Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress

Animals

Adult male Sprague-Dawley rats, weighing 240–290 g, were procured from the University Laboratory Animal Center of National Cheng Kung University (NCKU). Animal experiments were conducted after approval and in accordance with the strict guidelines of the Subcommittee on Research Animal Care of NCKU University Medical Center, and the standards meet the guidelines of the Taiwan National Institutes of Health.

Chemicals and reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Melatonin was dissolved in polyethylene glycol 400 (PEG 400) or dimethylsulfoxide (DMSO) (both from Sigma-Aldrich).

Neuronal cultures and oxygen and glucose deprivation (OGD) induced neuronal cell injury

According to a previously described method, cultured neurons were obtained from the cerebral cortices of 1-day-old Sprague-Dawley rats (36,37). Experiments were performed on cultured neurons between 7 and 10 days in vitro. OGD was achieved by inducing hypoxia and aglycemia according to the method (38,39). The OGD medium consisting of Hank's Balanced Salt Solution (HBSS) lacking glucose. The HBSS solution was bubbled with N₂ for 30 min to deplete glucose and oxygen from intracellular stores and extracellular space. Cultured neurons were pretreated with melatonin (10–500 µM) or control (0.1% DMSO) for 30 min, and then were subjected to OGD. After the deprivation period, cultured neurons were incubated in the culture medium under normal conditions (a humidified incubator with 5% CO₂ at 37°C). Each experiment consisted of three samples. Four independent instances were carried out for each experiment.

Transient MCA occlusion model and drug administration

Focal cerebral ischemia was induced in the rats by MCA occlusion using a modification of the intraluminal technique (26,27,35,40–42). Recirculation/reperfusion of cerebral blood flow was allowed by gently removing the monofilament carefully after 90 min of ischemia. In the sham-operated animals, all procedures except for the insertion of the nylon filament were carried out.

Animals were administered intravenously with either melatonin (5 mg/kg) or the same volume of PEG-saline at the start of reperfusion. In the first series of experiments, the animals treated with melatonin (5 mg/kg, n=5) or control (PEG-saline, n=6) were euthanized after 24 h following the ischemic insult then evaluated for brain infarction and neuronal damage. The second series of animals received melatonin (5 mg/kg, n=15) or control (n=15) upon reperfusion and were evaluated for ER stress-associated proteins by western blotting at 1 and 24 h post-reperfusion. The third series of animals received melatonin (5 mg/kg, n=19) or control (n=12) upon reperfusion and were then assessed for ER stress- and neuronal cell-associated proteins using immunofluorescence staining at 1 and 24 h post-reperfusion. Additionally, 15 animals received sham-operations to serve as nonischemic controls.

Animal sacrifice and quantification of ischemic damage

Rats subjected to MCA occlusion were perfused under anesthesia with cold phosphate-buffered saline (PBS) and then with 4% paraformaldehyde prepared in 0.1 M PBS for internal fixation. The subject brains were quickly removed, stored in the same fixative for 24 h, and sequentially immersed in 15 and 30% sucrose at 4°C for 48 h. They were then embedded in Optimal Cutting Temperature compound (OCT; Miles Inc., Elkhart, IN, USA) and frozen in liquid nitrogen. The brains were sectioned coronally on a cryostat (HM-500O; Microm International GmbH, Walldorf, Germany). Serial sections of 40 µm at eight preselected coronal levels with 1-mm intervals from the stereotaxic coordinates of the Bregma AP +2.22 to -4.78 mm were mounted on poly-L-lysine-coated slides and dried overnight at 37°C.

Brain infarction was determined by staining preselected brain slices with hematoxylin and eosin (H&E) stain. Under light microscopy, areas of neuronal perikarya displaying typical morphological features of ischemic damage were delineated. Infarction volume was measured using a computerized image analyzer (MCID Elite; Imaging Research Inc., St. Catharines, ON, Canada) and expressed as a percentage of the contralateral hemisphere volume (41).

