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Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test

Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test ANIMAL CELLS AND SYSTEMS 2019, VOL. 23, NO. 1, 10–17 https://doi.org/10.1080/19768354.2018.1557743 Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test a a b c a Yun-Hee Kim , Kuen-Su Lee , Young-Sung Kim , Yeon-Hwa Kim and Jae-Hwan Kim a b Department of Anesthesiology and Pain Medicine, Korea University Ansan Hospital, Ansan, Korea; Department of Anesthesiology and Pain Medicine, Korea University Guro Hospital, Seoul, Korea; Institute of Medical Science, Korea University Ansan Hospital, Korea University College of Medicine, Ansan, Korea ABSTRACT ARTICLE HISTORY Received 3 August 2018 Perioperative brain ischemia and stroke are leading causes of morbidity and mortality. Brief hypoxic Revised 3 October 2018 preconditioning is known to have protective effects against hypoxic-ischemic insult in the brain. Accepted 15 November 2018 Current studies on the neuroprotective effects of ischemic preconditioning are based on histologic findings and biomarker changes. However, studies regarding effects on memory are KEYWORDS rare. To precondition zebrafish to hypoxia, they were exposed to a dissolved oxygen (DO) Hypoxic preconditioning; concentration of 1.0 ± 0.5 mg/L in water for 30 s. The hypoxic zebrafish were then exposed to ischemia; memory; zebrafish 1.0 ± 0.5 mg/L DO until the third stage of hypoxia, for 10 min ± 30 s. Zebrafish were assessed for memory retention after the hypoxic event. Learning and memory were tested using the T-maze, which evaluates memory based on whether or not zebrafish moves to the correct target compartment. In the hypoxic preconditioning group, infarct size was reduced compared with the hypoxic-only treated zebrafish group; memory was maintained to a degree similar to that in the hypoxia-untreated group. The hypoxic-only group showed significant memory impairments. In this study, we used a hypoxic zebrafish model and assessed the effects of ischemic preconditioning not only on histological damages but also on brain function, especially memory. This study demonstrated that a brief hypoxic event has protective effects in hypoxic brain damage and helped maintain memory in zebrafish. In addition, our findings suggest that the zebrafish model is useful in rapidly assessing the effects of ischemic preconditioning on memory. Introduction protects from later, severe, ischemic insults (Miao et al. 2010). Traditional ischemic preconditioning models The brain is one of the organs that are particularly vulner- include rat and rodent models that are used to investi- able to ischemia. Due to their high metabolic rates, brain gate underlying mechanisms and neuroprotective strat- cells easily lose their function and die in response to egies (Pan et al. 2014). Due to the importance of hypoxia-induced ischemic insults (Murphy et al. 2008). ischemic research, there are many basic studies investi- Brain ischemia and stroke are known to be the leading gating the degree of tissue damage in ischemic precon- causes of morbidity and mortality worldwide (Hossmann ditioning models. However, the difficulty of assessing 2006; Wardlaw et al. 2010; Canazza et al. 2014). The inci- memory function limits research and clinical dence of stroke after non-cardiovascular and non-neuro- applications. logic surgeries is estimated to be 0.05–7%, and the The zebrafish is a relatively small, simple organism, incidence after cardiac surgeries is estimated to be 2– but because it is a vertebrate, a zebrafish gene is likely 10%. The mortality from perioperative stroke is high to resemble a mammalian or human gene, and similar (Parikh and Cohen 1993; Conlon et al. 2008; Zhou et al. genes may be associated with human-like function 2016), and, therefore, monitoring and prevention of (Howe et al. 2013). Zebrafish models are being increas- intraoperative cerebral ischemia are very important. ingly employed in neuroscience and can minimize bias Hypoxic preconditioning has a strong neuroprotective in experimental results by improving ease of setting effect against cerebral ischemic injuries and periopera- experimental conditions and observation efficiency tive cerebral ischemia/reperfusion injury (Sharp et al. (Shams et al. 2018). A previous study related to the 2004; Sinanović 2010). Ischemic preconditioning is a zebrafish behavior change upon ischemic insult phenomenon in which short-term, non-fatal ischemia showed that hypoxia might induce a change in CONTACT Jae-Hwan Kim anejhkim@korea.ac.kr Department of Anesthesiology and Pain Medicine, Korea University Ansan Hospital, 123, Jeokgeum-ro, Danwon-gu, Ansan-si, Gyeonggi-do 15355, Korea © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MOLECULAR & CELLULAR BIOLOGY ANIMAL CELLS AND SYSTEMS 11 zebrafish brain physiology and behavior (Braga et al. 2013). These findings showed the utility of zebrafish behavior change to assess brain function, especially learning and memory. Current ischemia studies are based on histologic findings and biomarker changes, and studies regarding brain functional effects, especially memory are rare. Therefore, we evaluated whether ischemic preconditioning could attenuate brain tissue damage and preserve memory using the T-maze behav- ior test in zebrafish under conditions of low dissolved oxygen (DO) (Kim et al. 2017). Materials and methods Animals This study was approved by the Ethical Committee on Animal Research at the Korea University College of Medi- cine (approval No. KOREA-2018-0020). In all experiments, adult zebrafish (4-6 months of age and 2.5–3.5 cm long) purchased from a local aquarium store (Jincheon, Chung- cheongbuk-do, Korea) were used. The zebrafish were short-finned wild-type and had a heterogeneous genetic background. They were kept in water at 28.5°C Figure 1. Schematic of the hypoxic chamber and procedure. (A) TM GasPak attached with lid of the hypoxic chamber. The hypoxic with a light cycle of 14 h and a dark cycle of 10 h in chamber was filled with 300 mL of system water. (B) The training aquarium containers and were fed brine shrimp twice a session was composed of 4 trials on consecutive days. The day. The aquarium container was equipped with a multi- memory test was administered 24 h after the last training trial. stage filtration system that had a sediment filter, post- Hypoxia was induced before the memory test and recovery carbon filter, fluorescent UV light, and sterilizing filter was allowed for 2 h. Hypoxic preconditioning was induced (Zebrafish AutoSystem, Genomic Design, Daejeon, before the hypoxia and recovery were allowed for 4 h. Korea). Treatment conditions and experimental groups Modeling hypoxia and hypoxic preconditioning in Animals were separated into four groups: Control, zebrafish zebrafish kept in normoxic conditions for 2 h; HYPOXIA TM (HYP), zebrafish subjected to