Journal of the Neurological Sciences 336 (2014) 74–82

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Ischemic preconditioning-induced neuroprotection against transient cerebral ischemic damage via attenuating ubiquitin aggregation Jae-Chul Lee a,1, In Hye Kim a,1, Geum-Sil Cho b, Joon Ha Park a, Ji Hyeon Ahn a, Bing Chun Yan c, Hyuk Min Kwon d, Young-Myeong Kim e, Seung Hwan Cheon f, Jun Hwi Cho f,g, Hui Young Lee g,h, Moo-Ho Won a,g,⁎, Jeong Yeol Seo i,⁎⁎ a

Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701 South Korea Department of Neuroscience, College of Medicine, Korea University, Seoul 136-705, South Korea c Institute of Integrative traditional & Western Medicine, Medical College, Yangzhou University, Yangzhou 25001, China d Department of Obstetrics and Gynecology, Kangwon National University Hospital, Chuncheon 200-701, South Korea e Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea f Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea g Institute of Medical Sciences, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea h Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea i Department of Emergency Medicine, Chuncheon Sacred Heart Hospital, College of Medicine, Hallym University, Chuncheon 200-702, South Korea b

a r t i c l e

i n f o

Article history: Received 17 July 2013 Received in revised form 16 September 2013 Accepted 7 October 2013 Available online 15 October 2013 Keywords: Ischemia–reperfusion Ischemic preconditioning Pyramidal neurons Delayed neuronal death Ubiquitin system Hippocampus

a b s t r a c t Ubiquitin binds to short-lived proteins, and denatured proteins are produced by various forms of injuries. In the present study, we investigated the effect of ischemic preconditioning (IPC) on free ubiquitin and its mutant form (ubiquitin+1) in the gerbil hippocampus induced by transient cerebral ischemia. The animals were randomly assigned to 4 groups (sham-operated-group, ischemia-operated-group, IPC plus (+)-sham-operated-group, and IPC + ischemia-operated-group). IPC was induced by subjecting gerbils to a 2 min of ischemia followed by 1 day of recovery. A significant loss of neurons was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia-operated-groups 5 days after ischemia–reperfusion (I–R). In all the IPC + ischemia-operated-groups, neurons in the SP were well protected. We found that strong ubiquitin immunoreactivity was detected in the SP in the sham-operated-group and the immunoreactivity was decreased with time after I–R. In all the IPC + ischemia-operated-groups, ubiquitin immunoreactivity in the SP was similar to that in the sham-operated group. Moderate ubiquitin+1 immunoreactivity was detected in the SP of the shamoperated-group, and the immunoreactivity was markedly increased 2 days after I–R. Five days after I–R, ubiquitin+1 immunoreactivity was very weak in the SP. In all the IPC + ischemia-operated-groups, ubiquitin+1 immunoreactivity in the SP was slightly decreased with time after I–R. Western blot analysis showed that, in all the IPC + ischemia-ischemia-groups, the levels of ubiquitin and ubiquitin+1 proteins were well maintained after I–R. In brief, our findings suggest that the inhibition of the depletion of free ubiquitin and the formation of ubiquitin+1 may have an essential role in inducing cerebral ischemic tolerance by IPC. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Transient global cerebral ischemia occurs when the blood supply to the brain is disrupted, resulting in oxygen and glucose deprivation of the tissues, which may cause irreversible brain damage [1]. In humans, selective neuronal damage/death in the brain can occur frequently after cardiac arrest and/or cardiopulmonary bypass surgery, which is a

⁎ Correspondence to: M.-H. Won, Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. Tel.: +82 33 250 8891; fax: +82 33 256 1614. ⁎⁎ Corresponding author. Tel.: +82 33 258 2378; fax: +82 33 258 2451. E-mail addresses: [email protected] (M.-H. Won), [email protected] (J.Y. Seo). 1 Jae-Chul Lee and In Hye Kim contributed equally to this article. 0022-510X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jns.2013.10.010

worldwide devastating health problem [2,3]. The Mongolian gerbil has been used as a good animal model to investigate the molecular mechanism of selective neuronal death following transient global cerebral ischemia [4,5] because about 90% of gerbils lack the communicating vessels between the carotid and vertebral circulations [6–8]. Thus, the bilateral occlusion of the carotid arteries essentially and completely eliminates the blood flow to the forebrain while completely sparing the vegetative centers of the brain stem. Ischemic preconditioning (IPC) is thought to occur in humans suffering transient ischemic attacks [9,10]. The IPC of the rodent brain with sublethal cerebral ischemia is also known to generate resistance to the subsequent lethal period of ischemia [11,12]. A 2-min period of cerebral ischemia in gerbils produces no appreciable neuronal damage in the hippocampus [12]. The IPC of the gerbil brain with this period

