The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vessels occlusion in rats

The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vessels occlusion in rats

    The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vesse...

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    The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vessels occlusion in rats Serdar Oruc, Y¨ucel G¨on¨ul, Kamil Tunay, Oya Akpinar Oruc, Mehmet Fatih Bozkurt, Erg¨un Karavelio˘glu, Erman Ba˘gcıo˘glu, Kerem Senol Cos¸kun, Sefa Celik PII: DOI: Reference:

S0024-3205(16)30248-X doi: 10.1016/j.lfs.2016.04.028 LFS 14869

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

1 March 2016 18 April 2016 21 April 2016

Please cite this article as: Oruc Serdar, G¨on¨ ul Y¨ ucel, Tunay Kamil, Oruc Oya Akpinar, Bozkurt Mehmet Fatih, Karavelio˘glu Erg¨ un, Ba˘ gcıo˘glu Erman, Co¸skun Kerem Senol, Celik Sefa, The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral ischemia reperfusion injury induced by four vessels occlusion in rats, Life Sciences (2016), doi: 10.1016/j.lfs.2016.04.028

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ACCEPTED MANUSCRIPT The antioxidant and antiapoptotic effects of crocin pretreatment on global cerebral

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ischemia reperfusion injury induced by four vessels occlusion in rats

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Serdar Oruc1, Yücel Gönül2, Kamil Tunay3, Oya Akpinar Oruc3, Mehmet Fatih Bozkurt4,

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Ergün Karavelioğlu5, Erman Bağcıoğlu6, Kerem Senol Coşkun6, Sefa Celik7

Afyon Kocatepe University, School of Medicine, Department of Neurology, Afyonkarahisar

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Afyon Kocatepe University, School of Medicine, Department of Anatomy, Afyonkarahisar

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Afyon Kocatepe University, School of Medicine, Department of Emergency Medicine, Afyonkarahisar

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Afyon Kocatepe University, School of Veterinary Medicine, Department of Pathology, Afyonkarahisar

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Afyon Kocatepe University, School of Medicine, Department of Neurosurgery, Afyonkarahisar

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Afyon Kocatepe University, School of Medicine, Department of Psychiatry, Afyonkarahisar

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Afyon Kocatepe University, School of Medicine, Department of Biochemistry,Afyonkarahisar

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Corresponding Author Assist Prof Dr Serdar ORUC

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Afyon Kocatepe University School of Medicine Department of Neurology,Faculty Member Mail; [email protected] Gsm; +90 505 453 06 11 Afyon Kocatepe University School of Medicine. Ali Cetinkaya Campus

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- 03200-Afyonkarahisar/TURKEY

Phone:+9027224633 01 Fax:+9027224633 00

Afyon Kocatepe University Scientific Research Projects Unit supported the present study (project no: 13.TIP.01).

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ACCEPTED MANUSCRIPT ABSTRACT Aims: Cerebral ischemia reperfusion (IR) injury is a process in which oxidative and apoptotic mechanisms play a part. Neuroprotective agents to be found could work out well for the efficient and safe minimization of cerebral IR injury. Crocin is a strong antioxidant agent; however the influence of this agent on the experimental cerebral

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ischemia model has not been studied extensively and thus it is not well-known. The objective of our study was to

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investigate the antioxidant, antiapoptotic and protective effects of crocin on the global cerebral IR induced by four-vessel occlusion.

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Main methods: A total of 30 adult female Sprague-Dawley rats were equally and randomly separated into three groups as follows: Sham, IR and IR+Crocin (40 mg/kg/day orally for 10 days). 24 hours after electrocauterization of bilateral vertebral arteries, bilateral common carotid arteries were occluded for 30 mins and reperfused for 30 mins. Oxidative stress parameters (TAS, TOS, OSI), Haematoxylin&Eosin staining,

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caspase-3 and hypoxia-inducible factor-1 alpha (HIF-1α) expressions and TUNEL methods were investigated. Key findings: There was a significant difference between the IR and Sham groups by means of OSI level, histopathological scoring, caspase-3, HIF-1α and TUNEL-positive cell parameters. We have also observed that

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pre-treatment with crocin reduced these parameter levels back to the baseline. Significance: The data obtained from the present study suggest that crocin may exert antiapoptotic, antioxidant and protective effects in IR-mediated brain injury induced by four-vessel occlusion. To the best of our

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knowledge, this would be the first study to be conducted in this field.

