Original research paper
3′,4′-Dihydroxyflavonol attenuates spatial learning and memory impairments in global cerebral ischemia
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Mehmet Oz1 , Enver Ahmet Demir 1, Merve Caliskan 1, Rasim Mogulkoc 1, Abdulkerim Kasım Baltaci 1, K. Esra Nurullahoglu Atalik 2 1
Department of Physiology, Faculty of Medicine, University of Selcuk, Konya, Turkey, 2Department of Pharmacology, Faculty of Meram Medicine, University of Necmettin Erbakan, Konya, Turkey
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Objectives: In the present study, effects of 3′ ,4′ -dihydroxyflavonol (DiOHF) on anxiety-like behavior, and learning and memory were investigated in a model of transient global cerebral ischemia and reperfusion. Methods: The animals were assigned to sham-operated, ischemia, and two DiOHF-treated (10 mg/kg i.p.) groups. DiOHF was administered at 1 hour before and immediately after the ischemia. Male rats were subjected to bilateral common carotid artery occlusion to induce acute cerebral ischemia for 20 minutes, followed by reperfusion for 7 days. The openfield, elevated plus maze (EPM), and Morris water maze tests were used to evaluate the effects of DiOHF treatment on ischemia-induced locomotor activity, anxiety-like behavior, and spatial and recognition memory impairments, respectively. Results: In the open field test, locomotor activity in the ischemic rats was not altered 6 days after the ischemia, nor was anxiety-like behavior, which was evaluated with the EPM (P > 0.05). In the water-maze test, cerebral ischemia significantly decreased the exploration time in the target quadrant, and the platform crossing counts were lower (P < 0.05) in the probe trial test; this memory impairment was significantly improved by DiOHF applied 1 hour before and immediately after ischemia (P < 0.05). Discussion: All together, these findings suggest that DiOHF reverses spatial learning and memory deficits resulting from transient global ischemia but has no significant effect on anxiety-like behavior. Keywords: DiOHF, Stroke, Learning and memory, Anxiety-like behavior
Introduction
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Transient global cerebral ischemia is a cerebrovascular condition that diminishes the cerebral blood flow up to 10%.1 Transient cardiac arrest, severe dysrhythmias, and intracerebral arterial occlusions are the most common causes of both global and focal cerebral ischemia in humans.2 Ischemia leads to cerebral injury by inducing a set of complex pathophysiological effects; consequently, secondary neural death (also called late-on set neural death) begins due to oxidative stress, excitotoxicity, and zinc toxicity.3,4 Neural cell death following transient global ischemia is more apparent, especially in ischemia-susceptible regions such as the hippocampus and striatum.5 Learning and memory abnormalities are evident behavioral and cognitive problems stemming from cerebral ischemia. It is well known that spatial learning and memory Correspondence to: Mehmet Oz, Department of Physiology, Faculty of Medicine, University of Selçuk, Selçuklu 42075, Konya, Turkey. Email: [email protected]
© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830514Y.0000000159
are substantially dependent on the hippocampal formation.6 In particular, hippocampal CA1 pyramidal neural damage is complicated by disturbances in cognitive functions, including spatial orientation and learning and memory.7–9 Although there is an increasing amount of cellular and molecular pathophysiological data about ischemia-related cognitive disturbances, the rate of development of novel drugs to treat postischemic stroke is not so high. Thus, health professionals need new treatment options that offer minimum adverse effects, and natural molecules are remarkable candidates to alleviate stroke. Flavonoids are the most common phenolic compounds in the human diet, and fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, and wine are the major sources of flavonoids.10 Traditionally, the health-related benefits of these compounds have been mainly attributed to their antioxidant capacity, involving radical scavenging and metal chelation.11–13 The synthetic flavonol 3′ ,4′ -dihydroxyflavonol
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DiOHF in cerebral I/R-induced cognitive alteration
(DiOHF) has three hydroxyl groups at its heterocyclic ring and a catechol group at ring B that exhibits robust antioxidant activity.14 This structure permits DiOHF to improve the superoxide dismutase capacity and blood flow at the ischemic site15,16 and makes it an efficient cytoprotective agent. Additionally, DiOHF explicitly alleviates striatal and cortical damage in moderate stroke in rats,17 and this finding indicates its relevance for consideration as a potential therapeutic agent to improve the neurological outcomes. Therefore, this model may be useful for evaluating the cerebroprotective effect on transient ischemia. Thus, in the present study, we investigated the efficiency of a single dose of DiOHF before or after ischemic onset in transient global cerebral ischemia in rats by assessing the locomotor activity, anxietylike behavior, and learning and memory using the open field (OF), elevated plus maze (EPM), and Morris water maze (MWM) tests, respectively.
Materials and methods Animals and groups
Transient global ischemia The animals were fasted for 12 hours, but they were allowed free access to water before surgery. A transient global cerebral ischemia model was prepared by bilaterally occluding the common carotid arteries (2VO), as described by Singh and Chopra 2013,18 with minor modifications. Briefly, the rats were anesthetized using a ketamine HCl (60 mg/kg) and xylazine HCl (10 mg/kg) cocktail injected intraperitoneally; the animals were then placed in the supine position. A midline ventral incision was made in the neck. The left and right common carotid arteries were exposed and carefully separated from the vagus nerve and surrounding tissues, then occluded using a traumatic microsurgical clips. The clips were then removed after 20 minutes, and the carotid blood flow through the arteries was inspected before the incision was sutured. Physiological saline (5 ml) heated to 37°C was injected subcutaneously to compensate for fluid loss during the anesthesia. Sham-operated animals, which served as controls, were subjected to the same surgical procedures but without occlusion of the carotid arteries. The rectal temperature was maintained at 37 ± 0.5°C with a heating lamp during and after the ischemia. After stroke induction, the rats were allowed to recover from anesthesia and surgery for 3 days before the behavioral studies.