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using a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept on the thermal incubator (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature of animals until the animals were euthanized. The animals were divided into four groups: (1) sham-operated-group (n = 12): the bilateral common carotid arteries were exposed, no ischemia was given (sham-operation) in the animals; (2) ischemia-operated-group (n = 12 at each time point): the animals were given a 5 min lethal ischemic insult 24 h after sham-operation (3) IPC plus(+)-sham-operated-group (n = 12): the animals were subjected to a 2 min sublethal ischemic insult; and (4) IPC+ischemia-operated-group (n=12 at each time point): the animals were pretreated with a 2 min sublethal ischemia 1 day prior to a 5 min lethal ischemia. Animals in groups 2 and 4 were given recovery times of 1 day, 2 days, and 5 days. This preconditioning paradigm has been proven to be very effective at protecting neurons against ischemia in this ischemic model [12].

of ischemia, however, which is followed by 1–7 days of reperfusion, protects against neuronal damage following subsequent longer periods of ischemia that usually kill the pyramidal neurons of the hippocampal CA1 region [12]. This phenomenon has been termed “ischemic tolerance,” and its mechanism has yet to be defined. Ubiquitin, a low-molecular-weight heat shock protein, binds to shortlived proteins and denatured proteins produced by various forms of injury, and ubiquitinated proteins are degraded by protease in an ATP-dependent manner [13]. Therefore, the loss of ubiquitin leads to an accumulation of abnormal proteins and may affect cellular structures and functions [14]. On the other hand, mutant-ubiquitin-form (ubiquitin+ 1) protein is a mutant protein produced by aberrant transcripts formed as a result of dinucleotide deletion in the open reading frame of mRNA, termed “molecular misreading” [15]. Ubiquitin+1 protein itself inhibits the proteasomal degradation of cellular proteins and can induce cell toxicity [16,17]. The ubiquitin system plays a key role in various stress conditions, such as ischemia [18], glutamate toxicity [19], Alzheimer's disease [20], and Parkinson's disease [21]. Protein degradation following ischemic damage may activate the ubiquitin system, which is directly involved in the intracellular hydrolytic degradation of unfolded and/or nonfunctional proteins [22]. In addition, the accumulation of ubiquitin+1 protein in the neurons is considered the hallmark of proteasomal dysfunction in neurodegenerative disorders [23]. It has been reported that the impairment of the ubiquitin–proteasome system leads to neuronal death [24], and it was shown herein that naive and mutant forms of ubiquitin were changed in the gerbil hippocampus induced by transient global cerebral ischemia [25]. There have been few studies, however, regarding alterations of free ubiquitin and ubiquitin+1 protein in the brain following IPC. Thus, the contribution of free ubiquitin and ubiquitin+1 protein to the delayed neuronal death in the gerbil hippocampus after transient global cerebral ischemia following IPC was also examined in the present study.

Sham-operated-, ischemia-operated-, IPC + sham-operated- and IPC + ischemia-operated- animals (n = 7 at each time point) were decapitated at 1 day, 2 days and 5 days after ischemia–reperfusion), because pyramidal neurons in the hippocampal CA1 region do not die until 2–3 days and die 4–5 days after ischemia–reperfusion. The animals were anesthetized with pentobarbital sodium and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M12phosphate-buffer (PB, pH 7.4). The brains were removed and postfixed in the same fixative for 6 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Germany) into 30 μm coronal sections, and they were then collected into six-well plates containing PBS.

2. Materials and methods

2.4. Cresyl violet (CV) staining

2.1. Experimental animals

To examine the neuronal death in the hippocampal CA1 region in each group using CV staining, the sections were mounted on gelatincoated microscopy slides. Cresyl violet acetate (Sigma, MO, USA) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid was added to this solution. The sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kato, Japan).

Male Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center, Kangwon National University, Chuncheon, South Korea. Gerbils were used at 6 months (B.W., 65–75 g) of age. The animals were housed in a conventional state under adequate temperature (23 °C) and humidity (60%) control with a 12-h light/12-h dark cycle, and were provided with free access to food and water. The procedures for animal handling and care 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 Academies Press, 8th Ed., 2011), and they were approved by the Institutional Animal Care and Use Committee (IACUC) at Kangwon University. All of the experiments were conducted to minimize the number of animals used and the suffering caused by the procedures used in the present study. 2.2. Induction of transient cerebral ischemia The animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. Under an operating microscope, a ventral neck incision was made and the bilateral common carotid arteries were gently exposed. Ischemia was induced by occluding the arteries with non-traumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope. After 2 min or 5 min of occlusion, the aneurysm clips were removed from the common carotid arteries. The body (rectal) temperature under free-regulating or normothermic (37 ± 0.5 °C) conditions was monitored with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA) and maintained

