Printer Friendly

The beneficial effect of melatonin in brain endothelial cells against oxygen-glucose deprivation followed by reperfusion-induced injury.

1. Introduction

Stroke is the third most frequent worldwide cause of adult death [1,2]. Specifically, about 80% of all strokes are ischemic, resulting from arterial occlusion in the brain [1]. Reperfusion after occlusion results in serious brain injury, due to overproduction of reactive oxygen species (ROS), calcium overload [3, 4], and blood-brain barrier (BBB) injury [5]. Finally, in ischemic stroke, the brain is damaged because of hypoxia and oxidative stress [6-10]. Reactive oxygen species (ROS) play a key role in the pathogenesis of many diseases, including central nervous system (CNS) diseases [11-14]. During ischemic stroke, the excessive generation of ROS leads to inflammation and cell apoptosis [15-21] and induces mitogen-activated protein kinase (MAPK) signaling [22-24]. c-Jun N-terminal kinase (JNK), one of the MApKs, is activated by a variety of cell stresses, including hyperosmotic shock, hypoxia, and ROS [25, 26]. JNK plays key roles in apoptosis and inflammation [27, 28]. JNK signaling is activated by inflammatory cytokines and promotes neuronal cell death [29]. Endothelial cells are also damaged by activation of JNK signaling, in response to oxidative stress [30]. Several studies have demonstrated that, in hypoxia and a state of reoxygenation, cells induce apoptotic signaling through JNK and p38 MAPK [31, 32]. The BBB controls the exchange of materials between blood and the brain and plays an important role in the homeostatic regulation of the brain microenvironment [33]. The tight junctions between capillary endothelial cells, which form an essential structural component of the BBB [34], include membrane proteins like occludin [35] and claudins [36, 37]. Several studies have suggested that hypoxia causes alterations of the tight junction proteins Claudin 5, occludin, ZO-1, and ZO-2, which affect BBB permeability [38, 39]. In addition, vascular endothelial growth factor (VEGF) is an inducer of vascular leakage [40] and is also known as vascular permeability enhancing factor [41, 42]. During ischemia, VEGF interacts with receptors for VEGF on the ischemic vessels and contributes to disruption of the BBB [43,44]. Zhang el al. demonstrated that inhibition of VEGF reduces BBB permeability [43]. Melatonin is synthesized in the pineal gland and has been known to function as an antioxidant [45]. Melatonin reduces the cellular toxicity of ROS in ischemia and reperfusion (I/R) brain injury [46]. In an in vivo cerebral ischemia model, several researches have demonstrated that melatonin treatment reduces brain damage in the setting of ischemia or hypoxia-induced injury [47, 48]. In vitro, melatonin protects primary neuronal cells from apoptotic death [49] and enhances survival of human neuroblastoma cells [50] in the setting of oxygen-glucose deprivation- (OGD-) induced injury. Furthermore, melatonin suppresses VEGF expression in cancer cells [51, 52] and inhibits serum VEGF levels in patients [53]. In the present study, we investigate whether melatonin protects brain endothelial cells against oxygen-glucose deprivation followed by reperfusion- (OGD/R-) induced injury. We show that melatonin reduces the generation of ROS, prevents disruption of the BBB by stabilizing expression of tight junction proteins and suppressing VEGF expression, and attenuates phosphorylation of JNK, a mediator of cellular apoptosis. Therefore, our results suggest that melatonin is important in protecting the BBB against cerebral ischemic damage.

2. Materials and Methods

2.1. Cell Culture. Murine brain endothelial cells (bEnd.3 cells; ATCC, VA, USA) were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone Laboratories, UT, USA), supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone Laboratories, UT, USA) and 100 units/mL of penicillin/streptomycin (Hyclone Laboratories, UT, USA), at 37[degrees]C in a humidified atmosphere in the presence of 5% C[O.sub.2] [54]. bEND.3 cells were used at 13 passages in this study.

2.2. Oxygen-Glucose Deprivation (OGD) and Reperfusion. Confluent cells were transferred to an anaerobic chamber (Forma Scientific, OH, USA) ([O.sub.2] tension, 0.1%) and washed three times with PBS. Then, culture medium was replaced with deoxygenated, glucose-free balanced salt solution, and cells were incubated for 6 h. Following oxygen-glucose deprivation (OGD) injury, cells were incubated for 18 h under normal growth conditions, with or without drug treatment [55].

2.3. Drug Treatment. Melatonin was purchased from Sigma (Sigma, MO, USA) and dissolved in ethanol. An equivalent volume of ethanol (final: 0.01%) or water was added to control and all melatonin-containing wells. bEnd.3 cells were exposed to 1-100 nM melatonin for 24 h before OGD/R injury. The present study consisted of four groups: (1) normal control (NC), bEnd.3 cells cultured with normal media without OGD injury; (2) experimental control (EC), bEnd.3 cells cultured in nontreated medium for 18 h after 6 h of OGD injury; (3) 10 nM melatonin (Mel 10 nM), bEnd.3 cells treated with 10 nM melatonin for 24 h before 6 h of OGD injury; these cells were then cultured in nontreated medium for 18 h; (4) 100 nM melatonin (Mel 100 nM): bEnd.3 cells were also treated with 100 nM melatonin (100 nM melatonin group) for 24 h before 6 h of OGD injury. These cells were then cultured in nontreated medium for 18 h. In Akt inhibitor groups, we treated 100 nM Akt inhibitor (Sigma, MO, USA) together with melatonin.

2.4. Hoechst 33258 and Propidium Iodide (PI) Staining. Cell viability was evaluated by staining bEnd.3 cells with Hoechst 33258 dye (Sigma, MO, USA) and propidium iodide (PI; Sigma, MO, USA). Hoechst dye was added to the culture medium (2-3 [micro]g/mL) and samples were then incubated at 37.8[degrees]C for 30 min. PI solution was then added (2-5 [micro]g/mL) just before cells were observed with a microscope (BX51; Olympus) equipped with epifluorescence and a UV filter block. PI-positive cells were counted as dead cells [56].

2.5. Cell Viability Assay. bEnd.3 cells (2 x 105 cells/mL) were seeded in 98-well plates to monitor all experiment conditions, including pretreatment, OGD injury, and reperfusion. Next, cells were rinsed twice with phosphate-buffered saline (PBS), and culture medium was replaced with serum-free medium and 100 [micro]L 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) (Sigma, MO, USA) solution (5 mg/mL in PBS) per well. After 1 h of incubation, medium was removed and dimethyl sulfoxide (DMSO) was added to solubilize the purple formazan product of MTT treatment. The supernatant from each well was analyzed using an ELISA plate reader (Labsystems Multiskan MCC/340; Fisher Scientific, PA, USA) at a wavelength of 570 nm, with background subtraction at 650 nm. All experiments were repeated at least three times. Cell viability in the control medium, without any treatment, was represented as 100%. Cell viability was reported as a relative value, compared to the control group.

2.6. Lactate Dehydrogenase (LDH) Assay. Cytotoxicity in all treatment groups was quantified by measuring the amount of LDH released into the culture medium from OGD/R-injured cells [57, 58]. LDH release (cytotoxicity %) was calculated by dividing the value at the experimental time point by the maximum value. The maximum LDH release was measured after freezing each culture at -70[degrees]C overnight, followed by rapid thawing, which induced nearly complete cell damage.