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Male Wistar albino rats weighing 350–400 g were purchased from the Necmettin Erbakan University Experimental Medicine Research and Application Center, and all experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80–23, revised 1996). The animals were kept in cages of no more than four animals, under constant temperature (21 ± 2°C) and humidity (50 ± 10%) with a 12-hour light/dark cycle (lights on 07:00–19:00), and tap water, and standard rat chow were offered ad libitum. Behavioral tasks were conducted between 10:00 and 14:00. Experimental procedures were approved by the local animal ethical committee. Each experimental protocol was statistically designed to use the minimal number of animals. Rats were randomly divided into four groups of six rats each: sham-operated (S), animals subjected to the anesthetic and surgical procedures of transient global cerebral ischemia without occlusion of the cerebral blood flow and receiving the DiOHFvehicle ( peanut oil and 5% DMSO) 1 hour before the surgical procedures; ischemia (I), animals subjected to the transient global ischemia procedure and receiving the DiOHF vehicle ( peanut oil and 5% DMSO) 1 hour before ischemia; DiOHF + ischemia(DI), animals subjected to the transient global ischemia procedure and receiving DI˙OHF (10 mg/kg i.p.) 1 hour before ischemia; and ischemia + DiOHF(ID), animals subjected to transient global ischemia procedure and receiving DI˙OHF (10 mg/kg i.p.) immediately after the ischemia. DiOHF was obtained as a yellow crystalline material in a 100 mg vial from Indofine (Hillsborough, NJ,
USA). The ketamine and xylazine used for the surgical procedures were purchased as commercial preparations for veterinary use.
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Neurobehavioral tests Morris water maze
Spatial learning and memory abilities were evaluated with the MWM test.6 The rats were subjected to four trials per day on postoperative days 3–6, followed by the probe trial without the platform. The MWM consisted of a tank (150 cm in diameter, 60 cm in height) filled to a depth of 45 cm with water maintained at 25 ± 1°C by an automatic heater. An escape platform (10 × 10 cm) was hidden 2 cm below the surface of the water, which was darkened with nontoxic food paint, in a fixed location at the center of one of the quadrants (target quadrant). The water maze pool was divided into four quadrants, and the starting quadrant was randomized daily, with all rats using the same daily order. The rats were released into the maze head-up and facing the wall of the maze. If the rat failed to find the hidden platform within 90 seconds, it was placed on the platform. The animal was allowed to remain on the platform for 30 seconds and to rest for 1 minute after each trial. The starting point was changed after each trial. The escape latency (seconds), swimming path length (cm), and swimming speed (cm per second) were analyzed. Data of the trials
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Open field test
Elevated plus maze test
Fig. 2 shows the effects of DiOHF treatment on the EPM performance between the groups. The influence of 10 mg/kg DiOHF 1 hour before and immediately after the ischemia on anxiety-like behavior was assessed in the animals subjected to ischemia. The monitoring included recording the total distance traveled (Fig. 2A), velocity (Fig. 2B), amount of time spent in the closed arms (Fig. 2C), and number of closed-arm entries (Fig. 2D) in the maze. No significant effects were found regarding the total distance traveled, velocity, number of closed-arm entries, and amount of time spent in the closed arms of the EPM in both ischemic non-treated and ischemic DiOHFtreated rats (Fig. 2, P > 0.05).
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The OF test was allowed to assess the locomotor and exploration activity and anxiety. The apparatus was made of black-painted Plexiglas measuring 80 × 80 cm and was surrounded by 40-cm-high walls. The male rats were gently placed individually into the center of the OF and allowed to explore freely for 5 minutes. The total distance traveled, velocity, number of rearings and defecations, and time spent at the center were recorded by an automatic video tracking system. The field was cleaned with 70% ethanol after the end of each session.
carotid arteries for 5 minutes (Fig. 1). Compared with the sham group, the male Wistar rats exposed to transient global cerebral ischemia showed no significant differences in the total distance traveled (indicator of locomotor activity) (Fig. 1A), velocity (Fig. 1B), mean total time spent at the center (anxiety-like behavior) (Fig. 1C), and number of fecal boli (Fig. 1D, P > 0.05), similar to the DiOHF-treated animals. The frequency of rearing was significantly higher in all the ischemia-induced rats than in the sham group (Fig. 1E, P < 0.05), but there was no significant difference between the vehicle-treated ischemia group and the rats receiving DiOHF before or after the onset of ischemia (Fig. 1E, P > 0.05).
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for each rat in each daily session were averaged. After each training session, the rats were dried with a towel and returned to their cages. To assess spatial learning ability, the platform was removed from the pool, and the animals were subjected to the 90-second probe trial following the last training session. The proportion of time spent in the target quadrant, the number of platform crossings, the path in the target quadrant, and the swimming speed (cm per second) were monitored and recorded by a video camera linked to a computer-based image analyzer (EthoVision XT v.8.0, Noldus Information Tech., Wageningen, Netherlands).
DiOHF in cerebral I/R-induced cognitive alteration
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Elevated plus maze test
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Anxiolytic activity was measured using the EPM, which consisted of two open arms of 50 × 10 cm, two closed arms of 50 × 10 × 40 cm, and a center square of 10 × 10 cm, and it was performed with all arms at an elevation of 50 cm from the floor. Each rat was placed on the central square facing an open arm and allowed to freely explore the maze for 5 minutes. An animal was considered to have entered an arm when all four paws crossed onto the arm. The total distance traveled, velocity, number of entries to the closed arms, and total time spent in the closed arms were recorded for 5 minutes. After each animal, the apparatus was wiped clean using 70% ethanol and allowed to dry.
Statistical analyses
Statistical analyses were performed using the SPSS v.18 statistical software (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance and posthoc Tukey’s multiple comparison tests were used for normally distributed data, whereas the Kruskal–Wallis and Mann–Whitney U tests were used for data that was not normally distributed (statistical significance, P < 0.05).