2.3. Tissue processing for histology

2.5. NeuN immunohistochemistry To examine the neuronal damage in the hippocampal CA1 region after transient cerebral ischemia in each group, using NeuN immunohistochemistry, the sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min. The sections were next incubated with diluted mouse anti-NeuN (a neuron-specific soluble nuclear antigen) (diluted 1:1000, Chemicon International, Temecula, CA) overnight at 4 °C. Thereafter the tissues were exposed to biotinylated goat anti-mouse IgG and streptavidin peroxidase complex (Vector, Burlingame, CA). And they were visualized with 3,3′-diaminobenzidine in 0.1 M Tris–HCl buffer and mounted on the gelatin-coated slides. After dehydration the sections were mounted with Canada balsam (Kanto, Tokyo, Japan). 2.6. Fluoro-Jade B (F-J B) histofluorescence To confirm the neuronal death in the brain after transient forebrain ischemia in each group, using F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration) histofluorescence [26], the sections were first

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immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% F-J B (Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were placed on a slide warmer (approximately 50 °C), and then examined using an epifluorescent microscope (Carl Zeiss, Germany) with blue (450–490 nm) excitation light and a barrier filter. With this method neurons that undergo degeneration brightly fluoresce in comparison to the background [27].

2.7. Immunohistochemistry for ubiquitin and ubiquitin+1 In order to examine the accurate degree of immunohistochemical staining, immunohistochemistry was processed under the same conditions at designated times after ischemia–reperfusion. The sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min. They were next incubated with diluted rabbit anti-ubiquitin (diluted 1:1000, LabFrontier, Seoul, South Korea) or rabbit anti-ubiquitin+1 (diluted 1:1000, LabFrontier) overnight at 4°C and subsequently exposed to biotinylated goat anti-rabbit IgG and streptavidin peroxidase complex (diluted 1:200, Vector). They were then visualized by staining with 3,3′diaminobenzidine (Sigma, St. Louis, MO) in 0.1 M Tris–HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration, the sections were mounted with Canada balsam (Kanto). 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.

2.8. Cell counts All measurements were performed to insure objectivity in blind conditions, by two observers for each experiment, carrying out the measures of experimental samples under the same conditions. The studied tissue sections were selected with 120 μm interval according to anatomical landmarks corresponding to AP from AP −1.4 to −1.8 mm of gerbil brain atlas, and cell counts were obtained by averaging the counts from 20 sections taken from each animal. All NeuN- and F-J B-positive structures were taken from 3 layers (stratum oriens, pyramidale and radiatum in the hippocampus proper) through an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. The mean number of NeuN- and F-J B-positive cells was counted in a 250 × 250 μm square applied approximately at the center of the CA1 region. Cell counts were obtained by averaging the total cell numbers from each animal per group. Twenty sections per animal were selected to quantitatively analyze ubiquitin and ubiquitin+1 immunoreactivity. Cellular immunoreactivity of ubiquitin and ubiquitin+1 was graded in the hippocampal CA1 region. Digital images of the hippocampal CA1 region were captured with an AxioM1 light microscope (Carl Zeiss) equipped with a digital camera (Axiocam, Carl Zeiss) connected to a PC monitor. Semiquantification of the immunostaining intensity of ubiquitin and ubiquitin+1 was evaluated with a digital image analysis software (MetaMorph 4.01, Universal Imaging Corp.). The mean intensity of ubiquitin and ubiquitin+ 1 immunostaining in each ubiquitin and ubiquitin+ 1 immunoreactive cell was measured by a 0–255 gray scale system (white to dark signal corresponded from 255 to 0). Based on this approach, the level of immunoreactivity was scaled as −, ±, + or ++, representing no staining (gray scale value: ≥ 200), weakly positive (gray scale value: 150–199), moderate (gray scale value: 100–149), or strong (gray scale value: ≤ 99), respectively [28].