2.7. Determination of Intracellular ROS. The level of intracellular ROS in each treatment group was measured using a fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Invitrogen, CA, USA), as previously described [59]. Cells were plated at a density of 1 x [10.sup.6] cells/mL and treated with melatonin for 24 h. After melatonin pretreatment, OGD injury and reperfusion were conducted. Then, bEND.3 cells were treated with 5 [micro]M DCF-DA for 30 min at 37[degrees]C. After washing with PBS, fluorescence was measured with a microscope (Nikon TS100-F ECLIPSE) equipped with a CCD camera (Hamamatsu Photonics) [54].

2.8. Western Blot Analysis. After pretreatment, OGD injury, and restoration, cells were washed rapidly with ice-cold PBS, scraped, and collected. Cell pellets were lysed with ice-cold RIPA buffer (Sigma, MO, USA). The lysates were centrifuged at 13,200 rpm for 1h at 4[degrees]C to produce whole-cell extracts. Protein content was quantified using the BCA method (Pierce, IL, USA). Protein (20 [micro]g) was separated on a 10% SDS-polyacrylamide (PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin, prepared in Tris-buffered saline/Tween (TBS-T; 20 nM Tris (pH 7.2); 150 mM NaCl; 0.1% Tween 20), for 1h at RT, immunoblots were incubated overnight at 4[degrees]C with primary antibodies that specifically detect Akt (1: 2000, Cell Signaling, MA, USA), p-Akt (1: 2000, Cell Signaling, MA, USA), JNK (1: 2000, Cell Signaling, MA, USA), p-JNK (1 : 2000, Cell Signaling, MA, USA), Claudin 5 (1: 1000, Santa Cruz, CA, USA), VEGF (1:1000, Millipore, MA, USA), Bax (1:2000, Cell Signaling, MA, USA), or [beta]-actin (1:2000, Cell Signaling, MA, USA). Next, blots were incubated with HRP-linked anti-mouse and -rabbit IgG antibodies purchased from Abcam (Cambridge, MA, USA) for 1 h at RT. Enhanced chemiluminescence was performed by ECL (Pierce, IL, USA) [54].

2.9. Immunocytochemistry (ICC). The expression of VEGF and Claudin 5 in bEnd.3 cells was confirmed by immunocytochemistry. Cells in all experimental groups were washed three times with PBS, fixed with 4% paraformaldehyde for 3 h, and then washed with PBS. bEnd.3 cells were permeabilized with 0.025% Triton X-100 and blocked for 1 h at RT with dilution buffer (Invitrogen, CA, USA). Primary anti-rabbit VEGF (1 : 500, Millipore, MA, USA) and anti-rabbit Claudin 5 (1 : 500, Santa Cruz, CA, USA) antibodies were prepared in dilution buffer, added to samples, and incubated for 3 h at RT. Primary antibody was then removed and cells were washed three times for 3 min each with PBS. Later, samples were incubated with FITC-conjugated goat, anti-rabbit (1 : 200, Jackson Immunoresearch, PA, USA) or Rhodamine-conjugated donkey, or anti-rabbit secondary antibodies (1: 500, Millipore, MA, USA) for 2 h at RT. Cells were washed again three times for 3 min each with PBS and stained with 1 [micro]g/mL 4',6-diamidino-2-phenylindole (DAPI) (1 : 100, Invitrogen, CA, USA) for 10 min at RT. Fixed samples were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, NY, USA).

2.10. Statistical Analysis. Statistical comparisons were performed using independent t-tests for two groups. SPSS software was used for all analyses. Data were expressed as mean [+ or -] S.E.M. of three independent experiments. Differences were considered significant at [sup.#]P < 0.1, [sup.*]P < 0.05, and **P < 0.001.

3. Results

3.1. Melatonin Attenuates the Cell Death of bEND.3 Cells after OGD/R-Induced Injury. To confirm the protective effect of melatonin on OGD/R-induced injury, we first conducted an MTT assay to check cell viability in all treatment groups (Figure 1(a)). Cell viability showed that the OGD/R injury exposed group exhibited decreased cell viability, compared to the normal control group (100% cell viability in the normal control group; 39% cell viability in the OGD/R injury exposed group). We checked the cell viability by pretreatment with melatonin 1nM to 100 nM. Cell viability in 1nM and 5nM melatonin pretreatment group was almost not different from the OGD/R injury exposed group. Treatment with 10 nM melatonin also did not change cell viability compared to the OGD/R injury exposed group (48% cell viability in the Mel 10 nM group). However, treatment with 100 nM melatonin obviously increased cell viability after OGD/Rinduced injury, compared to the normal control group (62% cell viability in the Mel 100 nM group) (Figure 1(a)). In addition, we evaluated cytotoxicity in bEND.3 cells following OGD/R injury using an LDH assay (Figure 1(b)). Cytotoxicity was 12% in the normal control group but was 28% in the OGD/R injury exposed group. Cytotoxicity in 1 nM and 5 nM melatonin pretreatment group was not largely different from the OGD/R injury exposed group. Treating cells with 10 nM melatonin resulted in 21% cytotoxicity and treating cells with 100 nM melatonin resulted in 18% cytotoxicity (Figure 1(b)). Considering cell viability and cytotoxicity data, we decided two concentrations of melatonin (10 nM melatonin concentration (among the low concentrations: 1 nM, 5 nM, and 10 nM) and 100 nM melatonin concentration (among the high concentrations: 50 nM, 100 nM)) to compare the effect of melatonin easily. We also conducted Hoechst/PI staining to check the dead cells in all groups (Figure 1(c)). Hoechst/PI staining images showed that only melatonin treatment groups were almost not different from the normal control group. PI-positive cells (dead cells) evidently were increased in the OGD/R injury exposed group, compared to the normal control group. 10 nM and 100 nM melatonin treatment promoted cell survival and inhibited cell death against OGD/R-induced injury. In the 100 nM melatonin treatment group, the protective effect of melatonin against OGD/R injury death in bEND.3 cells was more obvious than in the 10 nM melatonin treatment group (Figure 1(c)). Taken together, these findings suggest that melatonin attenuates OGD/R-induced damage in brain endothelial cells.

3.2. Melatonin Decreases OGD/R-Induced ROS Production. We measured ROS levels using DCF-DA reagent, a fluorescent dye that visualizes ROS. DCF-DA-positive cells increased after OGD/R. ROS levels in melatonin pretreatment groups (10 nM, 100 nM melatonin) were not largely different from ROS levels in the normal control group. In the OGD/R injury exposed group, ROS levels were evidently increased compared to the normal control group. This was partially blocked by pretreatment with 10 nM melatonin (Figures 2(a) and 2(b)). 100 nM melatonin pretreatment clearly decreased the number of DCF-DA-positive cells, compared to the OGD/R injury exposed group. This result suggests that melatonin inhibits OGD/R-induced ROS production in brain endothelial cells.

