Failure in neuroprotection of remote limb ischemic postconditioning in the hippocampus of a gerbil model of transient cerebral ischemia Jae-Chul Lee, Hyun-Jin Tae, Bai Hui Chen, Jeong Hwi Cho, In Hye Kim, Ji Hyeon Ahn, Joon Ha Park, Bich-Na Shin, Hui Young Lee, Young Shin Cho, Jun Hwi Cho, Seongkweon Hong, Moo-Ho Won, Chan Woo Park PII: DOI: Reference:

S0022-510X(15)02459-4 doi: 10.1016/j.jns.2015.09.371 JNS 14114

To appear in:

Journal of the Neurological Sciences

Received date: Revised date: Accepted date:

29 July 2015 5 September 2015 27 September 2015

Please cite this article as: Jae-Chul Lee, Hyun-Jin Tae, Bai Hui Chen, Jeong Hwi Cho, In Hye Kim, Ji Hyeon Ahn, Joon Ha Park, Bich-Na Shin, Hui Young Lee, Young Shin Cho, Jun Hwi Cho, Seongkweon Hong, Moo-Ho Won, Chan Woo Park, Failure in neuroprotection of remote limb ischemic postconditioning in the hippocampus of a gerbil model of transient cerebral ischemia, Journal of the Neurological Sciences (2015), doi: 10.1016/j.jns.2015.09.371

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ACCEPTED MANUSCRIPT Failure in neuroprotection of remote limb ischemic postconditioning in the hippocampus of a

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gerbil model of transient cerebral ischemia

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Jae-Chul Lee1, Hyun-Jin Tae2, Bai Hui Chen3, Jeong Hwi Cho1, In Hye Kim1, Ji Hyeon Ahn2, Joon Ha Park1, Bich-Na Shin3, Hui Young Lee4, Young Shin Cho5,6, Jun Hwi Cho6, Seongkweon Hong7,Soo Young Choi2,

Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South

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Moo-Ho Won1*, Chan Woo Park6*

Korea 2

Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University,

Chuncheon 24252, South Korea

Department of Physiology, College of Medicine, Hallym University, Chuncheon 24252, South Korea

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Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 24289, South

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Department of Emergency Medicine, Seoul Hospital, College of Medicine, Sooncheonhyang University, Seoul

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04401, South Korea

South Korea 7

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Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24289,

Department of General Surgery, School of Medicine, Kangwon National University, Chuncheon 24289, South

Korea

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- Jae-Chul Lee1 and Hyun-Jin Tae2 have contributed equally to this work.

*Corresponding authors:

Professor Moo-Ho Won, DVM, PhD: Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 24341, South Korea. TEL: +82-33-250-8891; FAX: +82-33-256-1614. E-mail: [email protected]

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ACCEPTED MANUSCRIPT Professor Chan Woo Park, MD, PhD: Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 24289, South Korea. TEL: 033-258-9371; FAX: 033-258-2451; E-mail:

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[email protected]

Keywords: Global cerebral ischemia; remote ischemic postconditioning; hippocampal CA1 neurons; delayed

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neuronal death; occlusion of hind limbs

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ACCEPTED MANUSCRIPT Abstract

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Remote ischemic postconditioning (RIPoC) has been proven to provide potent protection of the heart and brain

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against ischemia-reperfusion injury. However, despite the evidence of cerebral protection with RIPoC is compelling, RIPoC-mediated neuroprotection against transient cerebral ischemic insult is still mired in controversy. In this study, we examined the effect of RIPoC induced by sublathal transient hind limb ischemia

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on neuronal death in the hippocampus following 5 min of transient cerebral ischemia in gerbils. Animals were randomly assigned to sham-, ischemia-, sham plus (+) RIPoC- and ischemia+RIPoC-groups. RIPoC was

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induced by three cycles of 5-min and 10-min occlusion-reperfusion of both femoral arteries at predetermined points in time ( 0, 1, 3, 6, 12 and 24 h after transient cerebral ischemia). CV staining, F-J B histofluorescence staining and NeuN immunohistochemistry were carried out to examine neuroprotection in the RIPoC-mediated hippocampus 5 days after ischemia-reperfusion. In the ischemia-group, we found a significant loss of pyramidal

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cells in the stratum pyramidale (SP) of the hippocampal CA1 region at 5 days post-ischemia compared with the sham-group. In all of the ischemia+RIPoC-groups, the loss of pyramidal cells in the CA1 region at 5 days post-

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ischemia was not different from that in the ischemia-group. Our present findings indicate that RIPoC does not

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prevent hippocampal CA1 pyramidal cells from neuronal death induced by transient cerebral ischemia.