2.9. Western blot analysis To obtain the accurate data for changes in ubiquitin and ubiquitin+1 level in the hippocampal CA1 region after transient cerebral ischemia, sham-operated-, ischemia-operated-, IPC + sham-operated- and IPC + ischemia-operated-animals (n = 7 at each time point) were sacrificed at designated times (1 day, 2 days and 5 days after ischemia– reperfusion) and used for Western blot analysis. After sacrificing them and removing the hippocampus, it was transversely cut into 400 μm thicknesses on a vibratome (Leica), and the hippocampal CA1 region was dissected with a surgical blade. The tissues were homogenized in 50mM PBS (pH7.4) containing 0.1mM ethylene glycol bis (2-aminoethyl ether)-N,N,N′,N′ tetraacetic acid (EGTA) (pH 8.0), 0.2% Nonidet P-40, 10 mM ethylendiamine tetraacetic acid (EDTA) (pH 8.0), 15 mM sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM dithiothreitol (DTT). After centrifugation, the protein level was determined in the supernatants using a Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, Rockford, IL). Aliquots containing 20 μg of total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% SDS, 0.3% bromophenol blue and 30% glycerol. Then, each aliquot was loaded onto a 12.5% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Pall Crop, East Hills, NY). To reduce background staining, the membranes were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min and then with rabbit anti-ubiquitin (1:2000) or anti- ubiquitin+1 antiserum (1:2000), peroxidase-conjugated goat anti-rabbit IgG (Sigma) and an ECL kit (Pierce Chemical). The result of Western blot analysis was scanned, and the quantification of the analysis was done using Scion Image software (Scion Corp., Frederick, MD), which was used to count relative optical density (ROD): A ratio of the ROD was calibrated as %. 2.10. Statistical analysis Data are expressed as the mean ± SEM. Differences of the means among the groups were statistically analyzed by analysis of variance (ANOVA) with a post hoc Bonferroni's multiple comparison tests in order to elucidate ischemia-related differences among experimental groups. Statistical significance was considered at P b 0.05. 3. Results 3.1. Cresyl violet-positive (+) cells In this study, we used CV staining to examine the neuroprotective effects of IPC in the CA1 region of the gerbil. In the sham-operatedgroups, CV+ cells were easily detected in all the subregions of the hippocampus (Fig. 1A, B). Neurons in the stratum pyramidale were relatively large, pyramid-like or round in shape. In the ischemiaoperated-groups, the morphology and the number of CV+ cells were not changed until 2 days after ischemia–reperfusion (Fig. 1E, F, I, J). However, in the ischemia-operated-group at 5 days after ischemia– reperfusion, a few CV+ cells were found in the stratum pyramidale of the CA1 region compared to those of the sham-operated-group (Fig. 1M, N). Damaged cells were shrunken and contained dark and polygonal nuclei (Fig. 1N). In the IPC + sham-operated-group, CV+ cells were easily detected in all the subregions of the hippocampus, and the distribution pattern of CV+ cells in the stratum pyramidale of the CA1 region was similar to that in the sham-operated-group (Fig. 1C, D). In the IPC + ischemiaoperated-groups, the distribution pattern of CV+ cells in the stratum pyramidale was not changed compared with that in the IPC + shamoperated-group (Fig. 1G, H, K, L, O, P).

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Fig. 1. Histochemical staining for CV in the CA1 region of the ischemia-operated- (left two columns) and IPC + ischemia-operated- (right two columns) groups at sham (A–D), 1 (E–H), 2 (I–L) and 5 days (M–P) after ischemia–reperfusion. In the ischemia-operated-group, a few CV+ cells (arrows) are shown in the stratum pyramidale (SP) at 5 days after ischemia– reperfusion, while abundant CV+ cells (asterisk) are observed in the IPC + ischemia-operated-group. CA; cortical area, DG; dentate gyrus, SO; stratum oriens, SR; stratum radiatum. Scale bar = 800 (A, C, E, G, I, K, M, O) and 50 (B, D, F, H, J, L, N, P) μm.

3.2. NeuN+ neurons In the sham-operated-group, pyramidal neurons in the stratum pyramidale of the CA1 region were well immuno-stained with NeuN (Table 1, Fig. 2A). In the ischemia-operated-groups, no change in the number of NeuN+ neurons of the stratum pyramidale of the CA1 region was found until 2 days after ischemia–reperfusion (Table 1, Fig. 2C, E). However, 5 days after ischemia–reperfusion, a significant loss of NeuN+ neurons was observed in the CA1 of the ischemia-operated-group (Table 1, Fig. 2G). We examined the neuroprotective effect of IPC against delayed neuronal death in the pyramidal neurons of the stratum pyramidale of the CA1 region. The distribution pattern of NeuN+ neurons in the CA1 stratum pyramidale of the IPC + sham-operated-group was similar Table 1 Changes in the mean number of pyramidal neurons of the hippocampal CA1 region in the ischemia-operated- and IPC + ischemia-operated-groups. Time after I–R