3.3. Melatonin Prevents Degradation of Tight Junction Proteins against OGD/R Injury. To check the protective effect of melatonin on the integrity of tight junctions during OGD/R, we measured the level of Claudin 5, a tight junction protein, by immunocytochemistry (Figure 3(a)) and western blot analysis (Figure 3(b)). OGD/R stress obviously decreased the expression of Claudin 5 in the bEND.3 cells compared to the normal control (NC) group. The expression of Claudin 5 did not nearly change in the 10 nM melatonin treatment group, compared to the experimental control (EC) group which in exposed OGD/R injury. The expression of Claudin 5 was evidently attenuated by treatment with 100 nM melatonin (Figures 3(a) and 3(b)). This result shows that melatonin pretreatment protects degradation of Claudin 5 following OGD/R injury. Namely, melatonin may prevent deterioration of tight junctions in response to OGD/R-induced injury.

3.4. Melatonin Attenuates the Expression of VEGF after OGD/R-Induced Injury. We conducted immunocytochemistry (Figures 4(a) and 4(b)) and western blot analysis (Figure 4(c)) to confirm the expression of VEGF in all treatment groups. This result indicated that the expression of VEGF became considerably elevated after OGD/R injury in the bEND.3 cells. However, the expression of VEGF was reduced by melatonin treatment (both 10 nM and 100 nM melatonin pretreatment) (Figures 4(a) and 4(b)). This finding suggests that melatonin attenuates the expression of VEGF in brain endothelial cells following OGD/R-induced injury.

3.5. Melatonin Protects bEND.3 Cells via Akt Activation and JNK Suppression. To investigate whether Akt signaling was activated in OGD/R-induced stress, we first measured the phosphorylation status of Akt by western blot analysis (Figure 5(a)). Phosphorylation of Akt is associated with activation of Akt signaling and cell survival. Our result suggests that the protein expression of phosphor-Akt/Akt in the EC group is attenuated compared to the NC group. Expression of phosphor-Akt in the 10 nM melatonin treatment group did not nearly change compared to the EC group. However, expression of phosphor-Akt in the 100 nM melatonin treatment group was higher than in the EC group (Figure 5(a)). Next, we also examined the phosphorylation status of JNK by western blot analysis (Figure 5(b)), because the phosphorylation of JNK correlates with activation of apoptosis signaling. The expression of phosphor-JNK was decreased by melatonin treatment after OGD/R-induced injury. Pretreatment with 100 nM melatonin resulted in the obvious inhibition of JNK signaling whereas JNK activation in 10 nM melatonin pretreatment group was not largely different from the EC group (Figure 5(b)). These results suggest that melatonin 100 nM increases Akt activation and suppresses JNK activation. To confirm the relationship between melatonin and Akt signaling, we checked the expression of Bax by western blot analysis (Figure 5(c)). We confirmed that the protein expression of Bax in the bEND.3 cells was increased under OGD/R injury compared to the NC group. Also, 10 nM and 100 nM melatonin treatment reduced the protein expression of Bax under OGD/R injury. When we checked the expression of Bax in OGD/R injured bEND.3 cells with Akt inhibitor and melatonin pretreatment, we confirmed that Akt inhibitor pretreatment did not reduce the expression of Bax in melatonin pretreatment groups (Figure 5(d)). These findings indicate that melatonin may promote Akt signaling and suppress JNK signaling. Specifically, melatonin may attenuate the expression of Bax, known as an apoptotic protein through Akt activation in brain endothelial cells following OGD/R stress.

4. Discussion

Ischemic stroke causes oxidative stress in the brain as well as various neuropathological impairments [60]. BBB disruption is commonly observed in stroke patients [61, 62]. BBB damage is aggravated by reperfusion after ischemia [63]. ROS are generated during cerebral ischemia-reperfusion injury and lead to severe brain damage by promoting the cell apoptosis pathway [64, 65]. Also, ROS cause BBB hyperpermeability, brain edema, hemorrhage, and inflammation [66]. In the present study, we induced OGD/R injury, which is known as an appropriate in vitro model of stroke [67, 68], in brain endothelial cells to investigate the effect of ischemia-reperfusion injury. Recent research suggests that antioxidants attenuate oxidative damage induced byischemia-reperfusion injury by decreasing mechanisms of ROS production [69]. Previous researches have suggested that antioxidants preserve BBB disruption and attenuate ROS generation after cerebral ischemia reperfusion in vivo [70-72] and in vitro [73, 74]. Melatonin is known as an antioxidant [75], a powerful free radical scavenger [76-78], and the cellular protector against various oxidative stress-associated diseases [79, 80]. Several studies in animals have suggested that melatonin reduces cellular damage by decreasing ROS in ischemia-reperfusion injury [46, 81, 82] and ischemia-hypoxia injury [83]. In the present study, we confirmed that melatonin reduces OGD/R-induced ROS generation in brain endothelial cells and prevents cell death of brain endothelial cells following OGD/R injury. Hypoxia causes degradation of tight junction proteins, such as Claudin 3, ZO-1 and ZO-2, and occludin [38, 39]. Several studies have demonstrated that claudins are major proteins in tight junctions [84-87], which are essential structural components of the BBB [34]. And, Claudin 5 is an important molecule that promotes disruption of the BBB in hypoxic conditions [88]. Tao et al. have demonstrated that melatonin prevents degradation of ZO-1, a tight junction protein that protects against ischemic injury in endothelial cells [89]. To determine the protective effect of melatonin on impaired BBB function caused by ischemia reperfusion, we examined Claudin 5 protein expression in brain endothelial cells following OGD/R injury. Our findings suggest that melatonin may prevent BBB disruption during ischemia-reperfusion injury by inhibiting degradation of the Claudin 5 tight junction protein. Hypoxia results in increased paracellular permeability [38, 90-92], leading to formation of cerebral edema [93]. Hypoxia induces the expression of VEGF [94-97], which is considered as one of the most important factors that stimulates the formation of new blood vessels [94, 95]. VEGF increases the permeability of blood vessels [92, 98, 99] and leads to vasogenic edema [100-103]. Several studies have demonstrated that VEGF increases BBB permeability [99], while inhibition of VEGF reduces BBB permeability [43]. Melatonin protects BBB hyperpermeability and reduces brain edema in ischemic stroke [104,105]. Also, recent research has shown that melatonin reduces expression of VEGF in hypoxic damage [53,106-108]. In the present study, our results showed that melatonin reduced the expression of VEGF in brain endothelial cells following OGD/R-induced injury. In oxidative stress, ROS acts as an important mediator to activate the MAPK pathway [23, 24]. The phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling pathway is considered to be one of the cell survival pathways [109]. Many researches have demonstrated that Akt plays a major role in protection from cell death under oxidative stress [110-115] and attenuates ROS production, which protects cells [116]. In brain endothelial cells, Akt enhances cell survival and inhibits apoptosis [117-119]. Melatonin promotes Akt signaling to protect cells in response to stress [120]. In the present study, our result showed that melatonin enhanced Akt activation following OGD/R injury. This finding may indicate that melatonin protects brain endothelial cells via Akt activation in the setting of ischemia-reperfusion injury. In addition, Akt can protect cellular apoptosis by regulating a proapoptotic protein such as Bax [121-124]. Several studies demonstrated that melatonin may regulate the Bax expression and may be involved in the apoptosis signaling [125, 126]. In the present study, our results showed that melatonin may regulate the Bax expression through regulating Akt activation. Considering that Bax is the proapoptotic protein, melatonin may protect the apoptosis of brain endothelial cells through suppressing the expression of Bax in response to hypoxia and reperfusion stress. JNK signaling contributes to cellular apoptosis triggered by various stresses, including oxidized LDL, proinflammatory cytokines, or high glucose [127-129]. Specifically, excessive ROS generation is closely linked to JNK activation [130-132]. JNK activation triggers the mitochondrial apoptotic pathway [133, 134] and disrupts the BBB [135]. Several studies have shown that JNK inhibitors exert protective effects against ischemic injury in a rodent model [136-139]. In the present study, our findings suggest that melatonin attenuates JNK activation in OGD/R-exposed brain endothelial cells. This result indicates that melatonin may inhibit the death of brain endothelial cells via JNK suppression. In conclusion, melatonin protects brain endothelial cells against ischemic-reperfusion injury by reducing the production of ROS, by preserving tight junction proteins, by attenuating expression ofVEGF, and by regulating Akt activation and JNK suppression. Hence, this study suggests that melatonin may play as the protector on brain endothelial cells under brain hypoxic injury such as stroke. For application to the patients with stroke, this study has many limitations because of confirmation only in vitro study. However, these findings may provide the basic data for the further study on stroke.