hypoxia followed by 2 h A glass box attached to one pouch of GasPak (BD) was recovery; HYPOXIC PRECONDITIONING (HPC), zebrafish used as a closed hypoxia chamber (Figure 1(A)). Hypoxia subjected to hypoxic preconditioning followed by 4 h chambers were filled aquarium water that was pre-equi- recovery; and HPC + HYP, zebrafish subjected to a librated in the hypoxia chamber for at least one night sequence of hypoxic preconditioning, 4 h recovery, prior to zebrafish transfer to ensure appropriate hypoxia, and 2 h recovery (Figure 1(B)). After treatment, hypoxic condition. all zebrafish performed either the locomotor activity or Zebrafish showed a reliable sequence of behaviors, as T-maze experiments. To remove the brain, zebrafish described previously (Braga et al. 2013). The zebrafish in were anesthetized using MS-222 (tricaine, Sigma- the hypoxic group were exposed to hypoxic conditions Aldrich) and euthanized by decapitation. up to the third stage of the hypoxia (maintenance of opercular beats with brief movements), characterized by a critical, but non-lethal, condition. The zebrafish in TTC staining the hypoxic preconditioning group were exposed to hypoxic conditions for 30 s, until the first stage of 2,3,5-triphenyltetrazolium chloride (TTC) staining was hypoxia (swimming at the top). Following hypoxic incu- used to evaluate the activity of brain mitochondrial bation, zebrafish were removed from the hypoxia dehydrogenases. TTC staining was performed 2 or 4 h chamber and immediately transferred in a normoxia after the hypoxic or hypoxic preconditioning treatments. chamber. Whole brains were incubated, in darkness, with 1 mL of 12 Y.-H. KIM ET AL. 2% TTC (Sigma-Aldrich, St. Louis, MO, USA) phosphate buffer saline-based solution. For staining only, brains were incubated for 40 min at 37°C. After staining, TTC sol- ution was discarded and brains were placed in 4% paraf- ormaldehyde overnight. Images were taken the next day. For extracting, brains were incubated for 100 min at 37°C. TTC solution was discarded after staining and brains were gently rinsed with 2–3 drops of DMSO/ethanol (1:1) solution, and then placed in 1.5 mL tubes with 1 mL DMSO/ethanol solution, in darkness, overnight. The next day, brains were removed from the tubes prior to absorbance measurements by spectropho- tometer (Epoch, BioTek Instruments, USA). Brains were weighed (mg) before absorbance values were tested. Figure 2. Three-dimensional T-maze. The colors indicate the two Locomotor activity goal arms; red for the right arm and yellow for the left arm. The assessments of locomotor activities of the zebrafish included time spent mobile, meandering (absolute turn testing. During the memory test, there was no colored angle divided by the time mobile), and absolute turn cellophane or food reward in the T-maze. All the pro- angle (variations in the direction of the center point of cesses of the memory test were recorded with an the animal) in the total area of the T-maze where the OMEX camera and analyzed with an EthoVision XT zebrafish swam. Horizontal exploration represented the (Noldus) program. tendency of a zebrafish to explore whole areas. All data analyzed were from T-maze experiments. All locomotor activities were analyzed by EthoVision XT (Noldus) Statistical analysis program. All data were expressed as the mean (column) and stan- dard error of the mean (error bar). The T-maze data were T-maze experiment analyzed using the t-test or Mann–Whitney test. The locomotor activity data were analyzed using the one- For learning and memory, we used the protocol as way ANOVA with post hoc Bonferroni’s multiple compari- described previously, with minor modifications (Kim son test. All data were analyzed using SPSS 20.0 software. et al. 2017). All experiments were conducted between P values < 0.05 were regarded as significant. 10:00 and 16:00. The T-maze consisted of two arms and one stem. There was a start box (length 10 cm × width 10 cm × height 10 cm) on the bottom of the stem Results (50 cm × 10 cm × 10 cm) of the maze and it was divided by a transparent sliding door. Two target com- Forty-seven zebrafish were used in this study (11 control, partments (10 cm × 10 cm × 10 cm) were located at the 11 HYP, 12 HPC, and 13 HPC + HYP). The average time to end of both arms of the maze (20 cm × 10 cm × 10 cm) reach the third stage of hypoxia in the hypoxic chamber (Figure 2). Another transparent door was used to separ- was 10 min ± 30 s. ate the arms of the maze from the stem. The sleeves, TTC staining revealed deep red staining of the brains made of red or yellow–red cellophane, were designed of healthy zebrafish, while hypoxic-treated zebrafish to fit around the target compartments at the end of brains had more unstained areas by comparison. The each arm. ratio of absorbance to brain weight after TTC staining To minimize bias, all zebrafish were subjected to a was significantly less in the HYP group compared with habituation trial for 2 h before testing. Each zebrafish the other groups (Figure 3), while that of the HPC + underwent one trial per day during four consecutive HYP group was comparable to the control and HPC training days. During training periods, 20 µL of food groups (Figure 3). These findings showed that hypoxic (brine shrimp) was placed in the red cellophane com- preconditioning treatment significantly improved TTC partment before each zebrafish was placed into the absorbance comparing to the hypoxic-only group. start box. On the fifth day after the 4-day training The time spent mobile, absolute turn angle, and period, all experimental zebrafish underwent memory meandering showed no difference in all groups after ANIMAL CELLS AND SYSTEMS 13 in the compartment where red cellophane and food reward. However, hypoxic-treated zebrafish did not differentiate the distance moved in the compartment where red cellophane and food reward (Figure 5(B)) Zebrafish in the Control, HPC, and HPC + HYP groups increased the total number of entries in the compart- ment where red cellophane and food reward. However, the total number of entries did not differ between the right and left compartments in the HYP group (Figure 5 (C)). Figure 5(D) shows the movement trends for each group. Unlike the HYP group, the control, HPC, and HPC + HYP groups showed the preference for the red and reward compartment compared to the yellow com- partment, which was clearly shown in the moving trends. These findings consistently indicate that hypoxia elimin- ates the memory of training, and hypoxic precondition- ing prevents these adverse effects and preserves memory in zebrafish. Discussion Our findings suggest that hypoxic preconditioning pre- vents hypoxic-ischemic brain tissue and function damage, specifically, hypoxic-ischemic-induced memory deficits, as evaluated by the T-maze behavior test. Since brain hypoxia-ischemia is a common disorder with high Figure 3. Zebrafish brain injury detected by