Group Ischemia

Sham 1 day 2 days 5 days

IPC

NeuN+

F-J B+

NeuN+

F-J B+

369 ± 8.64 364 ± 7.15 356 ± 9.55 37 ± 6.34*

0 0 15 ± 2.5* 65 ± 8.45*

372 ± 6.28 362 ± 8.41 360 ± 7.69 298 ± 10.25*#

0 0 0 7 ± 2.85*#

+

to that in the sham-operated-group (Table 1, Fig. 2B). In the IPC + ischemia-operated-groups at 1, 2 and 5 days after ischemia– reperfusion, the distribution pattern of NeuN+ neurons in the stratum pyramidale was not significantly changed compared with that in the IPC + sham-operated-group (Table 1, Fig. 2D, F, H). 3.3. F-J B+ cells F-J B histofluorescence staining was also performed to examine neuronal degeneration in the CA1 region after ischemia–reperfusion. F-J B+ cells were not observed in the CA1 region of the shamoperated-groups and in the ischemia-operated-groups at 1 and 2 days after ischemia–reperfusion (Table 1, Fig. 3A, C, E). A significant increase of F-J B+ cells was observed in the stratum pyramidale of the CA1 region of the ischemia-operated-group 5 days after ischemia–reperfusion (Table 1, Fig. 3G). In the IPC + sham-operated-group, F-J B+ cells were not observed in the stratum pyramidale of the CA1 region, and F-J B+ cells were hardly observed in the IPC + ischemia-operated-groups at 1and 2 days after ischemia–reperfusion (Table 1, Fig. 3B, D, F). However, some F-J B+ cells, which were weakly stained with F-J B, were found in the stratum pyramidale of the CA1 region 5 days after ischemia–reperfusion (Table 1, Fig. 3H). 3.4. Ubiquitin immunoreactivity

+

The mean number of NeuN and F-J B cells is counted in a 250 × 250 μm square of the stratum pyramidale of the CA1 region after ischemia–reperfusion (I–R) (n = 7 per group; *P b 0.05, significantly different from the corresponding sham-group, #P b 0.05, significantly different from the respectively preceding group). IPC, ischemic preconditioning.

In the sham-operated-group, strong ubiquitin immunoreactivity was detected in the stratum pyramidale of the CA1 region (Table 2, Fig. 4A). In the ischemia-operated-group 1 day after ischemia–reperfusion, many

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Fig. 2. Immunohistochemistry for NeuN in the CA1 region of the ischemia-operated- (left column) and IPC + ischemia-operated- (right column) groups at sham (A, B), 1 (C, D), 2 (E, F) and 5 days (G, H) after ischemia–reperfusion. In the ischemia-operated-group, few NeuN-immunoreactive cells (arrows) are shown in the stratum pyramidale (SP) at 5 days after ischemia–reperfusion; the distribution pattern of NeuN+ in the stratum pyramidale (SP, asterisk) in the IPC + ischemia-operated-group is similar to that in the shamoperated-group. SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.

Fig. 3. F-J B histofluorescence staining in the CA1 region of the ischemia-operated- (left column) and IPC + ischemia-operated- (right column) groups at sham (A, B), 1 (C, D), 2 (E, F) and 5 days (G, H) after ischemia–reperfusion. In the ischemia-operated group, many F-J B+ cells are detected in the stratum pyramidale (SP, arrows) 5 days after ischemia–reperfusion. However, a few F-J B+ cells are detected in the IPC + ischemiaoperated-group 5 days after ischemia–reperfusion. SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.

neurons in the stratum pyramidale showed strong ubiquitin immunoreactivity (Table 2, Fig. 4C). Thereafter, ubiquitin immunoreactivity in the stratum pyramidale was apparently decreased with time after ischemia–reperfusion, while strong ubiquitin immunoreactivity was expressed in non-pyramidal cells in the CA1 region 2 days after ischemia–reperfusion (Table 2, Fig. 4E). Five days after ischemia–reperfusion, ubiquitin immunoreactivity was very weak in the stratum pyramidale of the CA1 region (Table 2, Fig. 4G). In the IPC + sham-operated-group, ubiquitin immunoreactivity was similar to that in the sham-operated group (Table 2, Fig. 4B). In the IPC + ischemia-operated-groups, ubiquitin immunoreactivity was not significantly changed until 5 days after ischemia–reperfusion (Table 2, Fig. 4D, F, H).

ubiquitin+ 1 immunoreactivity was very weak in the stratum pyramidale (Table 2, Fig. 5G). In the IPC+sham-operated-group, ubiquitin+1 immunoreactivity in the stratum pyramidale of the CA1 region was much higher than that in the sham-operated-group (Table 2, Fig. 5B). In the IPC + ischemiaoperated-groups, ubiquitin+1 immunoreactivity in the stratum pyramidale was slightly decreased with time after ischemia–reperfusion,

Table 2 Semi-quantifications of the immunoreactivity of ubiquitin and ubiquitin+ in the hippocampal CA1 region in the ischemia-operated- and IPC + ischemia-operated-groups. Antibody