http://dx.doi.org/10.1155/2014/639531

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF), which is funded by the Ministry of Science, ICT & Future Planning (2012-0005827).

References

[1] R. W. V. Flynn, R. S. M. MacWalter, and A. S. F. Doney, "The cost of cerebral ischaemia," Neuropharmacology, vol. 55, no. 3, pp. 250-256, 2008.

[2] K. P. Doyle, R. P. Simon, and M. P. Stenzel-Poore, "Mechanisms of ischemic brain damage," Neuropharmacology, vol. 55, no. 3, pp. 310-318, 2008.

[3] S. Kuroda and B. K. Siesjo, "Reperfusion damage following focal ischemia: pathophysiology and therapeutic windows," Clinical Neuroscience, vol. 4, no. 4, pp. 199-212, 1997

[4] T. Nakamura, H. Minamisawa, Y. Katayama et al., "Increased intracellular [Ca.sup.2+] concentration in the hippocampal CA1 area during global ischemia and reperfusion in the rat: a possible cause of delayed neuronal death," Neuroscience, vol. 88, no. 1, pp. 57-67, 1999.

[5] N. J. Abbott, A. A. K. Patabendige, D. E. M. Dolman, S. R. Yusof, and D. J. Begley, "Structure and function of the blood-brain barrier," Neurobiology of Disease, vol. 37, no. 1, pp. 13-25, 2010.

[6] J. Lee, M. C. Grabb, G. J. Zipfel, and D. W. Choi, "Brain tissue responses to ischemia," The Journal of Clinical Investigation, vol. 106, no. 6, pp. 723-731, 2000.

[7] J. P. MacManus, A. M. Buchan, I. E. Hill, I. Rasquinha, and E. Preston, "Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain," Neuroscience Letters, vol. 164, no. 1-2, pp. 89-92, 1993.

[8] S. Kihara, T. Shiraishi, S. Nakagawa, K. Toda, and K. Tabuchi, "Visualization of DNA double strand breaks in the gerbil hippocampal CA1 following transient ischemia," Neuroscience Letters, vol. 175, no. 1-2, pp. 133-136, 1994.

[9] G. M. Yan, R. P. Irwin, S. Z. Lin, M. Weller, K. A. Wood, and S. M. Paul, "Diphenylhydantoin induces apoptotic cell death of cultured rat cerebellar granule neurons," Journal of Pharmacology and Experimental Therapeutics, vol. 274, no. 2, pp. 983-990, 1995.

[10] F. C. Wilson, J. Harpur, T. Watson, and J. I. Morrow, "Adult survivors of severe cerebral hypoxia--case series survey and comparative analysis," NeuroRehabilitation, vol. 18, no. 4, pp. 291-298, 2003.

[11] H.-L. Hsieh and C.-M. Yang, "Role of redox signaling in neuroinflammation and neurodegenerative diseases," BioMed Research International, vol. 2013, Article ID 484613, 18 pages, 2013.

[12] J. Li, O. Wuliji, W. Li, Z. G. Jiang, and H. A. Ghanbari, "Oxidative stress and neurodegenerative disorders," International Journal of Molecular Sciences, vol. 14, no. 12, pp. 24438-24475, 2013.

[13] X. Wang, W. Wang, L. Li, G. Perry, H. G. Lee, and X. Zhu, "Oxidative stress and mitochondrial dysfunction in Alzheimer's disease," Biochimica et Biophysica Acta, vol. 1842, no. 8, pp. 1240-1247, 2013.

[14] M. H. Yan, X. Wang, and X. Zhu, "Mitochondrial defects and oxidative stress in Alzheimer disease and Parkinson disease," Free Radical Biology and Medicine, vol. 62, pp. 90-101, 2013.

[15] W. Paschen, "Role of calcium in neuronal cell injury: which subcellular compartment is involved?" Brain Research Bulletin, vol. 53, no. 4, pp. 409-413, 2000.

[16] P. H. Chan, "Reactive oxygen radicals in signaling and damage in the ischemic brain," Journal of Cerebral Blood Flow and Metabolism, vol. 21, no. 1, pp. 2-14, 2001.

[17] C. Iadecola and M. Alexander, "Cerebral ischemia and inflammation," Current Opinion in Neurology, vol. 14, no. 1, pp. 89-94, 2001.

[18] M. A. Petty and J. G. Wettstein, "Elements of cerebral microvascular ischaemia," Brain Research Reviews, vol. 36, no. 1, pp. 23-34, 2001.

[19] H. S. Noh, Y. Hah, R. Nilufar et al., "Acetoacetate protects neuronal cells from oxidative glutamate toxicity," Journal of Neuroscience Research, vol. 83, no. 4, pp. 702-709, 2006.

[20] B. Halliwell, "Free radicals, antioxidants, and human disease: curiosity, cause, or consequence?" The Lancet, vol. 344, no. 8924, pp. 721-724, 1994.

[21] T. Sugawara and P. H. Chan, "Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia," Antioxidants and Redox Signaling, vol. 5, no. 5, pp. 597-607, 2003.

[22] T. M. Millar, V. Phan, and L. A. Tibbles, "ROS generation in endothelial hypoxia and reoxygenation stimulates MAP kinase signaling and kinase-dependent neutrophil recruitment," Free Radical Biology and Medicine, vol. 42, no. 8, pp. 1165-1177, 2007

[23] J. A. McCubrey, M. M. LaHair, and R. A. Franklin, "Reactive oxygen species-induced activation of the MAP kinase signaling pathways," Antioxidants and Redox Signaling, vol. 8, no. 9-10, pp. 1775-1789, 2006.

[24] M. Torres and H. J. Forman, "Redox signaling and the MAP kinase pathways," BioFactors, vol. 17, no. 1-4, pp. 287-296,2003.

[25] M. A. Bogoyevitch, J. Gillespie-Brown, A. J. Ketterman et al., "Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart: p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion," Circulation Research, vol. 79, no. 2, pp. 162-173, 1996.

[26] K. R. Laderoute and K. A. Webster, "Hypoxia/reoxygenation stimulates Jun kinase activity through redox signaling in cardiac myocytes," Circulation Research, vol. 80, no. 3, pp. 336-344, 1997

[27] Z. Chen, T. B. Gibson, F. Robinson et al., "MAP kinases," Chemical Reviews, vol. 101, no. 8, pp. 2449-2476, 2001.

