real time PCR (qrtPCR) analysis was performed utilizing the real-time rotary analyzer (Corbett, Australia). A list of used primers and analyzed genes are presented in Table 1. Mean efficiencies and crossing point values for each gene was determined using LinRegPCR v. 11.0 (Ruijter et al., 2009) and normalized to values for a reference gene (ß-actin) in vehicle and melatonin groups with reference to sham group, using REST-2009 software (available at: http://www.gene-quantification.de/rest2009.html). Statistical analysis of relative gene expression results in real-Time PCR was performed using REST© freeware according to formula presented by Pfaffl et al (Pfaffl et al., 2002). 5.1.9. Histopathological evaluations and immunohistochemistry staining The rats were anesthetized 1 or 7 days after the MCA occlusion with Ketamine/Xylazine completely. After that, animals were exposed to cardiac perfusion with 4% PFA in phosphate buffered saline (PBS). After perfusion, brains were quickly removed and post fixed overnight in the same fixative and embedded in paraffin (Merck, Germany). Tissue blocks were sectioned on a microtome (Ultracut Reichert-Jung, Leica Microsystems, Germany) into 5 μm thick sections for hematoxylin-eosin (H&E) (Merck, Germany) and Nissl staining (cresyl violet, Merck, Germany) for histopatological evaluations. Moreover, immunohistochemistry staining for NeuN (Cat# MAB-377, Millipore, USA), Iba1 (Cat# 019-19741, Wako, Germany), glial fibrillary acidic protein (GFAP) (Cat# B-285, LS Bio, USA) and caspase-3 (active) (Cat# 3015-100, Bio vision, USA) was done. A list of used antibodies, manufactures and dilutions are given in Table 2. After heat-induced antigen retrieval (HIER), sections were incubated with 10% goat serum (Sigma, Germany) for 30 min. Then, slices were incubated overnight at 4°C with the respective primary antibodies. For blocking endogenous peroxidase, sections were incubated with 19

H2O2/PBS (0.3%) (Roth, Germany). Subsequently, the sections were incubated with the appropriate secondary antibodies followed by the ABC complex (Vector Labs, USA). Diaminobenzidine (DAB) (Dako, Denmark) was used as chromogen substrate. Finally, sections were counterstained with hematoxylin, dehydrated in graded alcohols, mounted and digitally recorded using a Nikon Eclipse 55i (Nikon, Germany). 5.2.

In Vitro

5.2.1. BV-2 cell line The mouse microglial cells, recombinant retrovirus (v-raf/v-mic) transformed; the BV-2 cell line was purchased from Banca biologica e cell factory (Banca biologica e cell factory, Italy). Cells were kept in a humidified environment at 37 ◦ C and 5% CO2 in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Germany) which supplemented with 10% hormone-free charcoalstripped FCS (CSFCS, Gibco, USA) and 0.5% penicillin–streptomycin (PS) (Invitrogen, Germany). The medium was changed every second day. Cells were sub-cultured at a level of approx. 80% confluence. 5.2.2. Cytotoxicity of melatonin To determine the cytotoxicity of melatonin on BV2 cells, the cell viability was performed using CellTiter-Blue® assay (Promega, Germany). The following concentrations of melatonin were used: 1 µM, 5 µM, 10 µM, 30 µM, 100 µM and 300 µM. The assay was performed used according to the manufacturer’s protocol. Data were acquired using a microplate reader Infinite M1000 PRO (Tecan, USA) and the fluorescence at 560/590 nm were recorded. The cell viability was estimated as a percent, ratio and compared with the control cells. Positive control was determined by using cell lysis solution.

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5.2.3. Hypoxic stimulation To generate hypoxic conditions, a self-constructed cube-shaped hypoxia chamber (28×14×26 cm) was flooded with inert nitrogen gas to replace the aerial oxygen 30 min prior to hypoxia incubation (Habib et al., 2014a, Habib et al., 2014b). Oxygen levels were constantly monitored using an oxygen-sensitive detector (Gox 100T, Greisinger Electronic GmbH, Germany) in the gas output tube. Exhausting air contained constant levels of oxygen between 0.1 to 0.3%. In order to maintain temperature at 37◦C, the chamber was surrounded and heated with water at 37◦C within a thin compartment between the walls of the chamber. 5.2.4. Evaluation of gene expression of melatonin receptor 1B (Mtnr1b) in BV-2 cells under hypoxic condition After detachment the BV2 cells by trypsin treatment (2.5% in PBS/EDTA) (Thermo Fisher Scientific, USA), they were seeded in 12-well cell culture dishes coated with Poly-D-lysine (Sigma, USA) for 24 h in DMEM supplemented with 0.5% PS. On the following day, the culture medium was changed to the starving medium; RPMI 1640 (Gibco, USA) with decreased glucose containing 0.5% CSFCS and 0.5% PS. The number of 400.000 cells was used for seeding. 24 h after cell seeding, BV-2 cells were exposed to hypoxia conditions lasting for 1, 2 and 3 h and subsequently to be exposed to oxygen reperfusion. After 24 h, RNA extraction was done. 5.2.5. Melatonin treatment BV-2 cells were seeded in 12-well cell culture dishes as described above. 24 h after using starving medium, we started the treatment with melatonin. Melatonin was diluted in pro analysis ethanol to give a final concentration of 30 µm in treatment medium (DMEM supplemented with 10% CSFCS and 0.5% PS). Then, after 24 h, BV-2 cells pretreated with melatonin were exposed