Histological analysis

The brain sections and cultured neurons were collected on poly-L-lysine coated slides for further processing. For immunohistochemistry, sections were permeabilized using Triton X-100 and sections were blocked in 1% serum for 1 h. After blocking, sections were incubated with rabbit anti-p-PERK antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit anti-eIF2 antibody (1:200; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-neuronal nuclei (NeuN) monoclonal antibody (1:1,000; eBioscience, Inc., San Diego, CA, USA), mouse anti-microtubule-associated protein-2 (MAP-2) monoclonal antibody (1:1,000; Santa Cruz Biotechnology, Inc.), and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:1,000; eBioscience, Inc.) at 4°C overnight. Appropriate secondary antibody conjugated with biotin (1:100) was then added, followed by FITC-conjugated streptavidin and Texas red-conjugated streptavidin (1:100) (both from Jackson ImmunoResearch, Inc., West Grove, PA, USA). Sections were counterstained for nucleus with 4′,-6-diamidino-2-phenylindole (DAPI) and then visualized under a fluorescence microscope (Olympus IX71; Olympus Optical Co., Ltd., Tokyo, Japan). The signals for FITC (green), Texas red (red), and DAPI (blue) were superimposed using Image Pro Plus 5.1 software (Media Cybernetics, Silver Spring, MD, USA).

Terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay

TUNEL assay was performed using an in situ cell death detection kit (Calbiochem, Merk Biosciences, Bad Soden, Germany) according to the manufacturer's protocol. Specifically, sections were fixed with 4% paraformaldehyde prepared in 0.1 M PBS and then incubated with 3% H₂O₂ at room temperature for 5 min. The sections were digested with fresh diluted proteinase K (1:200) at RT for 10 min. Then, TdT Equilibration buffer was added to each section and allowed to settle at RT for 30 min. Sections were incubated with TdT labeling reaction mixture at 37°C for 2 h in the dark. The nuclei were stained with DAPI. Sections were examined under the fluorescence microscope (Olympus IX71; Olympus Optical Co., Ltd.). The positive cells and total cells were counted for three fields at ×200 magnification. The results are presented as a ratio of positive cells to total cells.

Western blot analysis

Samples were obtained from cultured neurons and from the brain tissues of the contralateral, penumbral core, and ischemic core regions which were quickly dissected on dry ice after the animals were sacrificed. The 0.1 g of tissue was homogenized in 1 ml lysis buffer, containing 20 mM HEPES, 250 mM sucrose, 1 mM EDTA (pH 7.5), 20 mM EGTA (pH 7.5), 10 mM KCl, 250 mM MgCl₂ and a complete protease inhibitor on ice for 30 min. Homogenates were centrifuged at 800 × g for 15 min at 4°C. The nuclear extract pellets collected and stored at −80°C until used. The supernatant was carefully transferred to another centrifuge tube and was centrifuged at 100,000 × g for 1 h at 4°C. The resultant supernatant containing cytoplasma extract and ER extract pellets was harvested and stored at −80°C until used. After mixing with sodium dodecyl sulfate buffer and heating 10 min at 100°C, protein concentration was determined by bicinchoninic acid (BCA protein assay kit; Thermo Fisher Scientific, Waltham, MA, USA). For western blot analysis, an equal amount of protein (50 µg) was loaded in each well and subjected to 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). The separated proteins were then transferred onto polyvinylidene fluoride (PVDF) microporous membranes (IPVH00010; Millipore, Billerica, MA, USA) and blocked in 5% milk. The membranes were incubated with primary anti-p-PERK (1:400; Santa Cruz Biotechnology, Inc.), anti-eIF2α (1:1,000; Cell Signaling Technology, Inc.), ATF4 (1:300; Santa Cruz Biotechnology, Inc.), CHOP (1:300) cleaved caspase-3 (1:1,000) (both from Cell Signaling Technology, Inc.), actin (1:10,000; Chemicon International), LaminA/C (1:1,000; Santa Cruz Biotechnology, Inc.) antibodies, and finally incubated with horseradish peroxidase-conjugated anti-rabbit/mouse IgG (1:5,000; Chemicon International, Billerica, MA, USA). Bound antibodies were visualized with the Amersham ECL system (GE Healthcare Biosciences Corp., Piscataway, NJ, USA). A Luminescent Image Analyzer (Fujifilm LAS-3000; Fuji Photo Film Co., Tokyo, Japan) was used to measure optical densities.

Statistical analysis

Data are presented as the mean ± standard deviation of the mean. The significance of difference between means was assessed by Student's t-test (single comparisons) or by one-way analysis of variance (one-way ANOVA) with Fisher's protected least significant difference (LSD) post hoc comparison. P<0.05 was selected for statistical significance.