[28] J. M. Kyriakis and J. Avruch, "Sounding the alarm: protein kinase cascades activated by stress and inflammation," The Journal of Biological Chemistry, vol. 271, no. 40, pp. 24313-24316, 1996.

[29] P. Rockwell, J. Martinez, L. Papa, and E. Gomes, "Redox regulates COX-2 upregulation and cell death in the neuronal response to cadmium," Cellular Signalling, vol. 16, no. 3, pp. 343-353, 2004.

[30] D. J. Li, T. Zhao, R. J. Xin, Y. Y. Wang, Y. B. Fei, and F. M. Shen, "Activation of alpha7 nicotinic acetylcholine receptor protects against oxidant stress damage through reducing vascular peroxidase-1 in a JNK signaling-dependent manner in endothelial cells," Cellular Physiology and Biochemistry, vol. 33, no. 2, pp. 468-478, 2014.

[31] S. J. Harper and P. Lograsso, "Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38," Cellular Signalling, vol. 13, no. 5, pp. 299-310, 2001.

[32] K. Nozaki, M. Nishimura, and N. Hashimoto, "Mitogen-activated protein kinases and cerebral ischemia," Molecular Neurobiology, vol. 23, no. 1, pp. 1-19, 2001.

[33] N. J. Abbott, "Astrocyte-endothelial interactions and blood-brain barrier permeability," Journal of Anatomy, vol. 200, no. 6, pp. 629-638, 2002.

[34] P. Ballabh, A. Braun, and M. Nedergaard, "The blood-brain barrier: an overview: Structure, regulation, and clinical implications," Neurobiology of Disease, vol. 16, no. 1, pp. 1-13, 2004.

[35] M. Furuse, T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, and S. Tsukita, "Occludin: a novel integral membrane protein localizing at tight junctions," Journal of Cell Biology, vol. 123, no. 6, pp. 1777-1788, 1993.

[36] M. Furuse, K. Fujita, T. Hiiragi, K. Fujimoto, and S. Tsukita, "Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin," Journal of Cell Biology, vol. 141, no. 7, pp. 1539-1550, 1998.

[37] M. Furuse, H. Sasaki, and S. Tsukita, "Manner of interaction of heterogeneous claudin species within and between tight junction strands," Journal of Cell Biology, vol. 147, no. 4, pp. 891-903, 1999.

[38] K. S. Mark and T. P. Davis, "Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation," American Journal of Physiology: Heart and Circulatory Physiology, vol. 282, no. 4, pp. H1485-H1494, 2002.

[39] K. A. Witt, K. S. Mark, S. Hom, and T. P. Davis, "Effects of hypoxia-reoxygenation on rat blood-brain barrier permeability and tight junctional protein expression," American Journal of Physiology--Heart and Circulatory Physiology, vol. 285, no. 6, pp. H2820-H2831, 2003.

[40] K. S. Mark, A. R. Burroughs, R. C. Brown, J. D. Huber, and T. P. Davis, "Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature," The American Journal of Physiology: Heart and Circulatory Physiology, vol. 286, no. 1, pp. H174-H180, 2004.

[41] S. D. Croll, J. H. Goodman, and H. E. Scharfman, "Vascular Endothelial Growth Factor (VEGF) in seizures: a double-edged sword," Advances in Experimental Medicine and Biology, vol. 548, pp. 57-68, 2004.

[42] D. H. Dobrogowska, A. S. Lossinsky, M. Tarnawski, and A. W. Vorbrodt, "Increased blood-brain barrier permeability and endothelial abnormalities induced by vascular endothelial growth factor," Brain Cell Biology, vol. 27, no. 3, pp. 163-173, 1998.

[43] Z. G. Zhang, L. Zhang, Q. Jiang et al., "VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain," Journal of Clinical Investigation, vol. 106, no. 7, pp. 829-838, 2000.

[44] H. F. Dvorak, J. A. Nagy, D. Feng et al., "Vascular permeability factor/vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis," Current Topics in Microbiology and Immunology, vol. 237, pp. 97-132, 1999.

[45] B. Poeggeler, S. Saarela, R. J. Reiter et al., "Melatonin--a highly potent endogenous radical scavenger and electron donor: new aspects of the oxidation chemistry of this indole accessed in vitro," Annals of the New York Academy of Sciences, vol. 738, pp. 419-420, 1994.

[46] F. Dupuis, O. Regrigny, J. Atkinson et al., "Impact of treatment with melatonin on cerebral circulation in old rats," The British Journal of Pharmacology, vol. 141, no. 3, pp. 399-406, 2004.

[47] A. Wakatsuki, Y. Okatani, C. Izumiya, and N. Ikenoue, "Melatonin protects against ischemia and reperfusion-induced oxidative lipid and DNA damage in fetal rat brain," Journal of Pineal Research, vol. 26, no. 3, pp. 147-152, 1999.

[48] M. D. Ginsberg and R. Busto, "Rodent models of cerebral ischemia," Stroke, vol. 20, no. 12, pp. 1627-1642,1989.

[49] C. Harms, M. Lautenschlager, A. Bergk et al., "Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures," The FASEB Journal, vol. 14, no. 12, pp. 1814-1824, 2000.

[50] Z. Pei and R. T. F. Cheung, "Melatonin protects SHSY5Y neuronal cells but not cultured astrocytes from ischemia due to oxygen and glucose deprivation," Journal of Pineal Research, vol. 34, no. 3, pp. 194-201, 2003.

[51] D. Lv, P. Cui, S. Yao, Y. Xu, and Z. Yang, "Melatonin inhibits the expression of vascular endothelial growth factor in pancreatic cancer cells," Chinese Journal of Cancer Research, vol. 24, no. 4, pp. 310-316, 2012.

[52] M. Dai, P. Cui, M. Yu, J. Han, H. Li, and R. Xiu, "Melatonin modulates the expression of VEGF and HIF-1[alpha] induced by Co[Cl.sub.2] in cultured cancer cells," Journal of Pineal Research, vol. 44, no. 2, pp. 121-126, 2008.

[53] P. Lissoni, F. Rovelli, F. Malugani, R. Bucovec, A. Conti, and G. J. M. Maestroni, "Anti-angiogenic activity of melatonin in advanced cancer patients," Neuroendocrinology Letters, vol. 22, no. 1, pp. 45-47, 2001.

[54] H.-J. Jung, Y.-H. Jeon, K. K. Bokara et al., "Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways," Life Sciences, vol. 92, no. 1, pp. 42-50,2013.

[55] Z. Y. Mei, C. M. Chin, J. C. Yoon et al., "Agmatine inhibits matrix metalloproteinase-9 via endothelial nitric oxide synthase in cerebral endothelial cells," Neurological Research, vol. 29, no. 7, pp. 749-754, 2007.

[56] K. K. Bokara, K. H. Kwon, Y. Nho, W. T. Lee, K. A. Park, and J. E. Lee, "Retroviral expression of arginine decarboxylase attenuates oxidative burden in mouse cortical neural stem cells," Stem Cells and Development, vol. 20, no. 3, pp. 527-537, 2011.

[57] S. Hong, C. Y. Kim, J. E. Lee, and G. J. Seong, "Agmatine protects cultured retinal ganglion cells from tumor necrosis factor-alpha-induced apoptosis," Life Sciences, vol. 84, no. 1-2, pp. 28-32, 2009.