21

to hypoxia conditions lasting for 3 h and subsequently to be exposed to oxygen reperfusion. After 3, 6 and 24 h, RNA extraction was done. A schematic illustration of the protocol is presented in Fig. 2. 5.2.6. RNA isolation and semi-quantitative real-Time PCR The total RNA was extracted from BV-2 cells, using peqGold RNA TriFast (PeqLab, Germany) as previously described (Habib et al., 2013). RNA concentration was measured using a NanoDrop 1000 device (PeqLab, Germany). Complementary DNA of 1 μg of total BV-2 cells was synthesized using the M-MLV reverse transcription (RT) -kit and random hexanucleotide primers (Invitrogen, Germany). Semi qrtPCR analysis was performed using the MyIQ detection system (Biorad, Germany). Relative quantification was calculated by the ΔΔCt-method. Data were expressed as relative amount of the target gene to the amount of a reference gene (HPRT). The values of the control group were set to one. Data of interest are given as relative expression. A list of used primers and analyzed genes are presented in Table 1. 5.3.

Statistical analysis

Data are shown as arithmetic means±SEM calculated from three to six independent experiments depending on the read-out parameter and treatment. Relevant information is given in each figure legend. Statistical analysis of results was performed by a factorial analysis of variance (ANOVA). Generally, the ANOVA included assessment of treatment effect (i.e. the occurrence of differences in mean values between melatonin- and vehicle-fed rats), time- effects (The occurrence of daily changes) and of the interaction between treatment and time, from which inference about differences in timing between the experimental groups could be obtained. Posthoc Bonferroni’s multiple comparisons tests were applied to show which time points were significantly different within each experimental group to define the existence of peaks. P-values 22

lower than 0.05 were considered as evidence of statistical significance. All tests were performed by using GRAPH PAD PRISM 5.0 program (GraphPad Software, USA). Statistical analysis of relative gene expression results of brain tissue in real-Time PCR was performed using REST© freeware according to a formula presented by Pfaffl et al (Pfaffl et al., 2002). Declaration The authors have no conflict of interest to declare. Acknowledgements The authors thank Uta Zahn, Petra Ibold and Helga Helten for technical support in the Institute of Neuroanatomy, RWTH Aachen University. The authors as well thank the staffs in the National Brain Mapping Lab, Tehran, Iran for having allowed us to use their MRI scanner. Funding resources This work was supported by the Iran University of Medical Sciences [grant number 93-04-8725297] and the Institute of Neuroanatomy, RWTH Aachen University. References AJAMI, B., BENNETT, J., KRIEGER, C., TETZLAFF, W. & ROSSI, F. (2007) Local selfrenewal can sustain CNS microglia maintenance and function throughout adult life. Nature Neuroscience, 10, 1538–1543. BERKHEMER, O., FRANSEN, P., BEUMER, D., VAN DEN BERG, L., LINGSMA, H., YOO, A., SCHONEWILLE, W., VOS, J., NEDERKOORN, P., WERMER, M., VAN WALDERVEEN, M., STAALS, J., HOFMEIJER, J., VAN OOSTAYEN, J., LYCKLAMA À NIJEHOLT, G., BOITEN, J., BROUWER, P., EMMER, B., DE BRUIJN, S., VAN DIJK, L., KAPPELLE, L., LO, R., VAN DIJK, E., DE VRIES, J., DE KORT, P., VAN ROOIJ, W., VAN DEN BERG, J., VAN HASSELT, B., AERDEN, L., DALLINGA, R., VISSER, M., BOT, J., VROOMEN, P., ESHGHI, O., SCHREUDER, T., HEIJBOER, R., KEIZER, K., TIELBEEK, A., DEN HERTOG, H., GERRITS, D., VAN DEN BERG-VOS, R., KARAS, G., STEYERBERG, E., FLACH, H., MARQUERING, H., SPRENGERS, M., JENNISKENS, S., BEENEN, L., VAN DEN BERG, R., KOUDSTAAL, P., VAN ZWAM, W., ROOS, Y., VAN DER LUGT, A., VAN 23