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Keywords: Crocin, Cerebral ischemia-reperfusion injury, Hippocampus

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ACCEPTED MANUSCRIPT Introduction Cardiac arrest, asphyxia, shock, serious hypertension, brain injuries, surgeries related to the heart and thorax might be the cause for temporary global cerebral ischemia. (Madl and Holzer, 2004). The brain has limited ischemia tolerance in addition to being one of the most vulnerable organs to ischemia due to its restricted

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anaerobic metabolism and glycogen stores. Particularly, the hippocampal CA1 region, corpus striatum,

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pyramidal neurons of motor cortex, purkinje cells of cerebellum are affected more significantly by temporary and short-term ischemia compared to the other regions of the brain (Kirino et al., 1985). While the main goal of

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the post-ischemic period is to provide reperfusion, this condition does not always yield positive results. Reperfusion in tissues with ischemic injury leads to further damage particularly due to the effect of free oxygen radicals (Kukreja and Hess, 1992).

Apoptosis is one of the important mechanisms of delayed neural cell death in cerebral Ischemia-Reperfusion (IR)

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injuries (Brouns and De Deyn, 2009, Guan et al., 2009). Caspases play a central role in the development of apoptosis and they are members of the cystein proteinase family (Jin et al., 2003, Springer et al., 2001). The

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activation of Caspase 3, as one of these, results in degradation of many intracellular and cytoskelatal protein substrates. As a result, cellular damage and apoptotic cell death takes place (Mancini et al., 1998). There is a relationship between Caspase 3 as a mediator of loss of neurons and HIF-1α which binds functionally to the Caspase 3 gene promoter (Van Hoecke et al., 2007). It has been shown that with HIF stabilization, O2 sensitive

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organs such as the brain (Trollmann et al., 2014) and heart (Ong et al., 2014) can be protected via the reduction

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in inflammation and apoptosis. However, the neuroprotective mechanism of HIF-1 has not exactly been clarified (Ryou et al., 2015).

Cerebral IR injury is a process in which oxidative and apoptotic mechanisms play a part. Probable

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neuroprotective agents to be found could work out very well in the efficient and safe minimization of cerebral IR injuries. Nowadays, many anti-oxidant agents are being investigated to protect the brain against cerebral IR injury that occurs frequently in the clinics (Yaman et al., 2007). However, they have not yet earned significance to be used commonly in health care services since their mechanisms of action and possible clinical consequences not

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have

precisely.

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antioxidant

agents,

crocin,

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crocuses and gardenia flowers, is a highly water-soluble natural carotenoid (Alavizadeh and Hosseinzadeh, 2014). Crocin is one of the most important constituents in saffron (Crocus sativus L) (Gholamnezhad et al., 2013). The efficacy of crocin has been hitherto investigated in the various IR models in different tissues such as heart, skeletal muscle, renal, retinal and brain tissues (Dianat et al., 2014, Hosseinzadeh et al., 2009, Hosseinzadeh et al., 2005, Laabich et al., 2006, Sarshoori et al., 2014). Crocin has been shown to be an important antioxidant agent in these studies in addition to its anti-inflammatory, anti-apoptotic, antiatherosclerotic, anti-hyperlipidemic properties (Deslauriers et al., 2011, He et al., 2005, Lee et al., 2005, Mehri et al., 2012). However, the effect of crocin against cerebral IR has not been studied extensively and is not wellknown. To our knowledge, while the effect of crocin has been investigated only in global cerebral IR models induced by two-vessel occlusion so far (Zheng et al., 2007), it has not been studied on this IR model with fourvessel occlusion. Therefore, the objective of this study was to investigate the possible antioxidant and anti-apoptotic effects of crocin in global cerebral IR injury via biochemical (oxidative stress parameters), histopathological (Haematoxylin &Eosin staining), immunohistochemical (caspase-3 and HIF-1α staining) and TUNELmethods.

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ACCEPTED MANUSCRIPT Materials and Methods Afyon Kocatepe University Scientific Research Projects Unit supported the present study (project no:13.TIP.01). Animals The study protocol and experiemtal design was approved by the Ethical Committee for Animal Studies at Afyon

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Kocatepe University. Female Sprague-Dawley rats were used for this study. The mean weight values of rats

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were 300±20 g, the mean time for post-natal period was 8-12 weeks. Animals were kept at 24-26oC, 50-60% humidity and a 12 hr light/dark cycle prior to the experiments. Rats were fed with a standard diet and regular