Results Open field test We performed an OF test to examine the effects of DiOHF treatment on the anxiety-like behavior in rats subjected to occlusion of the bilateral common
Morris water maze test
Place navigation stage The total distance traveled, swimming velocity, and mean latency of finding the platform (escape latency) during the place navigation stage are shown in Fig. 3. In the sham group, the escape latencies showed a progressive reduction over the successive trials, similar to that of the DI and ID groups, but the values were non-significant; the vehicle-treated ischemic rats showed a slight, non-significant reduction of the escape latencies on the successive days of testing compared with the values for the sham group (Fig. 3A, P > 0.05). The total distance traveled (Fig. 3B) was significantly less for the sham group rats compared with the vehicle-treated ischemic rats only on the place navigation stage on day 4 (P < 0.05), and in this phase, it was observed that all the animals in all four groups could swim easily and that the swimming velocity was similar in all groups at all time points (Fig. 3C, P > 0.05).
Probe trial The total distance traveled, swimming velocity, mean time spent in the target quadrant, and number of crossings in the probe trial are shown in Fig. 4. The
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Figure 1 Effects of DiOHF treatment on the open field activity in rats subjected to global ischemia. The total distance traveled (A), velocity (B), time spent at the center (C), fecal pellets (D), and the number of rearings (E) in the open field. The data are expressed as the mean ± SD (n = 6). *P < 0.05 compared with the sham. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
probe trial was conducted by removing the platform and allowing the rat to swim for 90 seconds in search of the platform. The number of crossings over the platform location during the probe trial was lower in the ischemia group (P < 0.05) compared with that in the sham group (Fig. 4D). In addition, the animals treated with bilateral common carotid arterial occlusion showed a significant decrease in the exploration time in the target quadrant compared with the sham controls (Fig. 4C, P < 0.05). Those adverse results were reversed by 10 mg/kg DiOHF administered 1 hour before or immediately after the ischemia (Fig. 4C and D, P < 0.05). The swimming 4
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velocity and total distance traveled did not differ significantly (Fig. 4A and B, P > 0.05) among the three groups in this trial.
Discussion In the present study, we demonstrate for the first time that either pre-ischemic or post-ischemic DiOHF treatment improves spatial learning and memory, but not anxiety-like behavior, following transient global cerebral ischemia in rats. Rats exposed to transient global cerebral ischemia required more time to find the hidden platform, had fewer crossings over the platform area, and spent less time in the target quadrant.
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Figure 2 Analysis of performance in the elevated plus maze by ischemia-induced rats treated with DiOHF (10 mg/kg, i.p.). The total distance traveled (A), velocity (B), time spent in the closed arms (s) (C), and number of entries into the closed arms (D) in the elevated plus maze. The data are expressed as the mean ± SD (n = 6). No significant effects were found for any parameter of the elevated plus maze. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
Figure 3 Escape latency for the training trials (A), total distance traveled (B), and velocity (C) during the place navigation stage. The data are expressed as the means. *P < 0.05 compared with the sham group. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
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Figure 4 Effects of DiOHF on Morris water maze performance deficits induced by 20 minutes of global cerebral ischemia in rats (n = 6). (A) Total distance traveled, (B) velocity, (C) time in the target quadrant, and (D) number of crossings in the probe trial. The data are presented as the means ± SD. *P < 0.05 compared with the ischemia group. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
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Thus, this result demonstrates that ischemia caused severe disturbances of spatial learning and memory. In the present study, both pre-ischemic and delayed DiOHF administration increased the platform crossing count and the duration of time spent in the target quadrant. Additionally, the rats that underwent transient global cerebral ischemia showed increased rearing, which represents exploratory activity. We measured the effects of DiOHF on anxiety-like behavior in rats that were exposed to transient global cerebral ischemia with two different tests: EPM and OF. The OF test is recognized as a sensitive indicator of an animal’s ability to acclimate to a new environment. In the present study, the general locomotor activity, exploratory habits, and emotional behavior were assessed with the OF test. In the OF test, the duration spent in the center area was considered as the anxiety index, the total distance and velocity were accepted as indicators of locomotor activity, and the rearing count was assessed as an indicator of exploratory activity. In the EPM, rodents normally tend to avoid the open arms of the maze and prefer to stay in the closed arms, which have protective walls. The time spent in the open arms is used as an indicator of anxiety. If animals are highly anxious, the entrance count to the closed arms and the distance traveled decreases. The entrance count to all arms of the
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EPM is considered as an index of the locomotor activity.19 In general, locomotor activity is enhanced in vehicle-treated ischemic rodents compared with sham groups in the OF and EPM tests.20–22 In our study, the rearing count, which indicates exploratory behavior, increased in the ischemic rats compared with the sham group, but contrary to the above mentioned studies, there was no significant effect of cerebral ischemia in either the OF test or the EPM test in terms of locomotor activity. Two possible reasons may account for this result: (1) previous studies report that the ischemia-induced locomotor activity increment is more visible on the day after surgical intervention. The locomotor activity decreases gradually starting on the second day, and the difference between the sham and ischemia groups disappears by the sixth day.23,24 As similar to the method of Hamadate et al.,25 the OF test was conducted on the sixth day, and the EPM test was conducted on the seventh day. In our study, we demonstrated that ischemia did not alter the locomotor activity as assessed with the OF and EPM tests. (2) Girbovan et al.26 suggested that the handling of the rats during the drug administration before the OF test may affect their emotional reactivity, and the absence of ischemia-induced hyperactivity is associated with this situation. We conclude that the handling before the OF
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count and the duration in the target quadrant were higher in the DiOHF-treated rats compared with the vehicle-treated ischemic rats (P < 0.05). These results indicate that DiOHF can prevent cognitive dysfunction in rats with global cerebral ischemia/reperfusioninduced injury. Despite this argument, the precise mechanism of DiOHF concerning cognitive performance is not known because to the best of our knowledge, our present study is the first in the medical literature to investigate the effects of DiOHF on transient global ischemia-induced cognitive disturbances. However, there are some plausible explanations. Recent studies have demonstrated the cardioprotective effects of DiOHF, a synthetic flavonol. DiOHF administration during ischemia or immediately before reperfusion considerably reduces the infarct size.16,31,32 It has been reported that the cardioprotective properties of DiOHF in the isolated rat heart exposed to ischemiareperfusion arises from the antioxidant activity of this flavonol33 and the fact that DiOHF supplementation rapidly scavenges ROS and/or inhibits superoxide production.34 Moreover, DiOHF is more potent (in terms of vasodilation activity) than other flavonols such as quercetin and chrysin.35 The improvement of the spatial learning function by DiOHF supplementation might be attributable to (a) a reduction in the excessive generation of ROS and (b) the potency of DiOHF for vasorelaxation, which limits reperfusion injury. However, further studies are necessary to substantiate the above findings. In conclusion, the present study, which examined the neuroprotective effect of DiOHF in a rat model of transient global ischemia, suggests that both preischemic and post-ischemic DiOHF supplementation may provide useful strategies for recovery after brain ischemia-induced impaired cognitive function, but future studies should confirm this possibility and determine its mechanism of action.