TTC staining. (A) morbidity and mortality, there have been many studies TTC-stained zebrafish brain sections: Control, HPC, HYP, and on ischemia; however, no breakthrough therapy for ische- HPC + HYP. Scale bar, 20 μm. The square indicates an unstained mia has yet been established. Although many researchers area. (B) Spectrophotometric measurement (*p < .05, **p < .01, have made note of ischemic preconditioning, there are ***p < .001, each group n =5). difficulties in confirming its protective effects in animal Note: TTC = 2,3,5-triphenyltetrazolium chloride, control = Zebrafish kept in normoxic conditions for 2 h, HYP = Zebrafish subjected to hypoxia and kept models. Our report is the first to confirm protective in normoxic conditions for 2 h, HPC = Zebrafish subjected to hypoxic precon- effects of ischemic preconditioning against hypoxia- ditioning and kept in normoxic conditions for 4 h, HPC + HYP = Zebrafish first subjected to hypoxic preconditioning and kept in normoxic conditions for 4 h induced brain damage in zebrafish in terms of memory. and then subjected to hypoxia and kept in normoxic conditions for 2 h. In this study, we first evaluated the suitability of the zebrafish hypoxic preconditioning model. While previous 2 h of recovery time (Figure 4). These findings indicate studies have used a nitrogen perfusion method to set up TM that the results of our behavioral tests and subsequent the hypoxic chamber, we instead used GasPak . Under TM analysis are valid. the hypoxic condition induced by GasPak , TTC-defined The control and HPC groups spent significantly more brain damage in zebrafish was observed and became time in the red + reward compartment than in the yellow worse with increasing lengths of the hypoxic period. compartment compared with the HYP group (Figure 5 These findings indicate a clear correlation between (A), Control; ***p < .001, HPC; **p < .01). The HYP group hypoxia duration and brain damage. In addition, similarly spent time in both compartments, which indi- zebrafish behavioral impairments, which are known to cated loss of memory. The HPC + HYP group spent sig- be present in hypoxic conditions, have been observed nificantly more time in the red + reward compartment with the prolonged hypoxia in zebrafish (Braga et al. than in the yellow compartment, as did the control 2013). Therefore, our ischemic animal model showed group (Figure 5(A), HYP, ***p < .001). The control, HPC results consistent with previous studies, suggesting the and HPC + HYP groups had similar results in terms of dis- usefulness and effectiveness of this model for exper- tance moved and the total number of entries as well, iments designed to look at hypoxia and its effects. while the HYP group performed significantly differently A portable dissolved oxygen meter was used to in both of these measures (Figure 5(B,C)). Zebrafish in confirm whether the oxygen concentration had the Control, HPC, and HPC + HYP groups moved more reached 1.0 ± 0.5 mg/L DO in the hypoxia chambers. 14 Y.-H. KIM ET AL. Figure 4. Locomotor activities including (A) the total time spent mobile, (B) absolute turn angle and (C) meandering after 2 h of recov- ery time. There was no difference among the groups. Note: Control = zebrafish kept in normoxic conditions for 2 h, HYP = zebrafish subjected to hypoxia and kept in normoxic conditions for 2 h, HPC = zebrafish subjected to hypoxic preconditioning and then kept in normoxic conditions for 4 h, HPC + HYP = zebrafish subjected to hypoxic preconditioning and kept in normoxic conditions for 4 h and then subjected to hypoxia and kept in normoxic conditions for 2 h. When the zebrafish reached the third stage of hypoxia memory, and locomotor activities in zebrafish (Vignet (maintenance of opercular beats with brief movements), et al. 2013; Braida et al. 2014). we considered it severe hypoxia and conducted the Assessment of locomotor activities, including total experiment (Braga et al. 2016). Cerebral injury was eval- time spent mobile, turn angle, and meandering, is uated by TTC staining, which is a widely-used method useful in determining the presence or absence of abnor- to measure hypoxic brain damage (Yu and Li 2011). To mal movement of the zebrafish (Spink et al. 2001). Total quantify TTC staining and brain damage, the TTC stain time spent mobile represents the total time that the was extracted from the zebrafish brain and the absor- animal was mobile in the zone. Absolute turn angle indi- bance was measured using a spectrophotometric assay cates the sum of the absolute angle between each move- following standard procedures. The brains of the ment vector of the animal while it was inside the hypoxic preconditioning-treated zebrafish were stained compartment. Meander describes the change in direc- less than those of the control group, but were, overall, tion of movement of the animal relative to the distance more stained than those of the hypoxic-treated group. it moves; therefore, it can be used to compare the These results indicate that the hypoxic preconditioning amount of turning of animals traveling at different treatment was protective against hypoxic-ischemic speeds. brain injury. A previous study, which was instrumental in our In this study, we focused on the utility of zebrafish as a research design, showed that locomotor activities were behavioral model. Several zebrafish behavioral models not restored by 1 h after hypoxia, but were restored have been developed and showed usefulness in evaluat- after 3 h (Braga et al. 2013). In this study, zebrafish in ing cognitive function, learning, and memory (Levin and the hypoxic group were exposed to 10 min of hypoxia. Chen 2004; Collier et al. 2014). Sison and Gerlai showed After 2 h, the parameter values of locomotion activity, associative memory with visual perception in zebrafish including time spent mobile, absolute turn angle, and (2010). Furthermore, beyond simple associative learning, meandering, were not different from those in the spatial learning is present in zebrafish, pointing to the control group. In other words, the abnormal behavior validity of using the zebrafish model in behavioral neuro- of zebrafish due to hypoxic-ischemia insult was normal- science. Zebrafish display rapid and reliable condition- ized to show no difference from the control group’s ing, well-suited for neurobehavioral tests. Moreover, behaviors after 2 h of recovery time. If a zebrafish exhi- T-maze method may be useful to evaluate learning, bits abnormal behavior, this abnormal movement itself ANIMAL CELLS AND SYSTEMS 15 Figure 5. Recall ability of zebrafish in the color preference and food reward memory test. (A) Time spent, (B) distance moved and (C) the total number of entries of zebrafish in each compartment were measured in all groups. (D) The movement of each group of zebrafish and their transitions were tracked for a total of five minutes. (control, n = 11; HYP, n = 11; HPC, n = 12; HPC + HYP, n = 13). *p < .05, **p < .01, ***p < .001. Note: Control = Zebrafish kept in normoxic conditions for 2 h, HYP = Zebrafish subjected to hypoxia and kept in normoxic conditions for 2 h, HPC = Zebrafish subjected to hypoxic preconditioning and then kept in normoxic conditions for 4 h, HPC + HYP = Zebrafish subjected to hypoxic preconditioning and kept in normoxic conditions for 4 h and then subjected to hypoxia and kept in normoxic conditions for 2 h. can affect the evaluation behavior indices of zebrafish, so were assessed for memory retention. In this study, we that the memory function measured in T-maze becomes showed that memory was maintained more in the unreliable. Therefore, we evaluated the memory of ischemic preconditioning group than in the hypoxia- the zebrafish through behavioral assessment using only group. This confirms that ischemic preconditioning T-maze after 2 h. has the effect of reducing the memory impairment of The principal zebrafish T-maze study is based on zebrafish due to hypoxia. visual discrimination learning (Colwill et al. 2005). We Many clinical trials focus on remote ischemic precondi- used this protocol, using a T-maze apparatus with food tioning models because the brain is so sensitive to reward and color enhancement as per our previous ischemic stimulation that it is difficult to administer paper (Kim et al. 2017). In brief, the animal tested starts proper ischemic preconditioning directly to the brain. out at the bottom of the T (the start box on the edge While successful application of remote organ ischemic of the stem). To teach it to choose the ‘correct’ target preconditioning has been shown in the rat model, it (the red compartment during the training period), posi- was hard to induce remote preconditioning in zebrafish tive stimuli (food reward) are used to motivate the (Dave et al. 2006). Our study focuses on the effects of animal. This method evaluates memory based on ischemic preconditioning prior to global ischemia. whether or not the zebrafish moves to the ‘correct’ Because zebrafish are resistant to low oxygen levels, target compartment. On the fifth experimental day unlike other mammals, their physiological and behavioral after hypoxia and recovery periods, trained zebrafish response physiology and may differ from other species 16 Y.-H. KIM ET AL. that are unlikely to experience any hypoxic environment ORCID during adult life (Roesner et al. 2006). Young-Sung Kim http://orcid.org/0000-0001-8551-979X Other animal models of hypoxia show brain plasticity in stroke recovery with regards to activity-dependent rewir- ing and synapse strengthening (Murphy and Corbett References 2009). A previous zebrafish behavioral experiment (Braga Braga MM, Rico EP, Cordova SD, Pinto CB, Blaser RE, Dias RD, et al. 2013), mentioned above, showed rapid recovery of Rosemberg DB, Oliveira DL, Souza DO. 2013. Evaluation of behavioral impairment due to brain damage. Although spontaneous recovery of behavioral and brain injury this group suggest the occurrence of true recovery in the profiles in zebrafish after hypoxia. Behav Brain Res. Sep. 15;253:145–151. zebrafish hypoxic model, it is still unclear whether Braga MM, Silva ES, Moraes TB, Schirmbeck GH, Rico EP, Pinto hypoxic preconditioning leads to resistance to hypoxic CB, Rosemberg DB, Dutra-Filho CS, Dias RD, Oliveira DL, insult or promotion of damage recovery. In fact, our et al. 2016. Brain zinc chelation by diethyldithiocarbamate study considered rapid ischemic tolerance, not delayed tol- increased the behavioral and mitochondrial damages in erance. While rapid tolerance produces neuroprotection zebrafish subjected to hypoxia. Sci Rep. 6:20279. Braida D, Ponzoni L, Martucci R, Sparatore F, Gotti C, Sala M. within 1 h of the preconditioning event, independent of 2014. Role of neuronal nicotinic acetylcholine receptors new protein synthesis, classical or delayed ischemic toler- (nAChRs) on learning and memory in zebrafish. ance requires protein synthesis and changes in the Psychopharmacology. 231:1975–1985. genomic response after 24–72 h (Caldeira et al. 2014). Caldeira MV, Salazar IL, Curcio M, Canzoniero LM, Duarte CB. Further studies are required to investigate time courses 2014. Role of the ubiquitin-proteasome system in brain and events associated with recovery in more time ischemia: friend or foe? Prog Neurobiol. 112:50–69. Canazza A, Minati L, Boffano C, Parati E, Binks S. 2014. windows, including delayed ischemic tolerance. Experimental models of brain ischemia: a review of tech- To the best of our knowledge, this study is the first niques, magnetic resonance imaging, and investigational attempt to evaluate a behavioral model of hypoxic pre- cell-based therapies. Front Neurol. 5(19). conditioning in zebrafish. Zebrafish are easy to manage Collier AD, Khan KM, Caramillo EM, Mohn RS, Echevarria DJ. and our methods are easy to reproduce. Moreover, our 2014. Zebrafish and conditioned place preference: a transla- tional model of drug reward. Prog Neuropsychopharmacol protocol could be a useful method in future, similar Biol Psychiatry. 55:16–25. experiments, or in experiments designed to confirm Colwill RM, Raymond MP, Ferreira L, Escudero H. 2005. Visual the protective or toxic effects of other drugs in surgery discrimination learning in zebrafish (Danio rerio). Behav and anesthesia. Despite an abundance of research on Processes. 70:19–31. ischemic stroke, therapeutic development is slow. There- Conlon N, Grocott HP, Mackensen GB. 2008. Neuroprotection fore, it is necessary to develop and apply more research during cardiac surgery. Expert Rev Cardiovasc Ther. 6:503– models, and we believe our zebrafish model is a good Dave KR, Saul I, Prado R, Busto R, Perez-Pinzon MA. 2006. example. Finally, this study suggests that hypoxic pre- Remote organ ischemic preconditioning protect brain from conditioning can be evaluated by behavioral observation ischemic damage following asphyxial cardiac arrest. without examining brain tissue. Minimizing the damage Neurosci Lett. 404:170–175. to experimental animals, and reducing unnecessary Hossmann KA. 2006. Pathophysiology and therapy of exper- imental stroke. Cell Mol Neurobiol. 26:1057–1083. sacrifices, may be helpful in terms of animal ethics. 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Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test

Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test

Abstract

Perioperative brain ischemia and stroke are leading causes of morbidity and mortality. Brief hypoxic preconditioning is known to have protective effects against hypoxic-ischemic insult in the brain. Current studies on the neuroprotective effects of ischemic preconditioning are based on histologic findings and biomarker changes. However, studies regarding effects on memory are rare. To precondition zebrafish to hypoxia, they were exposed to a dissolved oxygen (DO) concentration of...