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water. Only water was given to the rats 12 hr prior to experiments. The whole protocol of the study was performed in accordance with the “Guidelines on the Care and Use of Animals for Scientific Purposes” developed by the National Health and Medical Research Council and “Institutional Administration Manual for Laboratory Animal Care and Use” prepared by the National Institute of Health (NİH issue no. 85–23, 1985

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revised). Experimental design

Rats were randomly divided into three groups (each group=10). Group 1 (Sham group) was fed with a standard

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diet for 10 days. Group 2 (ischemia, not treated by crocin –IR Group-) was also fed with a standard diet for 10 days. Group 3 (ischemia, treated by crocin –IR+Crocin Group-) was given a standard diet in addition to crocin administration (40 mg/kg/day) via a gastric tube for 10 days (Dianat et al., 2014)(Sigma Aldrich Chemical Co. St

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Lois, USA). The last dose was administered 12 hrs prior to the IR process. Global cerebral ischemia-reperfusion model

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Animals were starved for twelve hours prior to the surgical procedure.All animals were premedicated with intramuscular (İM) injections of 5 mg/kg Xylazine (Rompun, Bayer, Turkey). Rats were anesthetized with

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intramuscular injection of ketamine at 40 mg/kg dose (Ketalar, Parke-Davis, Eczacibasi, Turkey) followed by fixing their limbs to the operation tables, so that the sterilization of the environment would not be negatively affected. In accordance with the surgical procedure performed by Yaman et al. (Yaman et al., 2007), carotid arteries of the rats in Group 1 were explored by performing merely neck incisions which were left open for 30

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minutes. Carotid arteries were not occluded. During this period, no surgical procedure was applied and no drugs were administered. In groups 2 and 3, bilateral femoral veins and arteries were opened by a cannula (no:24) (Datex/Ohmeda S/5, Helsinki-Finland) through inguinal incision. Blood pressure and pulsation of the femoral artery were monitorized. To achieve the IR process in groups mentioned above; on first day, both vertebral arteries were electrocauterized at the level of the first cervical vertebra within the alar foramina. After the closure of incision, the rats were wakened. On second day (24 h later), the rats were again anesthetized. CCA were bilaterally occluded with aneurysm clips (Sugita, temporary aneurysm clip, Mizuho, Japan) for 30 min followed by 30 min of reperfusion. Mean pulse pressure was kept at 385/min, while the body temperature was maintained at 36.5-37.5ºC during ischemia. In addition, blood pressure was also maintained at normal levels throughout the ischemia process. The rats were sacrificed at the end of the procedure. Right after sacrification, the brains of the rats were removed immediately. The right hemibrains were reserved for pathological examination, while the left hemibrains were kept at -70° until the biochemistry assays were performed. Measurement of Total Oxidant Status (TOS)

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ACCEPTED MANUSCRIPT Serum levels of TOS were determined via automatic colorimetric measurement (Erel, 2005). In this system, oxidating agents present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by abundant glycerol molecules in the reaction. The ferricion forms a colored complex with xylenolorange in an acidic medium. The color intensity measured spectrophotometrically is proportional to the

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total amount of oxidant molecules in the sample. The assay was calibrated by hydrogen peroxide and the serum

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results were presented as micromolar hydrogen peroxide equivalent per litre (µmol H2O2 Equiv./L) and tissue results as micromolar hydrogen peroxide equivalent per gram proton (μmol H2O2 Eq/g prot).

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Measurement of Tissue Total Antioxidant Capacity (TAS)

The serum levels of TAS were determined by a novel automatic colorimetric measurement (Gonul et al., 2016). This method relies on the conversion of more stable ABTS (2,2’-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]) radical cation to its colorless neutral form as a result of reaction with antioxidants. This measurement

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yields perfectly reliable values less than 3% percent. The tissue TAS results were presented as mmol Trolox equivalent/g of protein and serum TAS results as mmolTroloxEquiv/L. Determination of Tissue Oxidative Stress Index (OSI)

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The ratio of TOS to TAS gives the degree of the oxidative stress index (OSI). The value of tissue OSI in the serum was calculated according to the following formulae: OSItissue(arbitrary unit)=TOS (µmol H2O2Eq/g prot)/TAS (mmol Trolox Eq/g prot)×100, OSI serum = [(TOS, μmol/L)/ (TAS, (mmolTroloxEquiv/L) x 100]

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(Gönül et al., 2016) Histopathologic assessment

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The brains were quickly placed in a neutral PBS-buffered formaldehyde solution after the experiments. They were then incubated for 48 hours and routine tissue monitoring was performed. This was followed by Paraffin