[58] S. K. Ahn, S. Hong, Y. M. Park, W. T. Lee, K. A. Park, and J. E. Lee, "Effects of agmatine on hypoxic microglia and activity of nitric oxide synthase," Brain Research, vol. 1373, pp. 48-54,2011.

[59] J. A. Royall and H. Ischiropoulos, "Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2[O.sub.2] in cultured endothelial cells," Archives of Biochemistry and Biophysics, vol. 302, no. 2, pp. 348-355, 1993.

[60] C. Nyakas, B. Buwalda, and P. G. M. Luiten, "Hypoxia and brain development," Progress in Neurobiology, vol. 49, no. 1, pp. 1-51, 1996.

[61] J. Zhou, H. Kong, X. Hua, M. Xiao, J. Ding, and G. Hu, "Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice," NeuroReport, vol. 19, no. 1, pp. 1-5, 2008.

[62] L. L. Rubin and J. M. Staddon, "The cell biology of the blood-brain barrier," Annual Review of Neuroscience, vol. 22, pp. 11-28, 1999.

[63] G. J. del Zoppo, R. von Kummer, and G. F. Hamann, "Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke," Journal of Neurology Neurosurgery and Psychiatry, vol. 65, no. 1, pp. 1-9, 1998.

[64] R. Hardeland, "Antioxidative protection by melatonin: multiplicity of mechanisms from radical detoxification to radical avoidance," Endocrine, vol. 27, no. 2, pp. 119-130, 2005.

[65] G. Wu, R. L. Xue, J. R. Lv, W. Li, and X. M. Lei, "Fas and TNFR1 expressions after cerebral ischemia and reperfusion in rats: association with cell apoptosis and the effects of Bcl-2 overexpression," Journal of Southern Medical University, vol. 31, no. 8, pp. 1298-1303, 2011.

[66] V. Li, T. Brustovetsky, and N. Brustovetsky, "Role of cyclophilin D-dependent mitochondrial permeability transition in glutamate-induced calcium deregulation and excitotoxic neuronal death," Experimental Neurology, vol. 218, no. 2, pp. 171-182, 2009.

[67] P. Lipton, "Ischemic cell death in brain neurons," Physiological Reviews, vol. 79, no. 4, pp. 1431-1568,1999.

[68] A. V. Andjelkovic, S. M. Stamatovic, and R. F. Keep, "The protective effects of preconditioning on cerebral endothelial cells in vitro," Journal of Cerebral Blood Flow and Metabolism, vol. 23, no. 11, pp. 1348-1355, 2003.

[69] M. I. Cervantes, P. M. de Oca Balderas, J. de Jesus GutierrezBanos et al., "Comparison of antioxidant activity of hydroethanolic fresh and aged garlic extracts and their effects on cerebral ischemia," Food Chemistry, vol. 140, no. 1-2, pp. 343352, 2013.

[70] X.-C. Wang, Y.-C. Zhang, N. Chatterjie, I. Grundke-Iqbal, K. Iqbal, and J. Wang, "Effect of melatonin and melatonyl-valpromide on [beta]-amyloid and neurofilaments in N2a cells," Neurochemical Research, vol. 33, no. 6, pp. 1138-1144, 2008.

[71] J. Li, H. Yin, X. Gu, Y. Zhou, W. Zhang, and Y. Qin, "Melatonin protects liver from intestine ischemia reperfusion injury in rats," World Journal of Gastroenterology, vol. 14, no. 48, pp. 7392-7396, 2008.

[72] J. Wang, J. Shen, Q. Gao et al., "Ischemic postconditioning protects against global cerebral ischemia/reperfusion-induced injury in rats," Stroke, vol. 39, no. 3, pp. 983-990, 2008.

[73] Y. C. Li, H. J. Lin, J. H. Yang et al., "Baicalein-induced apoptosis via endoplasmic reticulum stress through elevations of reactive oxygen species and mitochondria dependent pathway in mouse-rat hybrid retina ganglion cells (N18)," Neurochemical Research, vol. 34, no. 3, pp. 418-429, 2009.

[74] S. Ozbal, B. U. Ergur, G. Erbil, I. Tekmen, A. Bagryank, and Z. Cavdar, "The effects of [alpha]-lipoic acid against testicular ischemia- reperfusion injury in rats," The Scientific World Journal, vol. 2012, Article ID 489248, 8 pages, 2012.

[75] R. J. Reiter, "Antioxidant actions of melatonin.," Advances in pharmacology, vol. 38, pp. 103-117, 1997

[76] F. Tutunculer, S. Eskiocak, U. N. Basaran, G. Ekuklu, S. Ayvaz, and U. Vatansever, "The protective role of melatonin in experimental hypoxic brain damage," Pediatrics International, vol. 47, no. 4, pp. 434-439, 2005.

[77] D. Tan, R. J. Reiter, L. C. Manchester et al., "Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger," Current Topics in Medicinal Chemistry, vol. 2, no. 2, pp. 181-197, 2002.

[78] O. Celik and M. Naziroglu, "Melatonin modulates apoptosis and TRPM2 channels in transfected cells activated by oxidative stress," Physiology & Behavior, vol. 107, no. 3, pp. 458-465, 2012.

[79] P. D. Ray, B. W. Huang, and Y. Tsuji, "Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling," Cellular Signalling, vol. 24, no. 5, pp. 981-990, 2012.

[80] E. Kubo, N. Miyoshi, M. Fukuda, and Y. Akagi, "Cataract formation through the polyol pathway is associated with free radical production," Experimental Eye Research, vol. 68, no. 4, pp. 457-464,1999.

[81] Y. J. Lim, S. Zheng, and Z. Zuo, "Morphine preconditions purkinje cells against cell death under in vitro simulated ischemia-reperfusion conditions," Anesthesiology, vol. 100, no. 3, pp. 562-568, 2004.

[82] M. I. C. Alonso-Vale, S. Andreotti, S. B. Peres et al., "Melatonin enhances leptin expression by rat adipocytes in the presence of insulin," American Journal of Physiology-Endocrinology and Metabolism, vol. 288, no. 4, pp. E805-E812, 2005.

[83] R. E. Hartman, J. M. Lee, G. J. Zipfel, and D. F. Wozniak, "Characterizing learning deficits and hippocampal neuron loss following transient global cerebral ischemia in rats," Brain Research, vol. 1043, no. 1-2, pp. 48-56, 2005.

[84] K. Morita, H. Sasaki, K. Fujimoto, M. Furuse, and S. Tsukita, "Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis," Journal of Cell Biology, vol. 145, no. 3, pp. 579-588,1999.

[85] S. Liebner, A. Fischmann, G. Rascher et al., "Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme," Acta Neuropathologica, vol. 100, no. 3, pp. 323-331, 2000.

[86] S. Liebner, U. Kniesel, H. Kalbacher, and H. Wolburg, "Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells," European Journal of Cell Biology, vol. 79, no. 10, pp. 707-717, 2000.

[87] A. Lippoldt, U. Kniesel, S. Liebner et al., "Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood-brain barrier endothelial cells," Brain Research, vol. 885, no. 2, pp. 251-261, 2000.