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ACCEPTED MANUSCRIPT 1. Introduction

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Ischemic conditioning is regarded to be an efficacious protective strategy for brain ischemic injury, and thus, it

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attracts extensive interest [1-3]. This phenomenon is termed cerebral “ischemic tolerance”, although its basic mechanisms underlying cerebral ischemic tolerance are not fully understood yet [4]. Ischemic preconditioning (IPC) in the brain has been firmly established as a strategy to reduce ischemia-reperfusion injury [5]. Recently,

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we also demonstrated that IPC applied 1 day before 5 min of ischemia endowed the survival of 95% of pyramidal cells in the hippocampal CA1 region after transient cerebral ischemia [6, 7]. However, the application

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of IPC has been greatly restricted due to difficulty in predicting cerebral ischemic insult. Therefore, ischemic postconditioning (IPoC) confers post-ischemic neuroprotection when performed immediately or shortly after reperfusion [8-10]. Furthermore, classical IPC has been conducted in the same organs that received lethal ischemia [11, 12] and its clinical application has been limited due to the risk that applies an additional ischemia

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to the same organs, such as the brain or heart. For this reason, ischemic conditioning has been extended to remote ischemic conditioning including remote ischemic preconditioning (RIPC) and remote ischemic

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postconditioning (RIPoC), which are induced in distant non-vital organs [13-16]. After the first report of RIPC in the heart by Przyklenk et al. [17], who demonstrated intramyocardial

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protection across different coronary artery territories, many studies have shown that RIPC for ischemia– reperfusion injury is reproduced in other organs including the liver [18, 19] and kidney [20, 21]. Similarly,

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multiple subsequent studies have also shown positive effects of RIPC in some animal models of cerebral ischemic insults [13, 22, 23]. RIPC represents an interesting alternative to cerebral preconditioning because it is significantly less invasive and thus it has a much higher potential for clinical application; however, the application of RIPC in patients experiencing an unpredictable ischemic insults must be restricted due to difficulty in predicting cerebral ischemic insults. Intriguingly, RIPoC, a fascinating new intervention, has recently emerged as an effective way to ameliorate ischemia-reperfusion injury in the heart, lung and hind limb [24-26]. As for cerebral ischemia, RIPC in a rat model of focal ischemia, which was firstly reported by Ren et al. [13], reduces infarct volume and improves neurologic functions. With this protective concept of RIPC, RIPoC has also been demonstrated by a number of groups using some animal models of transient focal ischemia [15, 27, 28], cardiac arrest [29] and neonatal hypoxic ischemia [30, 31]. However, it remains controversial whether RIPoC could protect neurons from

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ACCEPTED MANUSCRIPT neuronal damage/death, although protective effects of RIPoC have been demonstrated in humans and animals. Furthermore, it is scientifically unclear whether RIPoC inhibits neuronal damage/death after transient cerebral

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ischemia, which shows different pathophysiology from that in focal cerebral ischemia. Thus, the aim of the

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present study was to determine whether RIPoC protect neurons from ischemic insult in the hippocampus induced by transient cerebral ischemia using the gerbil, which is a good animal of transient cerebral ischemia [7, 32]. RIPoC used in this study was performed by a series of brief occlusion and release of both femoral arteries

2. Materials and Methods

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2.1. Experimental animals and groups

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after transient cerebral ischemia.

We used the progeny of male Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental

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Animal Center, Kangwon National University, Chunchon, South Korea. Gerbils were used at 24 weeks (Body weighting 65 – 75 g) of age. All the experimental protocols were approved by the Institutional Animal Care and

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Use Committee (IACUC) at Kangwon University and adhered to guidelines that are in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National

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Academies Press, 8th Ed., 2011).

Experimental animals were divided into nine groups (n = 7 at each point in time) in experiment 1 and 2, respectively, as shown in Fig 1. Experiment 1 was subjected to 5-min cerebral ischemia and 3 cycles of 5-min RIPoC according to the scheduled time interval. Meanwhile, experiment 2 was subjected to 5-min cerebral ischemia and 3 cycles of 10-min RIPoC according to the scheduled time interval: (1) sham-group, which was given no cerebral ischemia; (2) sham+RIPoC-group, which was subjected to RIPoC without cerebral ischemia; (3) ischemia-group, which was subjected to cerebral ischemia without RIPoC; (4) ischemia+RIPoC (0 h)-group, which was subjected to RIPoC immediately after cerebral ischemia; (5) ischemia+RIPoC (1 h)-group, which was subjected to RIPoC 1 h after cerebral ischemia; (6) ischemia+RIPoC (3 h)-group, which was subjected to RIPoC 3 h after cerebral ischemia; (7) ischemia+RIPoC (6 h)-group, which was subjected to RIPoC 6 h after

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ACCEPTED MANUSCRIPT cerebral ischemia; (8) ischemia+RIPoC (12 h)-group, which was subjected to RIPoC 12 h after cerebral

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ischemia; (9) ischemia+RIPoC (24 h)-group, which was subjected to RIPoC 24 h after cerebral ischemia.