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Figure Legends

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Figure 1. The schematic illustration shows the protocol of tMCAO surgery and insertion of intra-arterial catheter in rats. Figure 2. The schematic illustration shows the treatment protocol of in vitro hypoxia experiments. Figure 3. The surgical procedures for making tMCAO model in rats and validation of animal modeling by MRI scanning and histological staining. (A): Surgical procedures: finding the external carotid artery (ECA) (a), cutting ECA and inserting a microfilament into the common carotid artery (CCA) (b), turning it to internal carotid artery (ICA) and (d) Pushing microfilament to ICA about 19-20 mm until reach to middle cerebral artery (MCA) (c). After 35 min of MCA occlusion, removing the microfilament and inserting a catheter in ICA for intraarterial injection of melatonin or vehicle (e). tMCAO Wistar rat 2h after surgery with spontaneous and continuous ipsilateral circling behavior (f). (B): Representative T2-weighted MRI images in rats with tMCAO after 24 h. Quantification revealed brain edema volume. Data are mean±SEM (a). Hematoxylin and eosin staining (H&E) of brain rat after tMCAO surgery for determining infarct area. Scale bar 100 µm) (b). Nissl staining of brain rat after tMCAO surgery for determining infarct area. Scale bar 50 µm (c). Figure 4. Intra-arterial injection of

sham, vehicle or melatonin group after 1 or 7 days following transient middle cerebral artery occlusion (tMCAO). (B) Infarct volume in tMCAO rats treated with melatonin or vehicle. Data are mean±SEM, the number of animals in each group (n) = 6. Functional recovery

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was evaluated using cylinder test (C), and elevated body swing test (EBST) (D) after 0, 1 and 7 days following tMCAO. Figure 5. Quantitative RT-PCR data of neural genes in the cortex and striatum after melatonin treatment in tMCAO rats on days 1 and 7. Gene expression in the cortex and striatum on day 1 (a-b) and day 7 (c-d). Data of gene expression was normalized to values for a reference gene (ßactin) in vehicle and melatonin groups with reference to sham group. *** p<0.001. GFAP glial fibrillary acidic protein, VEGFA vascular endothelial growth factor A, BDNF brain-derived neurotrophic factor, MAP2 microtubule-associated protein 2, HSPA1A heat shock protein family A member 1A, ß-actin beta-actin. Figure 6. Representative hematoxylin and eosin (H&E), and Nissl stained paraffin-embedded tissue sections from contralateral and the ipsilateral rat brains subjected to transient middle cerebral artery occlusion (tMCAO) followed by their sacrifice at various time points after tMCAO at day 1 and day 7 in sham, tMCAO+vehicle and tMCAO+Melatonin groups. Scale bar 100 µm for H&E, Scale bar 300 µm for Nissl staining. Figure 7. Effects of melatonin on immunohistochemical (NeuN, Iba1, GFAP and active caspase3) characterizations of infarcts induced by a transient MCAO at day 1 and 7 in sham, tMCAO+vehicle and tMCAO+melatonin groups. NeuN Scale bar 100 µm (a). Iba1 Scale bar 50 µm (b). GFAP Scale bar 50 µm (c). Active caspase-3 Scale bar 50 µm (d). Figure 8. Expression of Mtnr1b in BV-2 cells before and after hypoxia stimulation (1h, 2h and 3h). The values of the control group were set to one: ** p<0.01, **** p<0.0001, Ψ p<0.001,  p<0.0001, Mtnr1b melatonin receptor 1B (a). BV-2 cell viability after exposure to different melatonin concentrations (1, 5, 10, 30, 100 and 300 µM) (b). IC50 of BV-2 cells after using 30

melatonin with different concentration (IC50=14.77µM R2=0.542) (c). Morphological changes of BV-2 cells under hypoxic condition after exposure to melatonin (30 µM) or vehicle. Scale bar 100 µm (d). Figure 9. Quantitative RT-PCR results of inflammation-Related Genes in hypoxic BV-2 cells after reperfusion (3h, 6h and 24h). Data are mean±SEM. Data on gene expression were normalized to corresponding HPRT. The values of the control group were set to one (there isn’t in the figure). a. Expression of IL1B: ≠ p<0.05, b. Expression of TNFA, c. Expression of TREM2: ** p<0.01, d. Expression of iNOS, e. Expression of CCL5: ** p<0.01, f. Fold change ratio of TREM2/iNOS mRNA of normoxia: * p<0.05, *** p<0.001, **** p<0.0001, Ψ p<0.01. IL1B interleukin 1 beta, TNFA tumor necrosis factor, TREM2 triggering receptor expressed on myeloid cells 2, iNOS nitric oxide synthase 2, CCL5 C-C motif chemokine ligand 5, HPRT hypoxanthine phosphoribosyltransferase 1. Table 1. Sequences of the primers used for analysis of BV-2 cell and brain tissue. BDNF brainderived neurotrophic factor, CCL5 C-C motif chemokine ligand 5, GFAP glial fibrillary acidic protein, HPRT hypoxanthine phosphoribosyltransferase 1, HSPA1A heat shock protein family A member 1A, IL1B interleukin 1 beta, iNOS nitric oxide synthase 2, MAP2 microtubuleassociated protein 2, Mtnr1b melatonin receptor 1B, ß-actin beta-actin, TNFA tumor necrosis factor, TREM2 triggering receptor expressed on myeloid cells 2, VEGFA vascular endothelial growth factor A. Table 2. List of antibodies, manufactures and dilutions used in this study

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