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test for 4 days at the time of dropping the animals into the MWM and the drying with a towel after the test may lead to this result. In contrast, no study in the medical literature has examined the impact of DiOHF administration on the anxiety-like behavior and spatial learning and memory in sham rats or animals exposed to transient global ischemia. In the present study, the administration of DiOHF before and after ischemia showed no effect on the anxietylike behavior in the OF and EPM tests. It is possible that DiOHF administration and ischemia can alter responses in various behavioral tests, and this requires further investigation. The MWM is a standard test used to evaluate spatial learning and memory that is related to the cognitive functions of the hippocampus and cortex.27 This test objectively and comprehensively reflects the cognitive levels of animals. Therefore, in this study, the MWM test was employed to assess the memory function of rats 3 days after transient global cerebral ischemia was induced. Transient global cerebral ischemia inlaboratory animals is known to lead to deficits in spatial learning and memory as assessed in the MWM.28,29 Our results show that the escape latencies of the vehicletreated ischemic rats on the place navigation stage were somewhat longer than in the sham group, but this change was not statistically significant. Additionally, the ischemic rats had an average velocity similar to sham-operated animals in both the place navigation stage and probe trials, and this finding demonstrates that the tested abnormalities were not linked to locomotor ability or alterations in coordination.30 However, in the spatial probe trial, the duration in the target quadrant was shorter for the vehicle-treated ischemic group than for the sham-operated rats, and the frequency of platform crossings was lower for the vehicle-treated ischemic group than for the sham group. The lower platform crossing counts and the shorter duration in the target quadrant show disturbances in spatial learning and memory in this maze. The present study confirms that a 20-minute bilateral common carotid arterial occlusion in the rats causes a significant impairment in learning and memory, as indicated by a much poorer performance in the MWM. Acute transient global ischemia in rodents causes the generation of reactive oxygen species, which are one of the main reasons for neural death, and decreases the endogenous antioxidant defense capacity.18 Therefore, the damaged redox balance after transient global brain ischemia may be one of the mechanisms contributing to the learning and memory impairment. However, in the present study, both pre-ischemic and post-ischemic DiOHF treatment improved the cognitive function, which was disturbed after 20-minute bilateral common carotid arterial occlusion. In the probe trial, both the crossing
DiOHF in cerebral I/R-induced cognitive alteration
Disclaimer statements Contributors Mehmet Oz and Enver Ahmet Demir designed and supervised the whole experiment, and prepared the manuscript. Merve Caliskan followed pre- and post-surgical animal care. Rasim Mogulkoc and Abdulkerim Kasim Baltaci conducted the experimental surgical procedures. K. Esra Atalik Nurullahoglu done the behavioral assessment. Funding None. Conflicts of interest None. Ethics approval All experimental procedures in our present study were approved by the Necmettin Erbakan University Experimental Medicine Research and Application Center Animal Ethical Committee (Decision Number: 2014-33).
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19 Karakas A, Coskun H, Kaya A, Kucuk A, Gunduz B. The effects of the intraamygdalar melatonin injections on the anxiety like behavior and the spatial memory performance in male Wistar rats. Behav Brain Res 2011;222(1):141–50. 20 Kanhere R, Anjana A, Anbu J, Sumithra M, K F H NA. Neuroprotective and antioxidant potential of terpenoid fraction from Hygrophila auriculata against transient global cerebral ischemia in rats. Pharm Biol 2013;51(2):181–9. 21 Milot M, Plamondon H. Ischemia-induced hyperactivity: effects of dim versus bright illumination on open-field exploration and habituation following global ischemia in rats. Behav Brain Res 2008;192(2):166–72. 22 Plamondon H, Khan S. Characterization of anxiety and habituation profile following global ischemia in rats. Physiol Behav 2005;84(4):543–52. 23 Block F, Schwarz M. Dextromethorphan reduces functional deficits and neuronal damage after global ischemia in rats. Brain Res 1996;741(1–2):153–9. 24 Karasawa Y, Araki H, Otomo S. Changes in locomotor activity and passive avoidance task performance induced by cerebral ischemia in Mongolian gerbils. Stroke 1994;25(3):645–50. 25 Hamadate N, Yamaguchi T, Sugawara A, Togashi H, Izumi T, Yoshida T, et al. Liposome-encapsulated hemoglobin ameliorates impairment of fear memory and hippocampal dysfunction after cerebral ischemia in rats. J Pharmacol Sci 2010;114(4):409–19. 26 Girbovan C, Morin L, Plamondon H. Repeated resveratrol administration confers lasting protection against neuronal damage but induces dose-related alterations of behavioral impairments after global ischemia. Behav Pharmacol 2012; 23(1):1–13. 27 Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006;1(2):848–58. 28 Cheng O, Li Z, Han Y, Jiang Q, Yan Y, Cheng K. Baicalin improved the spatial learning ability of global ischemia/ reperfusion rats by reducing hippocampal apoptosis. Brain Res 2012;1470:111–8. 29 Hu X, Zhang Y, Li W, Liu J, Li Y. Preconditioning with sevoflurane ameliorates spatial learning and memory deficit after focal cerebral ischemia-reperfusion in rats. Int J Dev Neurosci 2013; 31(5):328–33. 30 Han B, Wang Q, Cui G, Shen X, Zhu Z. Post-treatment of Bax-inhibiting peptide reduces neuronal death and behavioral deficits following global cerebral ischemia. Neurochem Int 2011;58(2):224–33. 31 Wang S, Dusting GJ, Woodman OL, May CN. Selective vasodilator and chronotropic actions of 3′ ,4′ -dihydroxyflavonol in conscious sheep. Eur J Pharmacol 2004;491(1):43–51. 32 Wang S, Fei K, Xu YW, Wang LX, Chen YQ. Dihydroxyflavonol reduces post-infarction left ventricular remodeling by preventing myocyte apoptosis in the non-infarcted zone in goats. Chin Med J 2009;122(1):61–7. 33 Qin CX, Williams SJ, Woodman OL. Antioxidant activity contributes to flavonol cardioprotection during reperfusion of rat hearts. Free Radic Biol Med 2011;51(7):1437–44. 34 Leo CH, Hart JL, Woodman OL. 3′ ,4′ -Dihydroxyflavonol reduces superoxide and improves nitric oxide function in diabetic rat mesenteric arteries. PLoS One 2011;6(6):e20813. 35 Woodman OL, Chan E. Vascular and anti-oxidant actions of flavonols and flavones. Clin Exp Pharmacol Physiol 2004; 31(11):786–90.