Melatonin reduced brain infarction following MCA occlusion at 24 h

The animals treated with melatonin or control did not have altered local cortical blood perfusion or core temperature during the course of surgery (data not shown). Relative to control, the brain infarct volumes of melatonin treated subjects were reduced by 1.77-fold (P<0.05) at 24 h after stroke (Fig. 1A and B). This translates to a melatonin-mediated decrease in cortex and striatum infract size by 2.12- and 1.58-fold, respectively, as well as an increase in the number of the surviving cortex neurons by 1.47-folds (Fig. 1C and D; P<0.05).

Effects of melatonin on the expression protein of p-PERK- p-elF2α-ATF4-CHOP signaling pathway in rats subjected to MCA occlusion

To study the effect of melatonin-inhibited ER stress resulting from ischemia reperfusion, we evaluated the expression of ER stress-associated proteins including p-PERK, p-eIF2α, ATF4 and CHOP in the contralateral, penumbra cortex and ischemic core, of rats after post-insult. Post-ischemic expression of p-PERK and p-eIF2α protein was induced in ischemic brain 1 h after reperfusion onset and then declined (data not shown). The results revealed that ischemia reperfusion increased p-PERK and p-eIF2α expression at penumbral and ischemic core regions, while the rats treated with melatonin showed lower levels in p-PERK and p-eIF2α, as compared with control groups (Fig. 2A and B). For the melatonin treated animals, there was a significant reduction of 31 and 36% p-PERK expression of the penumbral and ischemic core regions, respectively, as compared to the control groups (P<0.05) (Fig. 2A). Effective inhibition of p-eIF2α levels at the penumbral and ischemic core regions by 33 and 43%, respectively, was found in melatonin-treated animals compared to control groups (P<0.05) (Fig. 2B). Additionally, the reduction of p-PERK and p-eIF2α of 18 and 21% intensity, respectively, was found in the ischemia cortex of the melatonin-treatment animals compared to controls (P<0.05) (Fig. 2C and D). The immunofluorescence staining revealed the expression of p-PERK was localized on neuronal cells (NeuN positive cells) and glial cells (GFAP positive cells) in the ischemic brains (Fig. 2E).

For ER stress-induced apoptosis, the pro-apoptotic transcription factor CHOP plays a key role (43). CHOP is the downstream signal of the PERK-p-eIF2α-ATF4 pathway in unfolded protein response (44). The results revealed that post-ischemic expression of ATF4 and CHOP protein was induced in ischemic brain 1 h and reached peak at 24 h after reperfusion onset (data not shown). Compared with the control groups, significantly decreased ATF4 expression at ischemic core and CHOP expression at penumbra by 38 and 36%, respectively, were found in the melatonin-treated animals at 24 h after ischemia reperfusion (P<0.05) (Fig. 3A and B). The immunofluorescent assay results showed lower ATF4 and CHOP intensity in the ischemia cortex by 12 and 14%, respectively, in the melatonin-treated animals at 24 h after reperfusion onset (P<0.05) (Fig. 3C and D).

Effects of melatonin on the expression of p-PERK and p-elF2α proteins in cultured neurons after OGD injury

The western blot analysis showed that the expression of p-PERK and p-eIF2α proteins induced at 1 h following cultured neurons exposed to OGD, peaked at 2 h and then declined (Fig. 4A and B). Expressions of p-PERK and p-eIF2α were localized on neuronal cells (MAP2 positive cells) and glial cells (GFAP positive cells) (Fig. 4C and D). Furthermore, pretreatment with melatonin at 10–100 µM effectively inhibited expression of both p-PERK and p-eIF2α by 51–42 and 43–28%, respectively, after OGD-induced cultured neuron injury (P<0.05) (Fig. 5A and B). Immunofluorescent staining indicated that pretreatment with melatonin at 10–100 µM also significantly reduced the intensity of p-PERK and p-eIF2α by 26–41 and 37–31%, respectively, in the cultured neurons after OGD injury (P<0.05) (Fig. 5C and D).

The anti-apoptotic effects of melatonin in ischemic reperfusion

The results of TUNEL staining showed abundance of TUNEL-positive neurons in cultured neurons after OGD injury (Fig. 6A). Pretreatment with melatonin at 50 µM significantly reduced the number of TUNEL-positive neurons in the cultured neurons after OGD injury. Western blot analysis revealed that OGD injury markedly increased expression of cytochrome c and cleaved caspase-3 in the cultured neurons, while the neurons treated with melatonin at 50 µM showed significantly reduced cytochrome c and cleaved caspase-3 levels compared to control (Fig. 6B). These results indicate melatonin prevented neuronal apoptosis by decreasing cytochrome c and cleaved caspase-3 levels after OGD-induced injury.