Groups

Category

Time after ischemia/reperfusion Sham

1 day

2 days

5 days

Ubiquitin

Ischemia

CSP CSOR CSP CSOR CSP CSOR CSP CSOR

++ + ++ + + ± ++ ±

++ + ++ ++ ++ + + +

+ ++ ++ + ++ ± + +

± ± ++ ± ± + ± ++

3.5. Ubiquitin+1 immunoreactivity Moderate ubiquitin+1 immunoreactivity was detected in the stratum pyramidale of the CA1 region of the sham-operated-group (Table 1, Fig. 5A). Ubiquitin+1 immunoreactivity was slightly increased in the stratum pyramidale 1 day after ischemia–reperfusion, and the ubiquitin+1 immunoreactivity was markedly increased in the stratum pyramidale 2 days after ischemia–reperfusion(Table 2, Fig. 5C, E): at this time point after ischemia–reperfusion, strong ubiquitin+1 immunoreactivity appeared in the nuclei of the CA1 pyramidal cells based on morphology (Fig. 5E). Five days after ischemia–reperfusion,

IPC Ubiquitin+

Ischemia IPC

Immunoreactivity is scaled as −, ±, + or ++, representing no staining, weakly positive, moderate or strong, respectively. CSP, cells in stratum pyramidale; CSOR, cells in stratum oriens and radiatum. IPC, ischemic preconditioning.

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Fig. 4. Immunohistochemical staining for free ubiquitin in the ischemia-operated- (left column) and IPC + ischemia-operated- (right column) groups at sham (A, B), 1 (C, D), 2 (E, F) and 5 days (G, H) after ischemia–reperfusion. In the ischemia-operated-group, many neurons (arrows) in the stratum pyramidale (SP) show strong ubiquitin immunoreactivity, and its immunoreactivity is apparently decreased in the stratum pyramidale from 2 days after ischemia–reperfusion. In the IPC + ischemia-operatedgroups, ubiquitin immunoreactivity in the SP is maintained until 5 days after ischemia– reperfusion. SO, stratum oriens; SR, stratum radiatum. Scale bar = 50 μm.

Fig. 5. Immunohistochemical staining for ubiquitin+ in the ischemia-operated- (left column) and IPC + ischemia-operated- (right column) groups at sham (A, B), 1 (C, D), 2 (E, F) and 5 days (G, H) after ischemia–reperfusion. In the ischemia-operated-group, strong ubiquitin+ immunoreactivity (arrows) is detected in the stratum pyramidale (SP) 2 days after ischemia–reperfusion, and ubiquitin+ immunoreactivity is markedly decreased in the SP 5 days after ischemia–reperfusion. In the IPC + ischemia-operatedgroup, ubiquitin+ immunoreactivity is apparently decreased in the SP from 5 days after ischemia–reperfusion. SO; stratum oriens, SR; stratum radiatum (SR). Scale bar = 50 μm.

and the immunoreactivity was low 5 days after ischemia–reperfusion (Table 2, Fig. 5D, F, H).

4. Discussion

3.6. Protein levels of ubiquitin and ubiquitin+1 The result of western blot study showed that change patterns of ubiquitin levels in the hippocampus after ischemia–reperfusion were generally similar to the immunohistochemical change (Fig. 6). Ubiquitin protein level was significantly decreased from 2 days after ischemia– reperfusion, and was lowest 5 days after ischemia–reperfusion. In the IPC + sham-operated-group, ubiquitin protein level was similar to that in the sham-operated group, and, in the IPC + ischemia-operatedgroups, ubiquitin protein levels were not changed significantly after ischemia–reperfusion. The change pattern of ubiquitin+1 levels in the hippocampus after ischemia–reperfusion was also similar to the immunohistochemical change (Fig. 6). In the ischemia-operated-group, ubiquitin+1 protein level was significantly increased 2 days after ischemia–reperfusion, and its level was lowest 5 days after ischemia–reperfusion. In the IPC+sham-operated-group, ubiquitin+1 protein level was much higher than that in the sham-operated-group, and, in the IPC + ischemiaoperated-groups, ubiquitin+1 protein levels were not significantly changed compared with that in the IPC + sham-operated-group.

In the CNS, the neurons in the hippocampal CA1 region are selectively damaged even after a brief ischemic insult. This topographical heterogeneity is known as “selective vulnerability of the brain” [1]. Especially, the pyramidal neurons in the CA1 region do not die immediately but survive over several days. This unique process is termed “delayed neuronal death (DND).” IPC does not lead to neuronal death and can induce neuronal tolerance to a subsequent longer or lethal period of ischemia [29]. The preconditioning time period was determined by a previous report [12], where it was indicated that at least a 1-day interval between sublethal 2-min ischemia and lethal 5-min ischemia is necessary for the induction of the neuroprotection of the CA1 pyramidal neurons. In the present study, IPC was induced by subjecting gerbils to 2-min ischemia followed by 1-day recovery. It was found that in all the IPC+ischemia-operated groups, the pyramidal neurons in the stratum pyramidale of the CA1 region were well protected 5 days after ischemia–reperfusion. The ubiquitin–proteasome system is a major nonlysozymal system for degrading proteins in cells, and it virtually protects all cell types from diverse stressful pathological conditions by reducing the proteotoxicity, which results from newly synthesized polypeptides and denatured proteins, and by eliminating the irreparably damaged proteins [30,31]. Accumulated evidence has proven that ubiquitin is