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Mehmet Oz1 , Enver Ahmet Demir 1, Merve Caliskan 1, Rasim Mogulkoc 1, Abdulkerim Kasım Baltaci 1, K. Esra Nurullahoglu Atalik 2 1
Department of Physiology, Faculty of Medicine, University of Selcuk, Konya, Turkey, 2Department of Pharmacology, Faculty of Meram Medicine, University of Necmettin Erbakan, Konya, Turkey
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Objectives: In the present study, effects of 3′ ,4′ -dihydroxyflavonol (DiOHF) on anxiety-like behavior, and learning and memory were investigated in a model of transient global cerebral ischemia and reperfusion. Methods: The animals were assigned to sham-operated, ischemia, and two DiOHF-treated (10 mg/kg i.p.) groups. DiOHF was administered at 1 hour before and immediately after the ischemia. Male rats were subjected to bilateral common carotid artery occlusion to induce acute cerebral ischemia for 20 minutes, followed by reperfusion for 7 days. The openfield, elevated plus maze (EPM), and Morris water maze tests were used to evaluate the effects of DiOHF treatment on ischemia-induced locomotor activity, anxiety-like behavior, and spatial and recognition memory impairments, respectively. Results: In the open field test, locomotor activity in the ischemic rats was not altered 6 days after the ischemia, nor was anxiety-like behavior, which was evaluated with the EPM (P > 0.05). In the water-maze test, cerebral ischemia significantly decreased the exploration time in the target quadrant, and the platform crossing counts were lower (P < 0.05) in the probe trial test; this memory impairment was significantly improved by DiOHF applied 1 hour before and immediately after ischemia (P < 0.05). Discussion: All together, these findings suggest that DiOHF reverses spatial learning and memory deficits resulting from transient global ischemia but has no significant effect on anxiety-like behavior. Keywords: DiOHF, Stroke, Learning and memory, Anxiety-like behavior
Introduction
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Transient global cerebral ischemia is a cerebrovascular condition that diminishes the cerebral blood flow up to 10%.1 Transient cardiac arrest, severe dysrhythmias, and intracerebral arterial occlusions are the most common causes of both global and focal cerebral ischemia in humans.2 Ischemia leads to cerebral injury by inducing a set of complex pathophysiological effects; consequently, secondary neural death (also called late-on set neural death) begins due to oxidative stress, excitotoxicity, and zinc toxicity.3,4 Neural cell death following transient global ischemia is more apparent, especially in ischemia-susceptible regions such as the hippocampus and striatum.5 Learning and memory abnormalities are evident behavioral and cognitive problems stemming from cerebral ischemia. It is well known that spatial learning and memory Correspondence to: Mehmet Oz, Department of Physiology, Faculty of Medicine, University of Selçuk, Selçuklu 42075, Konya, Turkey. Email: [email protected]
© W. S. Maney & Son Ltd 2014 DOI 10.1179/1476830514Y.0000000159
are substantially dependent on the hippocampal formation.6 In particular, hippocampal CA1 pyramidal neural damage is complicated by disturbances in cognitive functions, including spatial orientation and learning and memory.7–9 Although there is an increasing amount of cellular and molecular pathophysiological data about ischemia-related cognitive disturbances, the rate of development of novel drugs to treat postischemic stroke is not so high. Thus, health professionals need new treatment options that offer minimum adverse effects, and natural molecules are remarkable candidates to alleviate stroke. Flavonoids are the most common phenolic compounds in the human diet, and fruits, vegetables, nuts, seeds, herbs, spices, stems, flowers, and wine are the major sources of flavonoids.10 Traditionally, the health-related benefits of these compounds have been mainly attributed to their antioxidant capacity, involving radical scavenging and metal chelation.11–13 The synthetic flavonol 3′ ,4′ -dihydroxyflavonol
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DiOHF in cerebral I/R-induced cognitive alteration
(DiOHF) has three hydroxyl groups at its heterocyclic ring and a catechol group at ring B that exhibits robust antioxidant activity.14 This structure permits DiOHF to improve the superoxide dismutase capacity and blood flow at the ischemic site15,16 and makes it an efficient cytoprotective agent. Additionally, DiOHF explicitly alleviates striatal and cortical damage in moderate stroke in rats,17 and this finding indicates its relevance for consideration as a potential therapeutic agent to improve the neurological outcomes. Therefore, this model may be useful for evaluating the cerebroprotective effect on transient ischemia. Thus, in the present study, we investigated the efficiency of a single dose of DiOHF before or after ischemic onset in transient global cerebral ischemia in rats by assessing the locomotor activity, anxietylike behavior, and learning and memory using the open field (OF), elevated plus maze (EPM), and Morris water maze (MWM) tests, respectively.