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embedding and sectioning into 4-5 micron thick slices on the adhesive slides with a microtome. Paraffin embedded sections were then processed histochemically for Haematoxylin&Eosin (H&E) staining. The histopathological scoring (HPS) of the hippocampus (cornu ammonis regions) was carried out by a pathologist who is unaware of the groups as follows: grade 0, no damage to any hippocampal subregion; grade 1, scattered

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ischemic neurons in the CA1 subregion; grade 2, moderate ischemic damage; grade 3, whole pyramidal cell damage in the CA1 subregion; and grade 4, extensive cell damage in all hippocampal regions (Lei et al., 2014). Immunohistochemical assessment The brains were stained by Caspase 3, HIF-1α and their markers immunohistochemically. For this purpose, the sections were deparaffinized and rehydrated. They were incubated with 3% Hydrogen Peroxide solution for 10 minutes in order to deactivate the endogenous peroxidase activity. Antigen detection was carried out by adding a citrate solution at pH 6.0 for 20 minutes in a pressure cooker adapted to the microwave. This was followed by serum blockage for 15 minutes and the addition of primary antibodiesof rabbit polyclonal for Caspase 3 (Thermo-Pierce PA516335, 1/50 dilution), as well as Hıf-1α (Abcam ab2185, 1/50 dilüsyon) and were incubated at room temperature for 2 hours. Afterwards, biotinylated Anti-Rabbit IgG (Vector, BA-1100, 1/200 dilution) was added and incubated at room temperature for 1 hour. After this step, AvidinBiotin peroxidase kit (Vector Inc, Vectastain Elite ABC Kit, PK-6100) was used and incubated at room temperature for half an hour. Peroxidase substrate, 3-amino-9-ethylcarbasol (AEC, Vector Inc, ImmPACT AMEC Red,SK-4285) was added and stained with Gill's (I) haematoxylin in order to visualize the boundones. The slides were then analyzed under

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ACCEPTED MANUSCRIPT a light microscope (Zeiss Axiolab.A1) by a pathologist blinded to the identity of the groups following the use of an aqueous adhesive which were photographed using a camera (Zeiss Axiocam ICc5). Immunohistochemically, CA1 regions of cornu ammonis of the brains stained with Caspase 3, HIF-1α were analyzed carefully. Staining of the neurons in CA1 region were graded as follows; no staining (0) if it is less than

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5%, slightly intense (1) if it is between %5 to %25, moderately intense (2) if it is between %25 to %50 and

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intense (3) if it is more than %50 (Mohamed et al., 2013). TUNEL method

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The method was carried out according to Manufacturer’s recommendations using In Situ Cell Death Detection Kit, POD (Roche, Cat. No. 1 684 817). Paraffin blocks of tissue slices on the APES coated adhesive slides were stained by TUNEL method. The sections removed from the cornu ammonis of the brains were deparaffinized in xylol and rehydrated in the series of 100%, 96%, 80%, 70% ethanol. Proteinase K (10 units/ml, pH 8.0) was

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added for ten minutes and washed into PBS in order to unblock the DNA breaks caused by formaldehyde. It was then incubated in the TUNEL mix containing florochrome at 37ºC for ten minutes and washed into PBS again. The sections were transferred to a dark room. PBS was added to each which were then covered with a coverglass

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and analyzed by a pathologist blinded to the identity of the groups under Zeiss Imager-A2 microscobe followed by counting TUNEL positive cells of CA1 regions in two different areas (Shamsaei et al., 2015). Statistical Analysis

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Established data were analyzed using SPSS for Windows 15.0 software (SPSS Inc., Chicago, Illinois, USA). Shapiro-Wilk test was used to reveal whether the groups have normal distribution status. Outcomes of

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descriptive statistics were presented as mean ± standard deviation or median (minimum-maximum) as per test of normality. Statistical comparison of the continuous variables were carried out using one-way analysis of variance

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(ANOVA) or Kruskal–Wallis tests according to the normality condition of the groups. Tukey test was applied for post-hoc analysis where ANOVA test yielded positive results. When the Kruskal-Wallis test was significant, Mann–Whitney U test was used to determine the prominent differences in binary comparasions. P values less

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than 0.05 were considered as statistically significant.

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ACCEPTED MANUSCRIPT Results Biochemical parameters When the TAS tissue levels were compared among groups, a statistically significant difference was found (Table 1, p=0.001). Although the TAS tissue levels showed a significant decrease in the IR group as per the Sham

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group, a significant increase was found in the IR+Crocin group compared to the IR group (P<0.004).