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2.2. Induction of transient cerebral ischemia and RIPoC

Transient cerebral ischemia was developed according as previously described [32]. In brief, the animals were

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anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. Ischemia was induced by occluding both common carotid arteries with non-traumatic aneurysm clips (Yasargil FE 723K, Aesculap,

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Tuttlingen, Germany). After 5 of occlusion, the aneurysm clips were removed from the arteries. The body (rectal) temperature under free-regulating or normothermic (37 ± 0.5ºC) conditions was monitored with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA, USA) and maintained using a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter,

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animals were kept on the thermal incubator (temperature, 23ºC; humidity, 60%) (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature of animals until the animals were sacrificed.

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RIPoC was conducted in both hind limbs by occluding and releasing both femoral arteries by three cycles of 5 or 10 min-RIPoC, as shown in Fig. 1, immediately (0), 1, 3, 6, 12 and 24 h after 5 min-cerebral ischemia. In

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brief, under anesthesia, both femoral arteries were exposed, and RIPoC was induced by clamping the femoral arteries for 5-min or 10-min with non-traumatic aneurysm clips (Yasargil, Germany); the occlusion of the

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femoral arteries was confirmed by oxygen saturation probe attached in foot (Nonin Medical Inc, Plymouth, MN, USA) and monitored continuously during RIPoC. The body temperature was also maintained normothermic (37 ± 0.5ºC) conditions.

The animals in each group were given recovery times of 5 days, as shown in Fig. 1, because pyramidal cells in the hippocampal CA1 region do not die until 3 days and begin to die from 4 days after ischemia-reperfusion [33, 34].

2.3. Tissue processing for histology

The animals in each group were deeply anesthetized with pentobarbital sodium and perfused through the left ventricle with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M

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ACCEPTED MANUSCRIPT phosphate-buffer (PB, pH 7.4). The brains were removed and post-fixed in the same fixative for 6 h. The brain tissues were embedded in tissue-freezing medium and serially sectioned into 30 µm coronal sections on a

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cryostat (Leica, Wetzlar, Germany).

2.4. Cresyl violet (CV) staining and Fluoro-Jade B (F-J B) histofluorescence

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To investigate the delayed neuronal damage in the hippocampus after ischemia-reperfusion, CV staining and F-J B histofluorescence were performed as previously described [32]. In brief, the sections were stained with 1.0%

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(w/v) cresyl violet acetate (Sigma-Aldrich, St. Louis, MO, USA) and dehydrated. They were then mounted with Canada balsam (Kanto chemical, Tokyo, Japan). For F-J B histofluorescence, the sections were immersed in a 0.0004% F-J B (Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were examined using an epifluorescent microscope (Carl Zeiss, Göttingen, Germany) with blue (450-490 nm) excitation light

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and a barrier filter.

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2.5. Immunohistochemistry for neuronal nuclei (NeuN)

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For immunohistochemical staining, the sections were carried out according to our previous method as previously described [34]. The brain sections were blocked with 10% normal goat serum in 0.05 M PBS

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followed by staining with primary mouse anti-NeuN (a neuron-specific soluble nuclear antigen) (diluted 1:1,000, Chemicon International, Temecula, CA, USA) overnight at 4°C. The sections were next incubated with the secondary antibodies (Vector Laboratories Inc., Burlingame, CA, USA) and were developed using Vectastain ABC (Vector Laboratories Inc.). And they were visualized with 3,3’-diaminobenzidine in 0.1 M TrisHCl buffer. In order to establish the specificity of the immunostaining, a negative control test was carried out with pre-immune serum instead of primary antibody. The negative control resulted in the absence of immunoreactivity in any structures.

2.6. Data analysis

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ACCEPTED MANUSCRIPT The brain sections were selected according to anatomical landmarks corresponding to AP from −1.4 to −2.2 mm of gerbil brain atlas. The number of CV-positive, NeuN-immunoreactive and F-J B-positive cells were counted

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in a 250×250 μm square, applied approximately at the center of the CA1 region in the stratum pyramidale. Cell

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counts were obtained by averaging the total cell numbers from each animal per group: A ratio of the count was calibrated as % of sham-group. Cell counts were obtained by averaging the total cell numbers from each animal

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per group

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2.7. Statistical analysis

All data are presented as mean ± SD. A multiple-sample comparison was applied to test the differences between groups and days. The differences between groups in same day were assessed by using one-way ANOVA and a

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Tukey's post hoc test. Statistical significance was considered at P

Failure in neuroprotection of remote limb ischemic postconditioning in the hippocampus of a gerbil model of transient cerebral ischemia.

Remote ischemic postconditioning (RIPoC) has been proven to provide potent protection of the heart and brain against ischemia-reperfusion injury. Howe...
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