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Fig. 6. Western blot analysis of ubiquitin and ubiquitin+ in the hippocampus derived from the ischemia-operated and IPC + ischemia-operated-groups. Relative optical density (ROD) as % values of immunoblot band is also represented (*P b 0.05, significantly different from the sham-operated group, #P b 0.05, significantly different from the pre-adjacent group). The bars indicate the means ± SEM.

involved in the complex regulation of the levels and functions of many proteins and signaling pathways involved in determining the cell fate [32] Ubiquitin is thought to be a stress protein that plays a key role in protecting cells under stress conditions. It is known that the ubiquitin system plays a key role in the stress response and is involved in various stress conditions, such as ischemia [33], glutamate toxicity [19], and heat shock stress [34]. Five minutes of global cerebral ischemia causes almost complete inhibition of protein synthesis in all the forebrain structures of the gerbil [35]. In addition, Nakagomi et al. [36] showed that the recovery rate of protein synthesis in the gerbil brain after IPC was more rapid than that in the nonpreconditioned gerbils. In the present study, it was found that in the ischemia-operated groups, the ubiquitin immunoreactivity was apparently decreased in the stratum pyramidale of the CA1 region with time after ischemia–reperfusion, and the immunoreactivity was hardly found 5 days after I–R. In contrast, the free ubiquitin immunoreactivity in all the IPC-ischemia-operated groups was maintained in the CA1 pyramidal neurons after ischemia–reperfusion. This finding is supported by a paper by Kato et al. [37], which reported that following sublethal cerebral ischemia, the ubiquitin immunoreactivity disappeared for a brief time and was then recovered in the surviving hippocampal CA1 neurons. On the other hand, it was reported that a prolonged decrease of free ubiquitin might inhibit the ubiquitin proteolytic pathway, which might lead to the impairment of the degradation of target proteins such as p53, cyclins, and IκB [38], which are directly related to ischemia-triggered neuronal apoptosis [39]. Moreover, the ubiquitin pathway might be directly involved in apoptosis [40]. These results indicate that the expression of ubiquitin may be necessary for neurons to survive ischemic damage. As mentioned earlier, ubiquitin+1 is a mutant protein produced by aberrant transcripts formed as a result of dinucleotide deletion in the open reading frame of mRNA, termed “molecular misreading” [41,42]. It does not participate in tagging abnormal proteins for degradation by proteasomes, and at high concentrations, it inhibits the proteasomal degradation of cellular proteins and can induce cell toxicity [17,43]. In the present study, it was found that the ubiquitin+1 immunoreactivity was strongly increased in the CA1 stratum pyramidale

2 days after ischemia–reperfusion, and that 5 days after ischemia– reperfusion, the immunoreactivity was very weak. This finding is supported by papers that showed that the ubiquitin+1 immunoreactivity was increased in the gerbil CA1 pyramidal neurons after ischemia–reperfusion, and that the immunoreactivity disappeared 5 days after ischemia–reperfusion [25]. The authors indicated that the selective accumulation of ubiquitin+1 protein in the dying neurons of the ischemic CA1 region might have been induced by proteasomal dysfunction after ischemic damage. On the other hand, a previous study reported that a reduction of proteasomal activity in the CA1 neurons was selectively found in gerbils after transient global ischemia [44]. Ubiquitin+1 is a substrate for proteasome, and ubiquitin+1 in the neurologically intact brain is efficiently degraded by the ubiquitin proteasome system [43]. In addition, several studies have shown that ubiquitin+1 accumulation in many disorders is associated with impairment of the ubiquitin proteasomal system [23,45]: the presence of an impaired proteasomal system leads to the accumulation of ubiquitin+1, and thus, ubiquitin+1 accumulation strongly indicates proteasomal dysfunction [23]. In contrast, proteasome inhibitors block ischemia-induced cell death, although part of this effect is attributed to the inhibition of toxic NF-κB signaling and inflammation [46]. Considered together, these studies provide a scenario where ischemic neuronal injury involves ubiquitin+1 protein and proteasomal dysfunction in the CA1 region. The present study is supported by the results from previous studies that showed that protein aggregation is a major pathological event contributing to neuronal death after focal brain ischemia, in particular, that leads to irreversible inhibition of protein biosynthesis in the neurons destined to die after focal ischemia [47–50]. In the present study, in all the IPC + ischemia-operated groups, the ubiquitin+1 immunoreactivity in the SP was not significantly changed with time after ischemia–reperfusion. Zhang et al. [51] reported that isoflurane preconditioning significantly reduced the ubiquitinconjugated protein aggregation in the CA1 neurons after ischemia; the biochemical analyses revealed that isoflurane preconditioning decreased the accumulation of ubiquitin-conjugated proteins and reduced the free ubiquitin depletion after brain ischemia. It was shown in this study that IPC maintained the ubiquitin immunoreactivity