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When the TOS tissue levels were compared among groups, a statistically significant difference was found (Table 1, p=0.001). The TOS tissue levels showed a significant increase in both the IR and the IR+Crocin groups as to

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the Sham group (P<0.004).

A statistically significant difference was found between the OSI tissue levels among the groups (Table 1, p<0.001). There was a significant increase in OSI tissue levels of the IR group in comparison with sham; pretreatment with crocin reduced these levels back to baseline (P<0.004). On the other hand, there was a significant

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increase in the IR+Crocin group in comparsion with the Sham group (P<0.004).

No statistically significant difference was determined among the groups as a result of TAS serum level comparisons (Table 2, p>0.05).

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A statistically significant difference was also determined between the TOS serum levels among the groups (Table 2, p=0.013). The TOS serum levels showed a significant increase in the IR group in comparison with the Sham group (P<0.02).

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A statistically significant difference was found (Table 2, p=0.003) between the OSI serum levels among the groups. There was a significant increase in OSI serum levels of the IR group in comparison with the sham group

Histopathologic Staining

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and pre-treatment with crocin reduced these levels back to the baseline (P<0.02).

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Ischemic lesions were observed in the majority of the IR group when CA1 region of the brain cornu ammonis was analyzed histopathologically using H&E staining. The pyramidal neurons were shrunk and pyknotic. Their cytoplasms got an eosinophilic colors well as their nucleoli were almost indistinguishable (Figure 1B). There was a significant increase in the scoring of these histopathological damages of the IR group in comparison with

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the sham group and pre-treatment with crocin reduced these levels back to the baseline (P<0.05). The whole scoring data for the groups have been given in Table 3. Immunohistochemical Staining and TUNEL method Immunohistochemically, the CA1 regions of brain cornu ammonis stained with Caspase 3 (Figure 2) and HIF-1α (Figure 3) antibodies were analyzed carefully. There was a significant increase in the scoring of Caspase-3, HIF1α expressions of the IR group in comparison with the sham group and pre-treatment with crocin reduced these levels back to the baseline (P<0.05). The scoring data of Caspase-3 and HIF-1α expressions have been presented in Figure 4 according to groups. There was a significant increase in the number of apoptotic cells stained with TUNEL method of the IR group in comparison with the sham group and pre-treatment with crocin reduced these levels back to the baseline (P<0.05) (Figure 5). The scoring data of apoptotic changes in hippocampal CA1 region using TUNEL method in accordance with groups was given in Table 3. Hippocampus sections were also subjected to TUNEL staining after IR in this study in order to analyze the apoptotic cell death of hippocampus CA1 zone induced by four-vessel occlusion. As shown in Figure 5B most hippocampal neurons were positive for TUNEL staining in the sections from the IR group. However,

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ACCEPTED MANUSCRIPT quantitative analyses indicated that the number of TUNEL-positive cells in the hippocampal region was significantly attenuated in animals pretreated with crocin in comparison with IR animals (Figure 5C). As a result, these data clearly indicate that crocin has the ability to inhibit the intrinsic apoptotic pathway.

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Discussion

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Four-vessel occlusion model is the most commonly used technique in global brain ischemia models. This technique consists of a two-stage surgery within a 24-hour interval that possibly leads to a more consistent

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reduction in cerebral blood flow and production of preconditioning effects. The two-stage surgery is invasive and technically demanding. Cerebral IR may occur with high morbidity after shock, brain injury and cardiac arrest (Atlasi et al., 2013) to mimic this clinical situation, we performed global cerebral IR via 4 vessels occlusion on rats.

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An increasing level of knowledge on understanding the mechanisms of the cerebral IR injury in recent years has been an important step in the development of neuropreotective strategies in order to protect the brain from this damage. That is why experimental studies have focused on reducing the oxidative damage which plays an

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important role in IR injury. Until this day, different agents have been tried on different cerebral IR models (Dai et al., 2015b, Yaman et al., 2007). Despite the fact that the efficiency of Crocin, which is a strong antioxidant, on other IR models has been investigated, its effect on the global cerebral IR model with 4 vessel occlusion has not

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been investigated so far. Our current study, therefore, focuses on the antioxidant, antiapoptotic and protective effects of crocin on the global cerebral IR.