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© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
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2151-2485
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1976-8354
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10.1080/19768354.2018.1557743
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Abstract

ANIMAL CELLS AND SYSTEMS 2019, VOL. 23, NO. 1, 10–17 https://doi.org/10.1080/19768354.2018.1557743 Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test a a b c a Yun-Hee Kim , Kuen-Su Lee , Young-Sung Kim , Yeon-Hwa Kim and Jae-Hwan Kim a b Department of Anesthesiology and Pain Medicine, Korea University Ansan Hospital, Ansan, Korea; Department of Anesthesiology and Pain Medicine, Korea University Guro Hospital, Seoul, Korea; Institute of Medical Science, Korea University Ansan Hospital, Korea University College of Medicine, Ansan, Korea ABSTRACT ARTICLE HISTORY Received 3 August 2018 Perioperative brain ischemia and stroke are leading causes of morbidity and mortality. Brief hypoxic Revised 3 October 2018 preconditioning is known to have protective effects against hypoxic-ischemic insult in the brain. Accepted 15 November 2018 Current studies on the neuroprotective effects of ischemic preconditioning are based on histologic findings and biomarker changes. However, studies regarding effects on memory are KEYWORDS rare. To precondition zebrafish to hypoxia, they were exposed to a dissolved oxygen (DO) Hypoxic preconditioning; concentration of 1.0 ± 0.5 mg/L in water for 30 s. The hypoxic zebrafish were then exposed to ischemia; memory; zebrafish 1.0 ± 0.5 mg/L DO until the third stage of hypoxia, for 10 min ± 30 s. Zebrafish were assessed for memory retention after the hypoxic event. Learning and memory were tested using the T-maze, which evaluates memory based on whether or not zebrafish moves to the correct target compartment. In the hypoxic preconditioning group, infarct size was reduced compared with the hypoxic-only treated zebrafish group; memory was maintained to a degree similar to that in the hypoxia-untreated group. The hypoxic-only group showed significant memory impairments. In this study, we used a hypoxic zebrafish model and assessed the effects of ischemic preconditioning not only on histological damages but also on brain function, especially memory. This study demonstrated that a brief hypoxic event has protective effects in hypoxic brain damage and helped maintain memory in zebrafish. In addition, our findings suggest that the zebrafish model is useful in rapidly assessing the effects of ischemic preconditioning on memory. Introduction protects from later, severe, ischemic insults (Miao et al. 2010). Traditional ischemic preconditioning models The brain is one of the organs that are particularly vulner- include rat and rodent models that are used to investi- able to ischemia. Due to their high metabolic rates, brain gate underlying mechanisms and neuroprotective strat- cells easily lose their function and die in response to egies (Pan et al. 2014). Due to the importance of hypoxia-induced ischemic insults (Murphy et al. 2008). ischemic research, there are many basic studies investi- Brain ischemia and stroke are known to be the leading gating the degree of tissue damage in ischemic precon- causes of morbidity and mortality worldwide (Hossmann ditioning models. However, the difficulty of assessing 2006; Wardlaw et al. 2010; Canazza et al. 2014). The inci- memory function limits research and clinical dence of stroke after non-cardiovascular and non-neuro- applications. logic surgeries is estimated to be 0.05–7%, and the The zebrafish is a relatively small, simple organism, incidence after cardiac surgeries is estimated to be 2– but because it is a vertebrate, a zebrafish gene is likely 10%. The mortality from perioperative stroke is high to resemble a mammalian or human gene, and similar (Parikh and Cohen 1993; Conlon et al. 2008; Zhou et al. genes may be associated with human-like function 2016), and, therefore, monitoring and prevention of (Howe et al. 2013). Zebrafish models are being increas- intraoperative cerebral ischemia are very important. ingly employed in neuroscience and can minimize bias Hypoxic preconditioning has a strong neuroprotective in experimental results by improving ease of setting effect against cerebral ischemic injuries and periopera- experimental conditions and observation efficiency tive cerebral ischemia/reperfusion injury (Sharp et al. (Shams et al. 2018). A previous study related to the 2004; Sinanović 2010). Ischemic preconditioning is a zebrafish behavior change upon ischemic insult phenomenon in which short-term, non-fatal ischemia showed that hypoxia might induce a change in CONTACT Jae-Hwan Kim anejhkim@korea.ac.kr Department of Anesthesiology and Pain Medicine, Korea University Ansan Hospital, 123, Jeokgeum-ro, Danwon-gu, Ansan-si, Gyeonggi-do 15355, Korea © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MOLECULAR & CELLULAR BIOLOGY ANIMAL CELLS AND SYSTEMS 11 zebrafish brain physiology and behavior (Braga et al. 2013). These findings showed the utility of zebrafish behavior change to assess brain function, especially learning and memory. Current ischemia studies are based on histologic findings and biomarker changes, and studies regarding brain functional effects, especially memory are rare. Therefore, we evaluated whether ischemic preconditioning could attenuate brain tissue damage and preserve memory using the T-maze behav- ior test in zebrafish under conditions of low dissolved oxygen (DO) (Kim et al. 2017). Materials and methods Animals This study was approved by the Ethical Committee on Animal Research at the Korea University College of Medi- cine (approval No. KOREA-2018-0020). In all experiments, adult zebrafish (4-6 months of age and 2.5–3.5 cm long) purchased from a local aquarium store (Jincheon, Chung- cheongbuk-do, Korea) were used. The zebrafish were short-finned wild-type and had a heterogeneous genetic background. They were kept in water at 28.5°C Figure 1. Schematic of the hypoxic chamber and procedure. (A) TM GasPak attached with lid of the hypoxic chamber. The hypoxic with a light cycle of 14 h and a dark cycle of 10 h in chamber was filled with 300 mL of system water. (B) The training aquarium containers and were fed brine shrimp twice a session was