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in the ischemic CA1 region and controlled the expression of ubiquitin+1 in this region. These results indicate that the accumulation of ubiquitin+1 in the CA1 pyramidal neurons is not only a consequence of impaired proteasomal function but also a cause of cell toxicity. In brief, the effects of IPC on the ubiquitin–proteasome system have yet to be studied, although the neuroprotective action of IPC has been extensively described. This study provides some evidence that IPC significantly eliminates neuronal death in the CA1 region in a gerbil model of transient cerebral ischemia. As viewed by immunohistochemistry, IPC markedly reduced the depletion of free ubiquitin and the formation of ubiquitin+1 protein in the CA1 pyramidal neurons after ischemia–reperfusion. The present study's findings suggest that IPC-facilitated neuroprotection may be mediated in part by the reduction of a lethal aggregation of ubiquitin+1 protein after ischemia–reperfusion. Conflict of interest The authors have no financial conflict of interest. Acknowledgments The authors would like to thank Mr. Seung Uk Lee for their technical help in this study. This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0010580), and by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1020420). References [1] Kirino T, Sano K. Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol 1984;62:201–8. [2] Horn M, Schlote W. Delayed neuronal death and delayed neuronal recovery in the human brain following global ischemia. Acta Neuropathol 1992;85:79–87. [3] Petito CK, Feldmann E, Pulsinelli WA, Plum F. Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology 1987;37:1281–6. [4] Malek M, Duszczyk M, Zyszkowski M, Ziembowicz A, Salinska E. Hyperbaric oxygen and hyperbaric air treatment result in comparable neuronal death reduction and improved behavioral outcome after transient forebrain ischemia in the gerbil. Exp Brain Res 2013;224:1–14. [5] Cao Y, Mao X, Sun C, Zheng P, Gao J, Wang X, et al. Baicalin attenuates global cerebral ischemia/reperfusion injury in gerbils via anti-oxidative and anti-apoptotic pathways. Brain Res Bull 2011;85:396–402. [6] Fukuchi T, Katayama Y, Kamiya T, McKee A, Kashiwagi F, Terashi A. The effect of duration of cerebral ischemia on brain pyruvate dehydrogenase activity, energy metabolites, and blood flow during reperfusion in gerbil brain. Brain Res 1998;792:59–65. [7] Janac B, Radenovic L, Selakovic V, Prolic Z. Time course of motor behavior changes in Mongolian gerbils submitted to different durations of cerebral ischemia. Behav Brain Res 2006;175:362–73. [8] Selakovic V, Korenic A, Radenovic L. Spatial and temporal patterns of oxidative stress in the brain of gerbils submitted to different duration of global cerebral ischemia. Int J Dev Neurosci 2011;29:645–54. [9] Weih M, Kallenberg K, Bergk A, Dirnagl U, Harms L, Wernecke KD, et al. Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain? Stroke 1999;30:1851–4. [10] Moncayo J, de Freitas GR, Bogousslavsky J, Altieri M, van Melle G. Do transient ischemic attacks have a neuroprotective effect? Neurology 2000;54:2089–94. [11] Kirino T, Nakagomi T, Kanemitsu H, Tamura A. Ischemic tolerance. Adv Neurol 1996;71:505–11. [12] Nakamura H, Katsumata T, Nishiyama Y, Otori T, Katsura K, Katayama Y. Effect of ischemic preconditioning on cerebral blood flow after subsequent lethal ischemia in gerbils. Life Sci 2006;78:1713–9. [13] Rechsteiner M. Ubiquitin-mediated pathways for intracellular proteolysis. Annu Rev Cell Biol 1987;3:1–30. [14] Wojcik C, Di Napoli M. Ubiquitin–proteasome system and proteasome inhibition: new strategies in stroke therapy. Stroke 2004;35:1506–18. [15] van Leeuwen FW, de Kleijn DP, van den Hurk HH, Neubauer A, Sonnemans MA, Sluijs JA, et al. Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients. Science 1998;279:242–7. [16] Lam YA, Pickart CM, Alban A, Landon M, Jamieson C, Ramage R, et al. Inhibition of the ubiquitin–proteasome system in Alzheimer's disease. Proc Natl Acad Sci U S A 2000;97:9902–6.

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