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Many complex phenomena such as deprivation in blood circulation, oxidative stress, inflammation and apoptosis in the cerebral IR injury lead to an irreversible damage or death of neurons (Danton and Dietrich, 2003). One of

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the main reasons behind IR injury is the ROS produced (Lu et al., 2015). Electron transport chain in the mitochondria is one of the most important sources in ROS production (Sanderson et al., 2013). Mitochondria are structures related to membrane potential, ATP, load of calcium and apoptotic pathways. Ischemia leads to mitochondrial dysfunction as well as to a rapid increase in reactive oxygen species (ROS) such as super oxide

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and hydrogen peroxide due to the re-introduction of oxygen during reperfusion (Ambrosio et al., 1991). Production of abundant amounts of ROS can contribute to cell membrane destruction directly by inducing lipid peroxidation (Chan, 2004). It is well documented that attenuating oxidative stress is important in evolving neuroprotective strategies to enhance neuronal survival after cerebral ischemia (Liang et al., 2008). It has also been shown that antioxidant agents reduce neurodegeneration in cerebral IR injury by leaving an effect on ROS (Vakili et al., 2014). Being one of these agents, along with the crocin’s antioxidant effect against the retinal (Chen et al., 2015, Qi et al., 2013) and heart (Dianat et al., 2014, Esmaeilizadeh et al., 2015). IR injuries, its antiapoptotic and protective effects have also been shown. As far as we know, very few number of investigations have been carried out on the effect of crocin on different cerebral models (Alavizadeh and Hosseinzadeh, 2014, Khazdair et al., 2015). It has been reported (Vakili et al., 2014) that crocin increases the activities of brain superoxide dismutase (SOD) and glutathione peroxidase (GPx) but reduces the levels of brain malondialdehyde (MDA) in the middle cerebral artery IR model. In a global cerebral IR model with two vessel occlusion (Zheng et al., 2007), the SOD and GSH-px activities decreased and MDA levels increased significantly in cortical microvascular homogenates. Crocin reversed the increase of this MDA levels and the decrease of SOD and GSH-px activities, thereby confirming its antioxidant role in the brain I/R. Our study is in parallel to literature

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ACCEPTED MANUSCRIPT and supports the hypothesis that crocin has the antioxidant property due to the levels of increasing serum TAS and the decreasing OSI. The brain tissue is also susceptible to oxidative damage because it consumes significant amounts of oxygen and because antioxidant enzyme activity of the brain is weak (Mohammadi et al., 2013).Therefore, different brain

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tissue cells are vulnerable to oxidative damage and histopathological changes due to free radical production

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following brain ischemia (Su et al., 2009). Dai etal. put forth that IR induced by four-vessel occlusion caused a clear loss of ordered cell morphology, pyknosis in the pyramidal cells which have lost their uniformed shape and

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increased the number of irregularly shaped cells (Dai et al., 2015a). Sun et al. indicated that IR injury provokes many histopathologic changes in the CA1 region of hippocampus such as disappearance of nucleolus and shrunkage neurons (Sun et al., 2016). In our study, ischemic damage was investigated by H&E staining, a method commonly used to identify the histopathological changes associated with the development of IR injury.

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In the present study, pyramidal neurons in hippocampal region were shrunken and pyknotic, their cytoplasms got a color alike eosinophils and nuclei were shrunken and almost invisible. These histopathological neurodegenerations were significantly reduced in crocin treated rats. These findings are in line with previous

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studies (Sarshoori et al., 2014) which put forth that crocin prevents cells from neurodegeneration. The hippocampal CA1 region is well known as one of the most vulnerable regions in the brain. Neuronal death in the hippocampal CA1 region occurs following transient ischemic insult. For oxidative stress, ROS involved in

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I/R injury is one of the primary factors resulting in neuronal loss (Chen et al., 2011). There was a compelling evidence in many studies that accumulation of ROS caused by IR leading to neuronal damage and increases the

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occurrence of apoptotic cell death in the brain (Manzanero et al., 2013). Caspase 3, an interleukin converting enzyme in this apoptotic cell death pathway, is the fundamental receiver and a reliable marker indicating

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apoptotic activity (Keane et al., 2001, Kobeissy et al., 2006). It has been shown by several reports that this marker increases following brain ischemia (Sakurai et al., 2003). We evaluated whether the antiapoptotic properties of crocin involve an inhibition of caspase-3 activity. Although we expected that crocin would inhibit increased caspase 3 expression, the antiapoptotic effect of crocin following global cerebral ischemia has been put