Materials and methods Animals and groups
Transient global ischemia The animals were fasted for 12 hours, but they were allowed free access to water before surgery. A transient global cerebral ischemia model was prepared by bilaterally occluding the common carotid arteries (2VO), as described by Singh and Chopra 2013,18 with minor modifications. Briefly, the rats were anesthetized using a ketamine HCl (60 mg/kg) and xylazine HCl (10 mg/kg) cocktail injected intraperitoneally; the animals were then placed in the supine position. A midline ventral incision was made in the neck. The left and right common carotid arteries were exposed and carefully separated from the vagus nerve and surrounding tissues, then occluded using a traumatic microsurgical clips. The clips were then removed after 20 minutes, and the carotid blood flow through the arteries was inspected before the incision was sutured. Physiological saline (5 ml) heated to 37°C was injected subcutaneously to compensate for fluid loss during the anesthesia. Sham-operated animals, which served as controls, were subjected to the same surgical procedures but without occlusion of the carotid arteries. The rectal temperature was maintained at 37 ± 0.5°C with a heating lamp during and after the ischemia. After stroke induction, the rats were allowed to recover from anesthesia and surgery for 3 days before the behavioral studies.
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Male Wistar albino rats weighing 350–400 g were purchased from the Necmettin Erbakan University Experimental Medicine Research and Application Center, and all experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications no. 80–23, revised 1996). The animals were kept in cages of no more than four animals, under constant temperature (21 ± 2°C) and humidity (50 ± 10%) with a 12-hour light/dark cycle (lights on 07:00–19:00), and tap water, and standard rat chow were offered ad libitum. Behavioral tasks were conducted between 10:00 and 14:00. Experimental procedures were approved by the local animal ethical committee. Each experimental protocol was statistically designed to use the minimal number of animals. Rats were randomly divided into four groups of six rats each: sham-operated (S), animals subjected to the anesthetic and surgical procedures of transient global cerebral ischemia without occlusion of the cerebral blood flow and receiving the DiOHFvehicle ( peanut oil and 5% DMSO) 1 hour before the surgical procedures; ischemia (I), animals subjected to the transient global ischemia procedure and receiving the DiOHF vehicle ( peanut oil and 5% DMSO) 1 hour before ischemia; DiOHF + ischemia(DI), animals subjected to the transient global ischemia procedure and receiving DI˙OHF (10 mg/kg i.p.) 1 hour before ischemia; and ischemia + DiOHF(ID), animals subjected to transient global ischemia procedure and receiving DI˙OHF (10 mg/kg i.p.) immediately after the ischemia. DiOHF was obtained as a yellow crystalline material in a 100 mg vial from Indofine (Hillsborough, NJ,
USA). The ketamine and xylazine used for the surgical procedures were purchased as commercial preparations for veterinary use.
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Neurobehavioral tests Morris water maze
Spatial learning and memory abilities were evaluated with the MWM test.6 The rats were subjected to four trials per day on postoperative days 3–6, followed by the probe trial without the platform. The MWM consisted of a tank (150 cm in diameter, 60 cm in height) filled to a depth of 45 cm with water maintained at 25 ± 1°C by an automatic heater. An escape platform (10 × 10 cm) was hidden 2 cm below the surface of the water, which was darkened with nontoxic food paint, in a fixed location at the center of one of the quadrants (target quadrant). The water maze pool was divided into four quadrants, and the starting quadrant was randomized daily, with all rats using the same daily order. The rats were released into the maze head-up and facing the wall of the maze. If the rat failed to find the hidden platform within 90 seconds, it was placed on the platform. The animal was allowed to remain on the platform for 30 seconds and to rest for 1 minute after each trial. The starting point was changed after each trial. The escape latency (seconds), swimming path length (cm), and swimming speed (cm per second) were analyzed. Data of the trials
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Open field test
Elevated plus maze test
Fig. 2 shows the effects of DiOHF treatment on the EPM performance between the groups. The influence of 10 mg/kg DiOHF 1 hour before and immediately after the ischemia on anxiety-like behavior was assessed in the animals subjected to ischemia. The monitoring included recording the total distance traveled (Fig. 2A), velocity (Fig. 2B), amount of time spent in the closed arms (Fig. 2C), and number of closed-arm entries (Fig. 2D) in the maze. No significant effects were found regarding the total distance traveled, velocity, number of closed-arm entries, and amount of time spent in the closed arms of the EPM in both ischemic non-treated and ischemic DiOHFtreated rats (Fig. 2, P > 0.05).
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The OF test was allowed to assess the locomotor and exploration activity and anxiety. The apparatus was made of black-painted Plexiglas measuring 80 × 80 cm and was surrounded by 40-cm-high walls. The male rats were gently placed individually into the center of the OF and allowed to explore freely for 5 minutes. The total distance traveled, velocity, number of rearings and defecations, and time spent at the center were recorded by an automatic video tracking system. The field was cleaned with 70% ethanol after the end of each session.
carotid arteries for 5 minutes (Fig. 1). Compared with the sham group, the male Wistar rats exposed to transient global cerebral ischemia showed no significant differences in the total distance traveled (indicator of locomotor activity) (Fig. 1A), velocity (Fig. 1B), mean total time spent at the center (anxiety-like behavior) (Fig. 1C), and number of fecal boli (Fig. 1D, P > 0.05), similar to the DiOHF-treated animals. The frequency of rearing was significantly higher in all the ischemia-induced rats than in the sham group (Fig. 1E, P < 0.05), but there was no significant difference between the vehicle-treated ischemia group and the rats receiving DiOHF before or after the onset of ischemia (Fig. 1E, P > 0.05).