[88] T. Koto, K. Takubo, S. Ishida et al., "Hypoxia disrupts the barrier function of neural blood vessels through changes in the expression of claudin-5 in endothelial cells," The American Journal of Pathology, vol. 170, no. 4, pp. 1389-1397, 2007.

[89] R. Tao, J. Huang, X. Shao et al., "Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin," Journal of Pineal Research, vol. 54, no. 3, pp. 271-281, 2013.

[90] T. J. Abbruscato and T. P. Davis, "Protein expression of brain endothelial cell E-cadherin after hypoxia/aglycemia: influence of astrocyte contact," Brain Research, vol. 842, no. 2, pp. 277-286, 1999.

[91] C. Chryssanthou, T. Palaia, G. Goldstein, and R. Stenger, "Increase in blood-brain barrier permeability by altitude decompression," Aviation Space and Environmental Medicine, vol. 58, no. 11, pp. 1082-1086, 1987

[92] H. J. Schoch, S. Fischer, and H. H. Marti, "Hypoxia-induced vascular endothelial growth factor expression causes vascular leakage in the brain," Brain, vol. 125, part11, pp. 2549-2557,2002.

[93] K. Hayashi, S. Nakao, R. Nakaoke, S. Nakagawa, N. Kitagawa, and M. Niwa, "Effects of hypoxia on endothelial/pericytic co-culture model of the blood-brain barrier," Regulatory Peptides, vol. 123, no. 1-3, pp. 77-83, 2004.

[94] E. J. Battegay, "Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects," Journal of Molecular Medicine, vol. 73, no. 7, pp. 333-346, 1995.

[95] M. Milkiewicz, E. Ispanovic, J. L. Doyle, and T. L. Haas, "Regulators of angiogenesis and strategies for their therapeutic manipulation," International Journal of Biochemistry and Cell Biology, vol. 38, no. 3, pp. 333-357, 2006.

[96] G. L. Semenza, "Hydroxylation of HIF-1: oxygen sensing at the molecular level," Physiology, vol. 19, no. 4, pp. 176-182, 2004.

[97] J. Josko and M. Mazurek, "Transcription factors having impact on vascular endothelial growth factor (VEGF) gene expression in angiogenesis," Medical Science Monitor, vol. 10, no. 4, pp. RA89-RA98, 2004.

[98] W. G. Mayhan, "VEGF increases permeability of the blood-brain barrier via a nitric oxide synthase/cGMP-dependent pathway," American Journal of Physiology--Cell Physiology, vol. 276, no. 5, part 1, pp. C1148-C1153,1999.

[99] P. P. Young, C. R. Fantz, and M. S. Sands, "VEGF disrupts the neonatal blood-brain barrier and increases life span after non-ablative BMT in a murine model of congenital neurodegeneration caused by a lysosomal enzyme deficiency," Experimental Neurology, vol. 188, no. 1, pp. 104-114, 2004.

[100] P. Pichiule, J. C. Chavez, K. Xu, and J. C. Lamanna, "Vascular endothelial growth factor upregulation in transient global ischemia induced by cardiac arrest and resuscitation in rat brain," Molecular Brain Research, vol. 74, no. 1-2, pp. 83-90, 1999.

[101] A. D. Sinor, S. M. Irvin, C. S. Cobbs, J. Chen, S. H. Graham, and D. A. Greenberg, "Hypoxic induction of vascular endothelial growth factor (VEGF) protein in astroglial cultures," Brain Research, vol. 812, no. 1-2, pp. 289-291, 1998.

[102] R. Walter, M. Maggiorini, U. Scherrer, J. Contesse, and W. H. Reinhart, "Effects of high-altitude exposure on vascular endothelial growth factor levels in man," European Journal of Applied Physiology, vol. 85, no. 1-2, pp. 113-117, 2001.

[103] D. O. Bates and S. J. Harper, "Regulation of vascular permeability by vascular endothelial growth factors," Vascular Pharmacology, vol. 39, no. 4-5, pp. 225-237, 2002.

[104] M. Ersahin, H. Z. Toklu, S. Cetinel, M. YUksel, B. C. Yeen, and G. Sener, "Melatonin reduces experimental subarachnoid hemorrhage-induced oxidative brain damage and neurological symptoms," Journal of Pineal Research, vol. 46, no. 3, pp. 324-332, 2009.

[105] H.-Y. Chen, T.-Y. Chen, M.-Y. Lee et al., "Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood-brain barrier permeability after transient focal cerebral ischemia in mice," Journal of Pineal Research, vol. 41, no. 2, pp. 175-182, 2006.

[106] C. Kaur, V. Sivakumar, J. Lu, F. R. Tang, and E. A. Ling, "Melatonin attenuates hypoxia-induced ultrastructural changes and increased vascular permeability in the developing hippocampus," Brain Pathology, vol. 18, no. 4, pp. 533-547, 2008.

[107] Y. Zhang, Q. Liu, F. Wang et al., "Melatonin antagonizes hypoxia-mediated glioblastoma cell migration and invasion via inhibition of HIF-1[alpha]," Journal of Pineal Research, vol. 55, no. 2, pp. 121-130, 2013.

[108] C. Kaur, V. Sivakumar, J. Lu, and E. A. Ling, "Increased vascular permeability and nitric oxide production in response to hypoxia in the pineal gland," Journal of Pineal Research, vol. 42, no. 4, pp. 338-349, 2007

[109] S. Maddika, S. R. Ande, S. Panigrahi et al., "Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy," Drug Resistance Updates, vol. 10, no. 1-2, pp. 13-29, 2007

[110] B. Wang, J. Shravah, H. Luo, K. Raedschelders, D. D. Y. Chen, and D. M. Ansley, "Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation," Biochemical and Biophysical Research Communications, vol. 389, no. 1, pp. 105-111, 2009.

[111] R. Kimura, M. Okouchi, H. Fujioka et al., "Glucagon-like peptide-1 (GLP-1) protects against methylglyoxal-induced PC12 cell apoptosis through the PI3K/Akt/mTOR/GCLc/redox signaling pathway," Neuroscience, vol. 162, no. 4, pp. 1212-1219, 2009.

[112] S. Lee, J. Shin, Y. Hong et al., "Beneficial effects of melatonin on stroke-induced muscle atrophy in focal cerebral ischemic rats," Laboratory Animal Research, vol. 28, no. 1, pp. 47-54, 2012.

[113] T. Jiang, Q. Chang, Z. Zhao et al., "Melatonin-mediated cyto-protection against hyperglycemic injury in Muller cells," PLoS ONE, vol. 7, no. 12, Article ID e50661, 2012.

[114] S. H. Lee, W. Chun, P. J. Kong et al., "Sustained activation of Akt by melatonin contributes to the protection against kainic acid-induced neuronal death in hippocampus," Journal of Pineal Research, vol. 40, no. 1, pp. 79-85, 2006.

[115] P. Kong, J. Byun, S. Lim et al., "Melatonin induees Akt phosphorylation through Melatonin receptor- and PI3K-dependent pathways in primary astrocytes," Korean Journal of Physiology and Pharmacology, vol. 12, no. 2, pp. 37-41, 2008.

[116] B. D. Manning and L. C. Cantley, "AKT/PKB signaling: navigating downstream," Cell, vol. 129, no. 7, pp. 1261-1274, 2007

[117] H. Zhou, L. Liu, Y. Zhang et al., "Glutathione prevents free fatty acids-induced oxidative stress and apoptosis in human brain vascular endothelial cells through Akt pathway," CNS Neuroscience and Therapeutics, vol. 19, no. 4, pp. 252-261, 2013.