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forth in many studies. It was also shown that crocin lowers the expression of caspase 3 against the retinal (Chen et al., 2015) and gastric IRinjury (Mard et al., 2015). However, crocin’s effect on caspase 3 pathway against brain IR injury has not been studied yet. In this study, caspase 3 expression was elevated following cerebral IR ınjury which is an indicator of increased apoptosis. On the other hand, reduced caspase 3 expresion by crocin treatment supports the hypothesis indicating that crocin has an antiapoptotic effect. Previous reports showed that Caspase-3 activity in the hippocampus increased significantly following ischemia and that there were many TUNEL positive neurons in the CA1 area (Bayat et al., 2012). TUNEL is a common method for detecting DNA fragmentation resulting from apoptotic signalling cascades. The assay relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme catalysing the addition of dUTPs that are secondarily labelled with a marker. It may also label cells that have underwent severe DNA damage (Bayat et al., 2012). Similar studies which have the same IR period of ours, showed that hippocampal CA1 (Yaman et al., 2007) and prefrontal cortex (Eser et al., 2011) regions cells; the number of apoptotic cells there was a siginifcant increase found in the IR group compared to Sham and pretreatment with antioxidant agents reducing these levels back to baseline. In the our current study, consistent with the literature; crocin’s inhibitory effect on caspase activation was founded along with a gross reduction in

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ACCEPTED MANUSCRIPT the amounts of apoptotic cells, as shown by TUNEL staining, in the vulnerable hippocampal CA1 region following four vessels oclusion. HIF-1 is an important transcription factor that plays a role in the cells’ adaptation to hypoxic conditions. HIF-1 is composed of oxygen-regulated 1α subunit and constitutively expressed 1β subunit. Constitutively synthesized

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HIF-1α is degraded quickly by propil hydroxilase in the presence of O2, iron and ascorbate (Schofield and

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Zhang, 1999). Hypoxia leads to translocation of HIF-1α hydroxilase to the nucleus by inhibiting it. It forms a complex in the nucleus with HIF-1β that activates HIF-1, formation of active HIF-1 initiates target gene

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transcription which is vital for adaptation to hypxia of cells (Jaakkola et al., 2001). HIF-1α is involved in ischemia/hypoxia-induced cell death events by activating the expression of various pro-apoptotic genes during severe or sustained ischemia/hypoxia (Wang et al., 2004). A previous study suggested that there was a causal relationship between HIF-1α and caspase-3 induction through HIF-1α functional binding to the caspase-3 gene

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promoter (Van Hoecke et al., 2007). However, the effect of crocin on brain IR damage by the mentioned pathway has not been investigated. Therefore, the mechanisms were still unclear and investigations were launched to explore the crocin-mediated antiapoptotic effects against global cerebral IR injury. In our study,

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treatment with crocin leds to decreased expression of HIF-1α, caspase-3 and TUNEL, indicating that administration of crocin protects the brain from apoptosis, probably by inhibiting HIF-1α and caspase-3 pathway.

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In summary, this study is demonstrating, for the first time, that crocin could protect neurons and reduces apoptosis by blocking the activation of the caspase-3, HIF-1α in the cerebral IR injury induced by four vessels

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occlusion.

This study has various limitations, one of which is due to the fact that crocin was administered at 40 mg/kg dose

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in an experimental ischemia induced by 30 mins. of carotid artery occlusion followed by 30 mins. reperfusion after 24 hours of vertebral artery electrocauterization. Regarding this, one should also keep in mind that possible oxidative, apoptotic and histopathologic alterations may be caused by the application of IR model settings with

Conclusion

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different durations and different crocin dose levels.

The data acquiredfrom our study indicate that crocin treatment may atteunate apoptosis, probably mediated by decreasing OSI induced by ROS generation and inhibiting the protein expression of HIF-1α, TUNEL and caspase-3 after cerebral ischemia. As a result, future studies with different crocin doses and IR durations may contribute to reduce the possible ischemic complications, treatment costs and recovery period during the management of clinical conditions due to global cerebral ischemia.

Acknowledgment Afyon Kocatepe University Scientific Research Projects Unit supported the present study (Project no:13.TIP.01).

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(arrows). (C) The IR+Crocin group (n=10): Ischemic changes are seen only in a few neurons (arrows).