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for each rat in each daily session were averaged. After each training session, the rats were dried with a towel and returned to their cages. To assess spatial learning ability, the platform was removed from the pool, and the animals were subjected to the 90-second probe trial following the last training session. The proportion of time spent in the target quadrant, the number of platform crossings, the path in the target quadrant, and the swimming speed (cm per second) were monitored and recorded by a video camera linked to a computer-based image analyzer (EthoVision XT v.8.0, Noldus Information Tech., Wageningen, Netherlands).
DiOHF in cerebral I/R-induced cognitive alteration
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Elevated plus maze test
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Anxiolytic activity was measured using the EPM, which consisted of two open arms of 50 × 10 cm, two closed arms of 50 × 10 × 40 cm, and a center square of 10 × 10 cm, and it was performed with all arms at an elevation of 50 cm from the floor. Each rat was placed on the central square facing an open arm and allowed to freely explore the maze for 5 minutes. An animal was considered to have entered an arm when all four paws crossed onto the arm. The total distance traveled, velocity, number of entries to the closed arms, and total time spent in the closed arms were recorded for 5 minutes. After each animal, the apparatus was wiped clean using 70% ethanol and allowed to dry.
Statistical analyses
Statistical analyses were performed using the SPSS v.18 statistical software (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance and posthoc Tukey’s multiple comparison tests were used for normally distributed data, whereas the Kruskal–Wallis and Mann–Whitney U tests were used for data that was not normally distributed (statistical significance, P < 0.05).
Results Open field test We performed an OF test to examine the effects of DiOHF treatment on the anxiety-like behavior in rats subjected to occlusion of the bilateral common
Morris water maze test
Place navigation stage The total distance traveled, swimming velocity, and mean latency of finding the platform (escape latency) during the place navigation stage are shown in Fig. 3. In the sham group, the escape latencies showed a progressive reduction over the successive trials, similar to that of the DI and ID groups, but the values were non-significant; the vehicle-treated ischemic rats showed a slight, non-significant reduction of the escape latencies on the successive days of testing compared with the values for the sham group (Fig. 3A, P > 0.05). The total distance traveled (Fig. 3B) was significantly less for the sham group rats compared with the vehicle-treated ischemic rats only on the place navigation stage on day 4 (P < 0.05), and in this phase, it was observed that all the animals in all four groups could swim easily and that the swimming velocity was similar in all groups at all time points (Fig. 3C, P > 0.05).
Probe trial The total distance traveled, swimming velocity, mean time spent in the target quadrant, and number of crossings in the probe trial are shown in Fig. 4. The
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Figure 1 Effects of DiOHF treatment on the open field activity in rats subjected to global ischemia. The total distance traveled (A), velocity (B), time spent at the center (C), fecal pellets (D), and the number of rearings (E) in the open field. The data are expressed as the mean ± SD (n = 6). *P < 0.05 compared with the sham. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
probe trial was conducted by removing the platform and allowing the rat to swim for 90 seconds in search of the platform. The number of crossings over the platform location during the probe trial was lower in the ischemia group (P < 0.05) compared with that in the sham group (Fig. 4D). In addition, the animals treated with bilateral common carotid arterial occlusion showed a significant decrease in the exploration time in the target quadrant compared with the sham controls (Fig. 4C, P < 0.05). Those adverse results were reversed by 10 mg/kg DiOHF administered 1 hour before or immediately after the ischemia (Fig. 4C and D, P < 0.05). The swimming 4
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velocity and total distance traveled did not differ significantly (Fig. 4A and B, P > 0.05) among the three groups in this trial.
Discussion In the present study, we demonstrate for the first time that either pre-ischemic or post-ischemic DiOHF treatment improves spatial learning and memory, but not anxiety-like behavior, following transient global cerebral ischemia in rats. Rats exposed to transient global cerebral ischemia required more time to find the hidden platform, had fewer crossings over the platform area, and spent less time in the target quadrant.
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Figure 2 Analysis of performance in the elevated plus maze by ischemia-induced rats treated with DiOHF (10 mg/kg, i.p.). The total distance traveled (A), velocity (B), time spent in the closed arms (s) (C), and number of entries into the closed arms (D) in the elevated plus maze. The data are expressed as the mean ± SD (n = 6). No significant effects were found for any parameter of the elevated plus maze. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
Figure 3 Escape latency for the training trials (A), total distance traveled (B), and velocity (C) during the place navigation stage. The data are expressed as the means. *P < 0.05 compared with the sham group. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
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Figure 4 Effects of DiOHF on Morris water maze performance deficits induced by 20 minutes of global cerebral ischemia in rats (n = 6). (A) Total distance traveled, (B) velocity, (C) time in the target quadrant, and (D) number of crossings in the probe trial. The data are presented as the means ± SD. *P < 0.05 compared with the ischemia group. I: ischemia; DI: DiOHF before ischemia; ID: ischemia before DiOHF.