composed of 4 trials on consecutive days. The day. The aquarium container was equipped with a multi- memory test was administered 24 h after the last training trial. stage filtration system that had a sediment filter, post- Hypoxia was induced before the memory test and recovery carbon filter, fluorescent UV light, and sterilizing filter was allowed for 2 h. Hypoxic preconditioning was induced (Zebrafish AutoSystem, Genomic Design, Daejeon, before the hypoxia and recovery were allowed for 4 h. Korea). Treatment conditions and experimental groups Modeling hypoxia and hypoxic preconditioning in Animals were separated into four groups: Control, zebrafish zebrafish kept in normoxic conditions for 2 h; HYPOXIA TM (HYP), zebrafish subjected to hypoxia followed by 2 h A glass box attached to one pouch of GasPak (BD) was recovery; HYPOXIC PRECONDITIONING (HPC), zebrafish used as a closed hypoxia chamber (Figure 1(A)). Hypoxia subjected to hypoxic preconditioning followed by 4 h chambers were filled aquarium water that was pre-equi- recovery; and HPC + HYP, zebrafish subjected to a librated in the hypoxia chamber for at least one night sequence of hypoxic preconditioning, 4 h recovery, prior to zebrafish transfer to ensure appropriate hypoxia, and 2 h recovery (Figure 1(B)). After treatment, hypoxic condition. all zebrafish performed either the locomotor activity or Zebrafish showed a reliable sequence of behaviors, as T-maze experiments. To remove the brain, zebrafish described previously (Braga et al. 2013). The zebrafish in were anesthetized using MS-222 (tricaine, Sigma- the hypoxic group were exposed to hypoxic conditions Aldrich) and euthanized by decapitation. up to the third stage of the hypoxia (maintenance of opercular beats with brief movements), characterized by a critical, but non-lethal, condition. The zebrafish in TTC staining the hypoxic preconditioning group were exposed to hypoxic conditions for 30 s, until the first stage of 2,3,5-triphenyltetrazolium chloride (TTC) staining was hypoxia (swimming at the top). Following hypoxic incu- used to evaluate the activity of brain mitochondrial bation, zebrafish were removed from the hypoxia dehydrogenases. TTC staining was performed 2 or 4 h chamber and immediately transferred in a normoxia after the hypoxic or hypoxic preconditioning treatments. chamber. Whole brains were incubated, in darkness, with 1 mL of 12 Y.-H. KIM ET AL. 2% TTC (Sigma-Aldrich, St. Louis, MO, USA) phosphate buffer saline-based solution. For staining only, brains were incubated for 40 min at 37°C. After staining, TTC sol- ution was discarded and brains were placed in 4% paraf- ormaldehyde overnight. Images were taken the next day. For extracting, brains were incubated for 100 min at 37°C. TTC solution was discarded after staining and brains were gently rinsed with 2–3 drops of DMSO/ethanol (1:1) solution, and then placed in 1.5 mL tubes with 1 mL DMSO/ethanol solution, in darkness, overnight. The next day, brains were removed from the tubes prior to absorbance measurements by spectropho- tometer (Epoch, BioTek Instruments, USA). Brains were weighed (mg) before absorbance values were tested. Figure 2. Three-dimensional T-maze. The colors indicate the two Locomotor activity goal arms; red for the right arm and yellow for the left arm. The assessments of locomotor activities of the zebrafish included time spent mobile, meandering (absolute turn testing. During the memory test, there was no colored angle divided by the time mobile), and absolute turn cellophane or food reward in the T-maze. All the pro- angle (variations in the direction of the center point of cesses of the memory test were recorded with an the animal) in the total area of the T-maze where the OMEX camera and analyzed with an EthoVision XT zebrafish swam. Horizontal exploration represented the (Noldus) program. tendency of a zebrafish to explore whole areas. All data analyzed were from T-maze experiments. All locomotor activities were analyzed by EthoVision XT (Noldus) Statistical analysis program. All data were expressed as the mean (column) and stan- dard error of the mean (error bar). The T-maze data were T-maze experiment analyzed using the t-test or Mann–Whitney test. The locomotor activity data were analyzed using the one- For learning and memory, we used the protocol as way ANOVA with post hoc Bonferroni’s multiple compari- described previously, with minor modifications (Kim son test. All data were analyzed using SPSS 20.0 software. et al. 2017). All experiments were conducted between P values < 0.05 were regarded as significant. 10:00 and 16:00. The T-maze consisted of two arms and one stem. There was a start box (length 10 cm × width 10 cm × height 10 cm) on the bottom of the stem Results (50 cm × 10 cm × 10 cm) of the maze and it was divided by a transparent sliding door. Two target com- Forty-seven zebrafish were used in this study (11 control, partments (10 cm × 10 cm × 10 cm) were located at the 11 HYP, 12 HPC, and 13 HPC + HYP). The average time to end of both arms of the maze (20 cm × 10 cm × 10 cm) reach the third stage of hypoxia in the hypoxic chamber (Figure 2). Another transparent door was used to separ- was 10 min ± 30 s. ate the arms of the maze from the stem. The sleeves, TTC staining revealed deep red staining of the brains made of red or yellow–red cellophane, were designed of healthy zebrafish, while hypoxic-treated zebrafish to fit around the target compartments at the end of brains had more unstained areas by comparison. The each arm. ratio of absorbance to brain weight after TTC staining To minimize bias, all zebrafish were subjected to a was significantly less in the HYP group compared with habituation trial for 2 h before testing. Each zebrafish the other groups (Figure 3), while that of the HPC + underwent one trial per day during four consecutive HYP group was comparable to the control and HPC training days. During training periods, 20 µL of food groups (Figure 3). These findings showed that hypoxic (brine shrimp) was placed in the red cellophane com- preconditioning treatment significantly improved TTC partment before each zebrafish was placed into the absorbance comparing to the hypoxic-only group. start box. On the fifth day after the 4-day training The time spent mobile, absolute turn angle, and period, all experimental zebrafish underwent memory meandering showed no difference in all groups after ANIMAL CELLS AND SYSTEMS 13 in the compartment where red cellophane and food reward. However, hypoxic-treated zebrafish did not differentiate the distance moved in the compartment where red cellophane and food reward (Figure 5(B)) Zebrafish in the Control, HPC, and HPC + HYP groups increased the total number of entries in the compart- ment where red cellophane and food reward. However, the total number of entries did not differ between the right and left compartments in the HYP group (Figure 5 (C)). Figure 5(D) shows the movement trends for each group. Unlike the HYP group, the control, HPC, and HPC + HYP groups showed the preference for the red and reward compartment compared to the yellow com- partment, which was clearly shown in the moving trends. These findings consistently indicate that hypoxia elimin- ates the memory of training, and hypoxic precondition- ing prevents these adverse effects and preserves memory in zebrafish. Discussion Our findings suggest that hypoxic preconditioning pre- vents hypoxic-ischemic brain tissue and function damage, specifically, hypoxic-ischemic-induced memory deficits, as evaluated by the T-maze behavior test. Since brain hypoxia-ischemia is a common disorder with high Figure 3. Zebrafish brain injury detected by TTC staining. (A) morbidity and mortality, there have been many studies TTC-stained zebrafish brain sections: Control, HPC, HYP, and on ischemia; however, no breakthrough therapy for ische- HPC + HYP. Scale bar, 20 μm. The square indicates an unstained mia has yet been established. Although many researchers area. (B) Spectrophotometric measurement (*p < .05, **p < .01, have made note of ischemic preconditioning, there are ***p < .001, each group n =5). difficulties in confirming its protective effects in animal Note: TTC = 2,3,5-triphenyltetrazolium chloride, control = Zebrafish kept in normoxic conditions for 2 h, HYP = Zebrafish subjected to hypoxia and kept models. Our report is the first to confirm protective in normoxic conditions for 2 h, HPC = Zebrafish subjected to hypoxic precon- effects of ischemic preconditioning against hypoxia- ditioning and kept in normoxic conditions for 4 h, HPC + HYP = Zebrafish first subjected to hypoxic preconditioning and kept in normoxic conditions for 4 h induced brain damage in zebrafish in terms of memory. and then subjected to hypoxia and kept in normoxic conditions for 2 h. In this study, we first evaluated the suitability of the zebrafish hypoxic preconditioning model. While previous 2 h of recovery time (Figure 4). These findings indicate studies have used a nitrogen perfusion method to set up TM that the results of our behavioral tests and subsequent the hypoxic chamber, we instead used GasPak . Under TM analysis are valid. the hypoxic condition induced by GasPak , TTC-defined The control and HPC groups spent significantly more brain damage in zebrafish was observed and became time in the red + reward compartment than in the yellow worse with increasing lengths of the hypoxic period. compartment compared with the HYP group (Figure 5 These findings indicate a clear correlation between (A), Control; ***p < .001, HPC; **p < .01). The HYP group hypoxia duration and brain damage. In addition, similarly spent time in both compartments, which indi- zebrafish behavioral impairments, which are known to cated loss of memory. The HPC + HYP group spent sig- be present in hypoxic conditions, have been observed nificantly more time in the red + reward compartment with the prolonged hypoxia in zebrafish (Braga et al. than in the yellow compartment, as did the control 2013). Therefore, our ischemic animal model showed group (Figure 5(A), HYP, ***p < .001). The control, HPC results consistent with previous studies, suggesting the and HPC + HYP groups had similar results in terms of dis- usefulness and effectiveness of this model for exper- tance moved and the total number of entries as well, iments designed to look at hypoxia and its effects. while the HYP group performed significantly differently A portable dissolved oxygen meter was used to in both of these measures (Figure 5(B,C)). Zebrafish in confirm whether the oxygen concentration had the Control, HPC, and HPC + HYP groups moved more reached 1.0 ± 0.5 mg/L DO in the hypoxia chambers. 14 Y.-H. KIM ET AL. Figure 4. Locomotor activities including (A) the total time spent mobile, (B) absolute turn angle and (C) meandering after 2 h of recov- ery time. There was no difference among the groups. Note: Control = zebrafish kept in normoxic conditions for 2 h, HYP = zebrafish subjected to hypoxia and kept in normoxic conditions for 2 h, HPC = zebrafish subjected to hypoxic preconditioning and then kept in normoxic conditions for 4 h, HPC + HYP = zebrafish subjected to hypoxic preconditioning and kept in normoxic conditions for 4 h and then subjected to hypoxia and kept in normoxic conditions for 2 h. When the zebrafish reached the third stage of hypoxia memory, and locomotor activities in zebrafish (Vignet (maintenance of opercular beats with brief movements), et al. 2013; Braida et al. 2014). we considered it severe hypoxia and conducted the Assessment of locomotor activities, including total experiment (Braga et al. 2016). Cerebral injury was eval- time spent mobile, turn angle, and meandering, is uated by TTC staining, which is a widely-used method useful in determining the presence or absence of abnor- to measure hypoxic brain damage (Yu and Li 2011). To mal movement of the zebrafish (Spink et al. 2001). Total quantify TTC staining and brain damage, the TTC stain time spent mobile represents the total time that the was extracted from the zebrafish brain and the absor- animal was mobile in the zone. Absolute turn angle indi- bance was measured using a spectrophotometric assay cates the sum of the absolute angle between each move- following standard procedures. The brains of the ment vector of the animal while it was inside the hypoxic preconditioning-treated zebrafish were stained compartment. Meander describes the change in direc- less than those of the control group, but were, overall, tion of movement of the animal relative to the distance more stained than those of the hypoxic-treated group. it moves; therefore, it can be used to compare the These results indicate that the hypoxic preconditioning amount of turning of animals traveling at different treatment was protective against hypoxic-ischemic speeds. brain injury. A previous study, which was instrumental in our In this study, we focused on the utility of zebrafish as a research design, showed that locomotor activities were behavioral model. Several zebrafish behavioral models not restored by 1 h after hypoxia, but were restored have been developed and showed usefulness in evaluat- after 3 h (Braga et al. 2013). In this study, zebrafish in ing cognitive function, learning, and memory (Levin and the hypoxic group were exposed to 10 min of hypoxia. Chen 2004; Collier et al. 2014). Sison and Gerlai showed After 2 h, the parameter values of locomotion activity, associative memory with visual perception in zebrafish including time spent mobile, absolute turn angle, and (2010). Furthermore, beyond simple associative learning, meandering, were not different from those in the spatial learning is present in zebrafish, pointing to the control group. In other words, the abnormal behavior validity of using the zebrafish model in behavioral neuro- of zebrafish due to hypoxic-ischemia insult was normal- science. Zebrafish display rapid and reliable condition- ized to show no difference from the control group’s ing, well-suited for neurobehavioral tests. Moreover, behaviors after 2 h of recovery time. If a zebrafish exhi- T-maze method may be useful to evaluate learning, bits abnormal behavior, this abnormal movement itself ANIMAL CELLS AND SYSTEMS 15 Figure 5. Recall ability of zebrafish in the color preference and food reward memory test. (A) Time spent, (B) distance moved and (C) the total number of entries of zebrafish in each compartment were measured in all groups. (D) The movement of each group of zebrafish and their transitions were tracked for a total of five minutes. (control, n = 11; HYP, n = 11; HPC, n = 12; HPC + HYP, n = 13). *p < .05, **p < .01, ***p < .001. Note: Control = Zebrafish kept in normoxic conditions for 2 h, HYP = Zebrafish subjected to hypoxia and kept in normoxic conditions for 2 h, HPC = Zebrafish subjected to hypoxic preconditioning and then kept in normoxic conditions for 4 h, HPC + HYP = Zebrafish subjected to hypoxic preconditioning and kept in normoxic conditions for 4 h and then subjected to hypoxia and kept in normoxic conditions for 2 h. can affect the evaluation behavior indices of zebrafish, so were assessed for memory retention. In this study, we that the memory function measured in T-maze becomes showed that memory was maintained more in the unreliable. Therefore, we evaluated the memory of ischemic preconditioning group than in the hypoxia- the zebrafish through behavioral assessment using only group. This confirms that ischemic preconditioning T-maze after 2 h. has the effect of reducing the memory impairment of The principal zebrafish T-maze study is based on zebrafish due to hypoxia. visual discrimination learning (Colwill et al. 2005). We Many clinical trials focus on remote ischemic precondi- used this protocol, using a T-maze apparatus with food tioning models because the brain is so sensitive to reward and color enhancement as per our previous ischemic stimulation that it is difficult to administer paper (Kim et al. 2017). In brief, the animal tested starts proper ischemic preconditioning directly to the brain. out at the bottom of the T (the start box on the edge While successful application of remote organ ischemic of the stem). To teach it to choose the ‘correct’ target preconditioning has been shown in the rat model, it (the red compartment during the training period), posi- was hard to induce remote preconditioning in zebrafish tive stimuli (food reward) are used to motivate the (Dave et al. 2006). Our study focuses on the effects of animal. This method evaluates memory based on ischemic preconditioning prior to global ischemia. whether or not the zebrafish moves to the ‘correct’ Because zebrafish are resistant to low oxygen levels, target compartment. On the fifth experimental day unlike other mammals, their physiological and behavioral after hypoxia and recovery periods, trained zebrafish response physiology and may differ from other species 16 Y.-H. KIM ET AL. that are unlikely to experience any hypoxic environment ORCID during adult life (Roesner et al. 2006). Young-Sung Kim http://orcid.org/0000-0001-8551-979X Other animal models of hypoxia show brain plasticity in stroke recovery with regards to activity-dependent rewir- ing and synapse strengthening (Murphy and Corbett References 2009). A previous zebrafish behavioral experiment (Braga Braga MM, Rico EP, Cordova SD, Pinto CB, Blaser RE, Dias RD, et al. 2013), mentioned above, showed rapid recovery of Rosemberg DB, Oliveira DL, Souza DO. 2013. Evaluation of behavioral impairment due to brain damage. Although spontaneous recovery of behavioral and brain injury this group suggest the occurrence of true recovery in the profiles in zebrafish after hypoxia. Behav Brain Res. Sep. 15;253:145–151. zebrafish hypoxic model, it is still unclear whether Braga MM, Silva ES, Moraes TB, Schirmbeck GH, Rico EP, Pinto hypoxic preconditioning leads to resistance to hypoxic CB, Rosemberg DB, Dutra-Filho CS, Dias RD, Oliveira DL, insult or promotion of damage recovery. In fact, our et al. 2016. Brain zinc chelation by diethyldithiocarbamate study considered rapid ischemic tolerance, not delayed tol- increased the behavioral and mitochondrial damages in erance. While rapid tolerance produces neuroprotection zebrafish subjected to hypoxia. Sci Rep. 6:20279. Braida D, Ponzoni L, Martucci R, Sparatore F, Gotti C, Sala M. within 1 h of the preconditioning event, independent of 2014. Role of neuronal nicotinic acetylcholine receptors new protein synthesis, classical or delayed ischemic toler- (nAChRs) on learning and memory in zebrafish. ance requires protein synthesis and changes in the Psychopharmacology. 231:1975–1985. genomic response after 24–72 h (Caldeira et al. 2014). Caldeira MV, Salazar IL, Curcio M, Canzoniero LM, Duarte CB. Further studies are required to investigate time courses 2014. Role of the ubiquitin-proteasome system in brain and events associated with recovery in more time ischemia: friend or foe? Prog Neurobiol. 112:50–69. 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Journal

Animal Cells and SystemsTaylor & Francis

Published: Jan 2, 2019

Keywords: Hypoxic preconditioning; ischemia; memory; zebrafish

References