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Figure 2: Caspase 3 staining in the CA1 region of the hippocampus (A) Sham group (n=10): few neurons showing a Caspase 3 positivity (arrow). (B) IR group (n=10): a majority of neurons are Caspase 3 positive

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(arrows). (C) IR+Crocin group (n=10): a few neurons showing a Caspase 3 positivity (arrows). Figure 3: HIF-1α staining in the CA1 region of hippocampus (A) Sham group (n=10): HIF-1α positive staining is present in few neurons (arrows). (B) IR group (n=10): almost in every ischemic neurons showing an intense,

in a few neurons (arrows).

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diffuse HIF-1α positivity (arrows). (C) IR+Crocin group (n=10): Intense HIF-1α positive staining is present only

Figure 4: Scoring of the apoptotic changes seen in hippocampal CA1 region with Caspase 3 (A) and HIF-1α (B) antibodies. * : p<0.05 vs Sham, ▽: p<0.05 vs IR

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Figure 5: TUNEL staining in the CA1 region of the hippocampus (A) Sham group: Almost no TUNEL reaction positivity in neurons. (B) The IR group (n=10): very intense TUNEL positivity in almost all neurons. (C)

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IR+Crocin group (n=10): TUNEL reaction is positive in a few neurons (arrows).

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TABLES:

Table 1. Comparison of tissue oxidative stress parameters among the groups. †

One-way analysis of variance (ANOVA) and Tukey test with post hoc analysis, Kruskal-Wallis test and Mann–Whitney U-test

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Data are presented as mean  ±  standard deviation unless stated. The bold P-values show statistical significance (P<0.002).

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Same letters in the rows indicate statistical significance between the groups (P<0.05).

Table 2. Comparison of serum oxidative stress parameters among the groups † ‡

One-way analysis of variance (ANOVA) and Tukey test with post hoc analysis, Kruskal-Wallis test and Mann–Whitney U-test,

Data are presented in mean ±  standard deviation. Dark P values are statistically significant (P<0.05). Same letters in the rows indicate statistical significance between the groups (P<0.05).

Table 3. Pathological scoring among the groups. HPS: The histopathological scoring (HPS) of the hippocampus (cornu ammonis regions). TUNEL: The number of TUNEL positive cells in CA1 regions. One-way analysis of variance (ANOVA) and Tukey test with post hoc analysis, Data are presented in mean ±  standard deviation. Dark P values are statistically significant (P<0.001). Same letters in the rows indicate statistical significance between the groups (P<0.05).

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ACCEPTED MANUSCRIPT Table 1. Comparison of tissue oxidative stress parameters among the groups Groups

TAS

Sham

IR

IR+Crocin

0,438±0,114a

0,203±0,058a,b

0,418±0,144b

6,223±0,792a,c

8,429±0,909a

Eq/g prot)

8,364±0,570c

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TOS (μmol H2O2

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mmolTroloxe quivalent/g protein

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OSI 1387,09(868,86- 4088,32(3020,51-7148,02)a,b 2140,03(1249,61-3610,74)b,c Median 2240,53)a,c (min.-max.) † One-way analysis of variance (ANOVA) and Tukey test with post hoc analysis, ‡ Kruskal-Wallis test and Mann–Whitney U-test Data are presented as mean  ±  standard deviation unless stated. The bold P-values show statistical significance among groups (P<0.002). Same letters in the rows indicate statistical significance between the groups (P<0.004).

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P 0.001†

0.001† <0.001‡

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IR

IR+Crocin

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TAS 0,539±0,193 0,341±0,137 0,492±0,136 (pg/mg prot) TOS 8,244±1,163a 10,104±1,318a 9,421±0,937 (pg/mg prot) OSI 1752,521(934,643084,2(2090,02-6173,67)a,b 1854,736(1356,71Median a 2403,89) 3035,56)b (min.-max.) † One-way analysis of variance (ANOVA) and Tukey test with post hoc analysis, ‡ Kruskal-Wallis test and Mann–Whitney U-test, Data are presented in mean ±  Standard deviation. Dark P values are statistically significant among the groups (P<0.02). Same letters in the rows indicate statistical significance between the groups (P<0.02).

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P 0.052† 0.013† 0.003‡

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HPS TUNEL

0±0a,b 21,57±10,65

a,b

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IR+Crocin

2,42±0,53a,c

1,57±0,53b,c

176,14±53,63

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HPS: The histopathological scoring (HPS) of the hippocampus (cornu ammonis regions). TUNEL; The number of TUNEL positive cells in CA1 regions. One-way analysis of variance (ANOVA) and Tukey test with post hoc analysis, Data are presented in mean ±  Standard deviation. Dark P values are statistically significant (P<0.001). Same letters in the rows indicate statistical significance between the groups (P<0.05).

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