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Thus, this result demonstrates that ischemia caused severe disturbances of spatial learning and memory. In the present study, both pre-ischemic and delayed DiOHF administration increased the platform crossing count and the duration of time spent in the target quadrant. Additionally, the rats that underwent transient global cerebral ischemia showed increased rearing, which represents exploratory activity. We measured the effects of DiOHF on anxiety-like behavior in rats that were exposed to transient global cerebral ischemia with two different tests: EPM and OF. The OF test is recognized as a sensitive indicator of an animal’s ability to acclimate to a new environment. In the present study, the general locomotor activity, exploratory habits, and emotional behavior were assessed with the OF test. In the OF test, the duration spent in the center area was considered as the anxiety index, the total distance and velocity were accepted as indicators of locomotor activity, and the rearing count was assessed as an indicator of exploratory activity. In the EPM, rodents normally tend to avoid the open arms of the maze and prefer to stay in the closed arms, which have protective walls. The time spent in the open arms is used as an indicator of anxiety. If animals are highly anxious, the entrance count to the closed arms and the distance traveled decreases. The entrance count to all arms of the
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EPM is considered as an index of the locomotor activity.19 In general, locomotor activity is enhanced in vehicle-treated ischemic rodents compared with sham groups in the OF and EPM tests.20–22 In our study, the rearing count, which indicates exploratory behavior, increased in the ischemic rats compared with the sham group, but contrary to the above mentioned studies, there was no significant effect of cerebral ischemia in either the OF test or the EPM test in terms of locomotor activity. Two possible reasons may account for this result: (1) previous studies report that the ischemia-induced locomotor activity increment is more visible on the day after surgical intervention. The locomotor activity decreases gradually starting on the second day, and the difference between the sham and ischemia groups disappears by the sixth day.23,24 As similar to the method of Hamadate et al.,25 the OF test was conducted on the sixth day, and the EPM test was conducted on the seventh day. In our study, we demonstrated that ischemia did not alter the locomotor activity as assessed with the OF and EPM tests. (2) Girbovan et al.26 suggested that the handling of the rats during the drug administration before the OF test may affect their emotional reactivity, and the absence of ischemia-induced hyperactivity is associated with this situation. We conclude that the handling before the OF
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count and the duration in the target quadrant were higher in the DiOHF-treated rats compared with the vehicle-treated ischemic rats (P < 0.05). These results indicate that DiOHF can prevent cognitive dysfunction in rats with global cerebral ischemia/reperfusioninduced injury. Despite this argument, the precise mechanism of DiOHF concerning cognitive performance is not known because to the best of our knowledge, our present study is the first in the medical literature to investigate the effects of DiOHF on transient global ischemia-induced cognitive disturbances. However, there are some plausible explanations. Recent studies have demonstrated the cardioprotective effects of DiOHF, a synthetic flavonol. DiOHF administration during ischemia or immediately before reperfusion considerably reduces the infarct size.16,31,32 It has been reported that the cardioprotective properties of DiOHF in the isolated rat heart exposed to ischemiareperfusion arises from the antioxidant activity of this flavonol33 and the fact that DiOHF supplementation rapidly scavenges ROS and/or inhibits superoxide production.34 Moreover, DiOHF is more potent (in terms of vasodilation activity) than other flavonols such as quercetin and chrysin.35 The improvement of the spatial learning function by DiOHF supplementation might be attributable to (a) a reduction in the excessive generation of ROS and (b) the potency of DiOHF for vasorelaxation, which limits reperfusion injury. However, further studies are necessary to substantiate the above findings. In conclusion, the present study, which examined the neuroprotective effect of DiOHF in a rat model of transient global ischemia, suggests that both preischemic and post-ischemic DiOHF supplementation may provide useful strategies for recovery after brain ischemia-induced impaired cognitive function, but future studies should confirm this possibility and determine its mechanism of action.
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test for 4 days at the time of dropping the animals into the MWM and the drying with a towel after the test may lead to this result. In contrast, no study in the medical literature has examined the impact of DiOHF administration on the anxiety-like behavior and spatial learning and memory in sham rats or animals exposed to transient global ischemia. In the present study, the administration of DiOHF before and after ischemia showed no effect on the anxietylike behavior in the OF and EPM tests. It is possible that DiOHF administration and ischemia can alter responses in various behavioral tests, and this requires further investigation. The MWM is a standard test used to evaluate spatial learning and memory that is related to the cognitive functions of the hippocampus and cortex.27 This test objectively and comprehensively reflects the cognitive levels of animals. Therefore, in this study, the MWM test was employed to assess the memory function of rats 3 days after transient global cerebral ischemia was induced. Transient global cerebral ischemia inlaboratory animals is known to lead to deficits in spatial learning and memory as assessed in the MWM.28,29 Our results show that the escape latencies of the vehicletreated ischemic rats on the place navigation stage were somewhat longer than in the sham group, but this change was not statistically significant. Additionally, the ischemic rats had an average velocity similar to sham-operated animals in both the place navigation stage and probe trials, and this finding demonstrates that the tested abnormalities were not linked to locomotor ability or alterations in coordination.30 However, in the spatial probe trial, the duration in the target quadrant was shorter for the vehicle-treated ischemic group than for the sham-operated rats, and the frequency of platform crossings was lower for the vehicle-treated ischemic group than for the sham group. The lower platform crossing counts and the shorter duration in the target quadrant show disturbances in spatial learning and memory in this maze. The present study confirms that a 20-minute bilateral common carotid arterial occlusion in the rats causes a significant impairment in learning and memory, as indicated by a much poorer performance in the MWM. Acute transient global ischemia in rodents causes the generation of reactive oxygen species, which are one of the main reasons for neural death, and decreases the endogenous antioxidant defense capacity.18 Therefore, the damaged redox balance after transient global brain ischemia may be one of the mechanisms contributing to the learning and memory impairment. However, in the present study, both pre-ischemic and post-ischemic DiOHF treatment improved the cognitive function, which was disturbed after 20-minute bilateral common carotid arterial occlusion. In the probe trial, both the crossing
DiOHF in cerebral I/R-induced cognitive alteration
Disclaimer statements Contributors Mehmet Oz and Enver Ahmet Demir designed and supervised the whole experiment, and prepared the manuscript. Merve Caliskan followed pre- and post-surgical animal care. Rasim Mogulkoc and Abdulkerim Kasim Baltaci conducted the experimental surgical procedures. K. Esra Atalik Nurullahoglu done the behavioral assessment. Funding None. Conflicts of interest None. Ethics approval All experimental procedures in our present study were approved by the Necmettin Erbakan University Experimental Medicine Research and Application Center Animal Ethical Committee (Decision Number: 2014-33).
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