[118] I. Shiojima and K. Walsh, "Role of Akt signaling in vascular homeostasis and angiogenesis," Circulation Research, vol. 90, no. 12, pp. 1243-1250, 2002.

[119] G. Song, G. Ouyang, and S. Bao, "The activation of Akt/PKB signaling pathway and cell survival," Journal of Cellular and Molecular Medicine, vol. 9, no. 1, pp. 59-71, 2005.

[120] J. Bai, L. Dong, Z. Song et al., "The role of melatonin as an antioxidant in human lens epithelial cells," Free Radical Research, vol. 47, no. 8, pp. 635-642, 2013.

[121] Y. Zhang, X. Wang, H. Yang et al., "Kinase AKT controls innate immune cell development and function," Immunology, vol. 140, no. 2, pp. 143-152, 2013.

[122] B. A. Teicher, "Molecular targets and cancer therapeutics: discovery, development and clinical validation," Drug Resistance Updates, vol. 3, no. 2, pp. 67-73, 2000.

[123] Y. Huang, D. Wu, and W. Fan, "Protection of ginsenoside Rg1 on chondrocyte from IL-1beta-induced mitochondria-activated apoptosis through PI3K/Akt signaling," Molecular and Cellular Biochemistry, vol. 392, no. 1-2, pp. 249-257, 2014.

[124] X. Zhang, L. Bi, Y. Ye, and J. Chen, "Formononetin induces apoptosis in PC-3 prostate cancer cells through enhancing the Bax/Bcl-2 ratios and regulatingthe p38/Akt pathway," Nutrition and Cancer, vol. 66, no. 4, pp. 656-661, 2014.

[125] C. Xu, A. Wu, H. Zhu et al., "Melatonin is involved in the apoptosis and necrosis of pancreatic cancer cell line SW-1990 via modulating of Bcl-2/Bax balance," Biomedicine and Pharmacotherapy, vol. 67, no. 2, pp. 133-139, 2013.

[126] M. Mohseni, E. Mihandoost, A. Shirazi, Z. Sepehrizadeh, J. T. Bazzaz, and M. Ghazi-Khansari, "Melatonin may play a role in modulation of bax and bcl-2 expression levels to protect rat peripheral blood lymphocytes from gamma irradiation-induced apoptosis," Mutation Research--Fundamental and Molecular Mechanisms of Mutagenesis, vol. 738-739, no. 1, pp. 19-27, 2012.

[127] F. M. Ho, S. H. Liu, C. S. Liau, P. J. Huang, and S. Y. Lin-Shiau, "High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-JUN NH2-terminal kinase and caspase-3," Circulation, vol. 101, no. 22, pp. 2618-2624, 2000.

[128] W. Takabe, R. Li, L. Ai, F. Yu, J. A. Berliner, and T. K. Hsiai, "Oxidized low-density lipoprotein-activated c-Jun NH2-terminal kinase regulates manganese superoxide dismutase ubiquitination: implication for mitochondrial redox status and apoptosis," Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 30, no. 3, pp. 436-441, 2010.

[129] G. Garin, J. Abe, A. Mohan et al., "Flow antagonizes TNF-[alpha] signaling in endothelial cells by inhibiting caspase-dependent PKC[zeta] processing," Circulation Research, vol. 101, no. 1, pp. 97-105, 2007.

[130] H. M. Shen and Z. G. Liu, "JNK signaling pathway is a key modulator in cell death mediated by reactive oxygen and nitrogen species," FreeRadical Biology and Medicine, vol. 40, no. 6, pp. 928-939, 2006.

[131] B. Cai, X. Li, Y. Wang et al., "Apoptosis of bone marrow mesenchymal stem cells caused by homocysteine via activating jnk signal," PLoS ONE, vol. 8, no. 5, Article ID e63561, 2013.

[132] W. S. Choi, S. Y. Yoon, T. H. Oh, E. J. Choi, K. L. O'Malley, and Y. J. Oh, "Two distinct mechanisms are involved in 6-hydroxydopamineand MPP+-induced dopaminergic neuronal cell death: role of caspases, ROS, and JNK," Journal of Neuroscience Research, vol. 57, no. 1, pp. 86-94,1999.

[133] K. Chang, C. Hsu, S. Liu et al., "Cadmium induces apoptosis in pancreatic beta-cells through a mitochondria-dependent pathway: the role of oxidative stress-mediated c-Jun N-terminal kinase activation," PLoS ONE, vol. 8, no. 2, Article ID e54374, 2013.

[134] E. Shaulian and M. Karin, "AP-1 as a regulator of cell life and death," Nature Cell Biology, vol. 4, no. 5, pp. E131-E136, 2002.

[135] G. A. Rosenberg, "Matrix metalloproteinases in neuroinflammation," Glia, vol. 39, no. 3, pp. 279-291, 2002.

[136] Y. Gao, A. P. Signore, W. Yin et al., "Neuroprotection against focal ischemic brain injury by inhibition of c-Jun N-terminal kinase and attenuation of the mitochondrial apoptosis-signaling pathway," Journal of Cerebral Blood Flow and Metabolism, vol. 25, no. 6, pp. 694-712, 2005.

[137] Q.-H. Guan, D.-S. Pei, Y.-Y. Zong, T.-L. Xu, and G.-Y. Zhang, "Neuroprotection against ischemic brain injury by a small peptide inhibitor of c-Jun N-terminal kinase (JNK) via nuclear and non-nuclear pathways," Neuroscience, vol. 139, no. 2, pp. 609-627, 2006.

[138] Q. H. Guan, D. S. Pei, X. M. Liu, X. T. Wang, T. L. Xu, and G. Y. Zhang, "Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis," Brain Research, vol. 1092, no. 1, pp. 36-46, 2006.

[139] T. Borsellol, P. G. H. Clarkel, L. Hirt et al., "A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia," Nature Medicine, vol. 9, no. 9, pp. 1180-1186, 2003.

Juhyun Song, (1) So Mang Kang, (1,2) Won Taek Lee, (1) Kyung Ah Park, (1) Kyoung Min Lee, (3) and Jong Eun Lee (1,2)

(1) Department of Anatomy, Yonsei University College of Medicine, Seoul, Republic of Korea

(2) BK21 Plus Project for Medical Sciences and Brain Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea

(3) Department of Neurology, Seoul National University College of Medicine, Seoul, Republic of Korea

Correspondence should be addressed to Jong Eun Lee; jelee@yuhs.ac

Received 25 March 2014; Revised 7 June 2014; Accepted 20 June 2014; Published 14 July 2014

Academic Editor: Honglian Shi
COPYRIGHT 2014 Hindawi Limited
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Research Article
Author:Song, Juhyun; Kang, So Mang; Lee, Won Taek; Park, Kyung Ah; Lee, Kyoung Min; Lee, Jong Eun
Publication:Oxidative Medicine and Cellular Longevity
Article Type:Report
Date:Jan 1, 2014
Words:8602
Previous Article:Oxidative stress and histological changes in a model of retinal phototoxicity in rabbits.
Next Article:Dysregulation of histone acetyltransferases and deacetylases in cardiovascular diseases.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters