Abstract

Severe tissue hypoxia, resulting from cardiac dysfunction for example, is increasingly recognized as a trigger for neurobehavioral change. However, the impact of tissue hypoxia on anxiety-like behavior and disinhibition during development remains poorly understood. Zebrafish larvae provide an effective vertebrate model to evaluate how hypoxia influences behavior in early life. We assigned larvae to three groups: (1) normoxic control, (2) acute severe hypoxic exposure (HE) using cardiac dysfunction as evidence of exposure severity, or (3) HE plus 12 h exposure to 0.3 μmol AZ3 (p38 MAPK inhibitor). At one- and 2-weeks post-exposure we quantified survival, morphometrics, and behavioral measurements (emergence latency, thigmotaxis, zone occupancy, novel object approach, and velocity). Survival differed significantly among all groups (P < 0.001). Untreated HE reduced occupancy of the novel object zone 21% relative to controls at 2 weeks (P = 0.04), indicating diminished neophilia (attraction to novel objects). p38 MAPK inhibition increased body mass (P < 0.001 vs. controls) and prevented the length restriction seen in HE, but also increased by 17% the time spent in thigmotaxis zone compared to HE larvae alone (P = 0.04), consistent with increased anxiety-like behavior. Neither emergence latency nor velocity differed between groups, but both variables changed in the three groups after 1 week decreasing and increasing respectively. Novelty approach distance was unaffected by treatment. Thus, acute early-life hypoxic exposure in zebrafish—severe enough to induce cardiac injury among other forms of likely tissue damage—results in statistically significant but modest decreases in exploratory behavior. P38 MAPK inhibition stimulates growth compared to controls, and also decreases exploratory behavior compared to hypoxic exposure alone and promotes small anxiety-like responses without altering general locomotion. The results highlight the importance of integrating behavioral endpoints into hypoxia exposure research and suggest that targeting p38 MAPK may carry neurobehavioral trade-offs.

1 Introduction

Both the vertebrate heart and the brain are very high-energy demanding organs, relying on high oxygen requirements to produce sufficient ATP to sustain metabolic processes (Mir et al., 2025; Özugur et al., 2020; Ventura-Clapier et al., 2011; Watts et al., 2018). The tight coupling between neuronal activity and substrate delivery becomes disrupted when cerebral blood flow decreases (Kanat et al., 2003). Such disruption can arise for a variety of reasons, often a result of cardiac dysfunction. Systemic exposure to acute severe hypoxia in zebrafish larvae, resembling myocardial infarction, causes oxidative damage to multiple organs, including brain (Napolitano et al., 2018; Sawahata et al., 2021; Silva et al., 2016) and heart (Burggren et al., 2024; Zou et al., 2019). The cardiovascular parameters that contribute to neurological consequences extend far beyond simple “pump failure,” after MI, encompassing blood pressure dynamics, inflammatory signaling, barrier function, and cerebrovascular regulation—all of which must be considered when evaluating and treating humans with cardiac disease at risk for neurological complications.

We focused on larval zebrafish to investigate whether acute hypoxic injury during a critical developmental window—when cardiac and neural systems are actively maturing—establishes lasting behavioral phenotypes. Early-life exposure paradigms are particularly relevant for understanding developmental origins of adult disease and behavioral disorders.

One of the poorly explored impacts of the complex relationship between brain function and cardiac perfusion is subtle changes in behavior. Myocardial infarction has long been recognized as a condition inducing behavioral changes, for example inducing psychological stress and behavioral impairment in humans (Murphy et al., 2020; Murphy et al., 2015) and reducing exploratory behavior in experimental models (Schoemaker and Smitst, 1994). Presumably, the main effector is the severe tissue hypoxia resulting from cardiac dysfunction. However, little is known from the clinical perspective specifically regarding “boldness” in humans with cardiovascular diseases leading to tissue hypoxia. Anxiety-like behavior was evaluated in rats after myocardial infarction, with epigenetic disruption of the hippocampus being one of the mechanisms involved in increasing anxiety (Zhou et al., 2020).

Anxiety-like behavior and boldness represent two closely related yet distinct dimensions of animal personality and affective function (Khursigara et al., 2024). Anxiety is characterized by heightened caution, avoidance, and increased stress responses to perceived threats. Boldness reflects an individual’s propensity to take risks and explore novel environments. Boldness (and its counterpart, shyness) is included in a framework of 16 personality factors (Cattell and Mead, 2008). Boldness behavior is also associated with exploration in animal behavior (Dean et al., 2020; Hamilton et al., 2021; Ólafsdóttir and Magellan, 2016; Wisenden et al., 2011). We employed the novel object approach test, validated for measuring neophilia in 14–21 dpf zebrafish larvae, with maximal responses observed at 14 dpf. Neophilia—attraction to novel objects—serves as an established proxy for boldness in fish behavior, reflecting risk-taking propensity and exploratory drive (Dahlbom et al., 2011; Dean et al., 2020). Thigmotaxis (wall-hugging behavior) is validated as an anxiety-like measure in zebrafish larvae from 7 dpf onward (Richendrfer et al., 2012; Schnörr et al., 2012). These behavioral constructs are evolutionarily conserved across vertebrates, facilitating translation to mammalian systems.

Our main aim was to explore the link between acute systemic tissue hypoxia and subsequent chronic behavioral effects in the zebrafish larvae. We used cardiac arrest and associated myocardial damage as a proxy for hypoxia-induced systemic damage—essentially, modeling effects of human myocardial infarction, rather than chronic cardiac disease. We tested the hypothesis in the zebrafish larval model that acute systemic tissue hypoxia will cause subsequent chronic changes in behavior as the larvae continue to grow and develop. We also tested the effects on hypoxia-related behavioral alterations of p38 mitogen-activated protein kinase (p38 MAPK). P38 MAPK is involved in multiple body systems, responding mainly to stress, and inflammation (Wang et al., 2024; Zhang et al., 2007). P38 MAPK plays essential roles in cardiovascular, nervous, immune, hepatic, gastrointestinal, and reproductive systems (Han et al., 2020). This protein kinase has been proposed as a therapeutical target to induce cardiac regeneration, being a stress-responsive kinase that regulates cardiac inflammation, cell death, hypertrophy, and contractility (Bassi et al., 2008). However, there is still no supporting clinical evidence as a treatment for any inflammatory diseases (Wang et al., 2024). Furthermore, there are no studies addressing myocardial recovery plus behavioral evaluation (as part of potential side effects) involving protein kinases (Asih et al., 2020; Corrêa and Eales, 2012). Similar to its role in cardiac tissue, p38α is also the most extensively studied p38 isoform in the brain (Asih et al., 2020; Corrêa and Eales, 2012). Based on our previous experiments, where cardiac function increased in larvae experiencing myocardial hypoxic injury after p38 MAPK inhibition (Vazquez Roman, 2024) (article in preparation), we have analyzed this compound’s behavioral effects on larvae following systemic tissue hypoxia severe enough to cause cardiac damage and arrest. We tested the hypothesis that p38 inhibition will modify the behavioral changes (anxiety-like and boldness-like behaviors) in zebrafish larvae produced by hypoxic injury.

The zebrafish as a model offers a tractable in vivo system to jointly assess the effects of severe hypoxia on behavior. By quantifying thigmotaxis, emergence latency, swimming velocity, exploration, and novel-object approach at 1- and 2-weeks post-hypoxic exposure, our study sought to correlate acute severe tissue hypoxia, cardiac damage, pharmacologic recovery, and behavioral effects into an integrated framework that can inform mechanisms, side-effect profiles, and potential prognostic markers.

2 Material and method2.1 Zebrafish sources, husbandry and breeding

Adult wild-type (AB strain) zebrafish were obtained from a commercial supplier in Denton, Texas. All adult fish were maintained under the same controlled conditions: Aquarium water containing 60 mg of Instant Ocean Aquarium Sea Salt per 1 liter of Deionized (DI) Water, maintained at 28 ± 0.5°C, with 14:10 h light:dark photoperiod, fed twice a day (∼3% body weight) with Tetramin Tropical Flakes and once a day with frozen brine shrimp (Artemia sp.) on nauplii stage. Water quality parameters were assessed daily (pH 7–7.6, ammonia, nitrates and nitrites at ∼0 ppm). Healthy adult zebrafish (4–6 months old) were used as breeding stock in the Aquatic Facilities at the University of North Texas. Females and males (∼100 fish per tank) were kept together in large glass tanks (∼90 L).

Zebrafish typically breed when the laboratory lights turn on (08:00). During the night before the planned breeding, small breeding tanks with artificial plastic plants as enrichment were placed attached to the inside glass wall of the larger tanks. At breeding, adult fish came and swam above the breeding tanks. Hence, as a result of multiple breeding pairs, large numbers of fertilized eggs fell to the bottom of the breeding tanks and were collected and washed with aquarium water.

Viable embryos were separated from infertile eggs and reared in E3 media. All surviving embryos hatched at ∼4 dpf. The larvae were fed twice daily with Otohime™ A1 Starter larvae food, alternated with A2 at 14 dpf (PTAqua, Dublin, Ireland), sprinkled on the medium surface. Larvae were always held in approximately the same density (∼50 larvae/L) to avoid differences in growth that can emerge from crowding. They were reared in static tanks with water changed twice a day.

All experiments were conducted under University of North Texas Institutional Animal Care and Use Committee permit # 18005.

2.2 Hypoxia and p38 MAPK inhibitor exposures

Zebrafish larvae were divided into three groups at 7 dpf. The first group—designated the controls—was held in normoxic aquarium water and never exposed to hypoxia or p38 MAPK inhibitor treatment. Boldness in zebrafish can be dictated by both the strain of zebrafish and the test context (Mustafa et al., 2019). Consequently, a control group was used in every replicate to reduce the effect caused by strain/age of parents, or other extrinsic factors influencing variation as a confounding result.

The second group was exposed to acute severe ambient hypoxia (∼1 kPa) for a period of ∼20 min. This was a time judged to be sufficient to induce at least myocardial hypoxic injury. Both our preliminary experiments and previously published research showed that zebrafish larvae experience cardiac arrest after ∼20 min in severe hypoxic exposure (Figure 1; Burggren et al., 2024). This acute severe hypoxic exposure results not only in cardiac arrest, but also causes cardiomyocyte apoptosis, determined by observation of colocalization of molecular markers Cardiac Troponin T and Cleaved Caspase-3 (Burggren et al., 2024). Very likely additional tissues, including those of the brain and other neural tissue, are similarly damaged by this level of systemic hypoxia (Braga et al., 2013; Zeng et al., 2022). In the current experiment, pooled zebrafish larvae from the same clutch (∼100 larvae enclosed in a submerged glass chamber) were exposed to ∼1 KPa for ∼20 min. Following exposure, the pooled larvae were returned to normoxic water. Larvae under these acute severe hypoxic conditions were presumed to experience cardiovascular damage (if not cardiac arrest and possible subsequent death) due to the identical treatment as the larval zebrafish in the study of Burggren et al. (2024). This second group received no p38 MAPK inhibitor treatment.

Comparison time taken by zebrafish larvae to reach cardiac arrest during acute severe (1 KPa O2) hypoxic exposure. Cardiac arrest, readily observed in larval zebrafish through the translucent body wall, was used as a proxy for systemic hypoxic damage. Individual larval data are plotted. N values are indicated below each experiment. Measurements on each provided date are from larvae derived from a separate clutch of zebrafish eggs.

The third larval zebrafish group was exposed to acute severe hypoxia causing presumed severe hypoxic injury (HE) as indicated for the second group described above. Cardiac arrest and heart damage was used as a proxy for systemic tissue damage following acute severe hypoxic exposure. However, this population was also exposed to 0.3 μmol of the protein kinase p38 MAPK inhibitor (AstraZeneca compound AZ3, 503.6 g/mol), in their surrounding medium (E3 or aquarium water, depending upon age) during the first 12 h following acute severe hypoxic exposure. This dose of p38 MAPK inhibitor and length of exposure was chosen because other experiments revealed that this dose induced the highest recovery of cardiac function in HE larvae (Vazquez-Roman et al., in preparation). After 12 h of exposure to p38 MAPK inhibitor in E3 medium, treated larvae were placed in aquarium water until the day of testing.

2.3 Rearing conditions after hypoxic exposure and before behavioral trials

Immediately following acute severe hypoxic exposure and the resulting cardiac arrest and presumed myocardial hypoxic injury, all larvae were returned to normoxic E3 media, in which they were subsequently reared until day 8 dpf, at which point in development they were transferred to aquarium water. All larvae were fed with A1 starter larvae food twice daily; this feeding regime was alternated with A2 (Otohime™ PTAqua, Dublin, Ireland) after they reached 14 dpf. Larvae were not fed during the days of behavioral testing.

2.4 Survival assessment

Survival was assessed daily over a 14 day period, beginning from immediately before hypoxic exposure at Day 7, until the last day of behavioral trials on Day 21. These days of hypoxic exposure, expressed as dHE, are equivalent to days of development (days post fertilization, dpf) as follows: 1 dHE = 7 dpf or 1 week following hypoxic exposure, and 15 dHE = 21 dpf or 2 weeks following hypoxic exposure.

2.5 Morphometric variables

Immediately after conclusion of the behavioral trials, larvae were fixed in Z-Fix (Electron Microscopy Sciences, Hatfield PA, United States). Body mass was determined after fixation, following a standardized procedure (Vazquez Roman and Burggren, 2022). Body mass measurements were conducted using an analytical balance (MettlerToledo XA105 Dual Range§). Total body length was assessed after larval whose body mass was recorded. Photographs for determining body length were obtained with a microscope connected to a high-resolution camera and analyzed in ImageJ.

After assessment of body mass and total body length, evaluation of larvae growth was conducted using the formula for Fulton’s Condition Factor:

K = 100 (W/L3)

where “W” represents body mass (weight), and “L” the total length (Froese, 2006).

2.6 Behavioral testing: anxiety-like and boldness-like behavior

Anxiety-like and boldness-like behavior are common measurements in fish behavior and may be employed in behavioral neuroscience (Cueto-Escobedo et al., 2022; Dean et al., 2020; Hagen et al., 2024). Zebrafish larvae were assessed at two different time points to evaluate anxiety-like behavior and boldness-like behavior. Behavioral testing was conducted at 14–15 dpf (1 week after HE), and 21–22 dpf (2 weeks after HE) to assess differences between exposed larvae and control larvae at these two different developmental points. Different larvae were used for each group of experiments—i.e., there were no repeated measurements.

The behavioral arena was identical to that used from Dunton et al. (2025), with multiple chambers leading into the actual test arena. This apparatus consisted of a dark start chamber and a light start chamber—both attached to an acrylic arena (14 cm long × 6 cm wide × 2 cm depth) (Figure 2A). The overall apparatus was filled to a depth of ∼1.8 cm with water maintained at 28 ± 0.5°C. A high-speed video camera (Logitech C920 × HD Pro Webcam, Full HD) was located ∼30 cm above the arena to record videos of larval behavior. Opaque cardboard was placed strategically covering the surroundings of the recording site, to avoid any visual influence (including by the experimenter) on the larvae.

Testing arena and novel object. (A) Testing arena for novel object approach. NO, novel object; TZ, thigmotaxis zone; Z1, zone 1; Z2, zone 2. (B) Novel object seen from a lateral view.

For testing anxiety-like and boldness-like behaviors, an individual larva was placed into the dark start chamber. After a 3 min acclimation period to the dark start chamber, the opaque barrier separating was carefully removed without disturbing the larva. This enabled the larva potentially to swim into the attached light chamber. During a subsequent 3 min period, the individual larva was allowed to acclimate in the light chamber, and then the second opaque barrier separating light chamber and test arena was carefully removed to allow the novel object approach test to begin.

The novel object approach test was used to measure “neophilia,” the willingness to face new experiences and new environments. In fish this is a well-established proxy for boldness (Dahlbom et al., 2011; Dean et al., 2020; Hagen et al., 2024), with validation extending to larval stages at 14–21 dpf (Gjinaj et al., 2025). Boldness assessment was determined by the quantification of neophilia by determining the time spent near a novel object within a test arena. Thus, a longer time spent near the novel object signaled a greater degree of neophilia. The novel object comprised a tridimensional multicolored object formed from a 1 cm x 1 cm assembly of blue, yellow, green, and red LEGO “dots” ®. An array of colors was used to avoid bias related to color preference in zebrafish (Figure 2B). Multicolored LEGO® figurines are useful in examining fish behavior, as they serve as effective, easily acquired and standardized novel objects to test how fish respond to unfamiliar items in their environment (Hamilton et al., 2016, 2021).

Video recordings (VirtualDub Software) started when the second door was removed. Videos were recorded for a 10 min period. The start chamber was immediately blocked off after the individual larva emerged (Figure 2A). Larvae that did not emerge during the 10 min-trial were discarded from the behavioral analyses.

For all behavioral tests only “naïve” larvae were used—i.e., no larvae were experimented upon twice.

2.7 Behavioral video analyses

EthoVision XT Noldus software with the DanoVision extension was used to analyze video recordings of zebrafish larval behavior. Concentric circles were added in EthoVision to virtually divide the videos of the arena into four different zones based on the position of the novel object (Figure 2A). The first zone added was zone 3 (surrounding the novel object), and the subsequent zones were created with an enlargement factor of 2 and 3 for zones 2, and 1, respectively. The exploration behavior in a novel tank was also used to measure boldness-exploration (Mazué et al., 2015).

Length of time (s, seconds) spent in the thigmotaxis zone (arena walls) was quantified to evaluate anxiety-like behavior. Time to emerge from the chamber, time spent in the novel object zone, and average distance to the object were compared as a proxy for boldness in zebrafish larvae. Average swimming velocity, related to locomotion, was also quantified.

2.8 Statistics

Survival analyses were conducted using a Kaplan-Meier Survival Analysis: LogRank test. General linear models (GLM) were generated for assessing time spent in each zone (thigmotaxis zone, zones 1–3, from outer to inner zones, respectively), and average distance to the object. Time of emergence was used as a covariate with time spent on each zone, and distance from the novel object. Normality of each model was assessed with the Shapiro Wilk test. Data from models that were not normally distributed were rank-transformed. Two-way ANOVAs were generated for this model. A Tukey post-hoc test was performed to find differences among groups and across time after acute severe hypoxic exposure. Results were reported as estimated marginal means (EMMs) ± S.E.M. when covariant was significant in the two-way ANOVA. If covariant was not significant for the particular dependent variable, results were plotted as means ± S. E. M. Survival analyses were performed with SigmaPlot software (Systat Software, Inc.®, San Jose, CA). R-Studio was used to perform two-way ANOVAs for Fulton’s Condition Factor, and time to emerge from start chamber and velocity, and Two-Way ANOVAs with emergence time as a covariate (time spent in each zone, and average distance to the object).

A significance level of p < 0.05 was adopted for all tests. The logic flow of the experiments is shown in Figure 3.

Flowchart overview of the experiments.

3 Results3.1 Survival rate

Survival rate of the control population, the population experiencing tissue hypoxic damage (as evident from myocardial damage), and the hypoxic exposure + p38 MAPK inhibition population were determined based on the time after acute severe hypoxic exposure (Figure 4). Hypoxic exposure produced survival rates of 56% in the HE group and 60% in the HE + p38 MAPK inhibition group the day after hypoxic exposure. Two weeks after acute severe hypoxic exposure, a higher survival rate occurred in control groups (38%), followed by the HE group (16%), and lastly, the HE + p38 MAPK inhibition group (11%). All Pairwise Multiple Comparison Procedures revealed significant (P = < 0.001) differences among all group interactions: controls vs. HE, controls vs. HE + p38 MAPK inhibition, and HE vs. HE + p38 MAPK inhibition.

Survival assessment following acute severe systemic hypoxia—evident from myocardial hypoxic injury—and the effect of p38 MAPK inhibition in zebrafish larvae. All groups are significantly different from each other at one or more points following systemic hypoxic exposure (P < 0.001). See text for additional explanation.

3.2 Effects of acute severe hypoxic exposure on growth

Body mass was not significantly different among all three groups at 1 week after HE (P > 0.05). However, body mass was significantly higher in all larvae 2 weeks after HE compared to larvae 1 week earlier (P = 0.008), showing the normal effects of growth. Body mass in larvae with HE + p38 MAPK inhibition was significantly higher than controls 2 weeks after HE (P < 0.001) (Figure 5A). No significant effects in body mass after time occurred in controls and in the HE group.

Effects of acute severe systemic hypoxic exposure on subsequent growth in zebrafish larvae. N values are at the bottom of each bar. Legends for the three graphs are indicated in (C). (A) Larvae experiencing severe hypoxic exposure (HE) + p38 MAPK inhibition were significantly heavier than the control group at 2 weeks after severe hypoxic exposure (P < 0.001). Larvae experiencing hypoxic exposure + p38 MAPK inhibition were significantly heavier at 2 weeks after HE than 1 week after HE (P = 0.008). (B) Body length across different time points after HE was significantly longer in controls (P = 0.001), and HE + p38 MAPK inhibition (P = 0.0002) groups, at 2 weeks after myocardial hypoxic injury, when compared to same groups at 1 week after acute severe hypoxic exposure. Larvae with HE + p38 MAPK inhibition were significantly longer than larvae with acute severe hypoxic exposure alone at 2 weeks after exposure (P = 0.005). (C) Fulton’s Condition Factor and effect of HE + p38 MAPK inhibition in zebrafish larvae growth. Hypoxic exposure groups with and without p38 MAPK inhibition were significantly higher than controls at 2 weeks after exposure. No significant differences in Condition Factor occurred across different time points after acute severe hypoxic exposure (P = 0.78).

Total body length across different time points after HE was significantly longer in controls (P = 0.001), and HE + p38 MAPK inhibition groups (P = 0.0002), at 2 weeks after systemic hypoxic exposure (HE), when compared to same groups at 1 week after HE. Larvae with HE + p38 MAPK inhibition at 2 weeks after HE had a significantly longer body length than larvae with HE only (P = 0.005) (Figure 5B).

Regarding the effect of HE and p38 MAPK inhibition in zebrafish larvae growth, HE groups with and without p38 MAPK inhibition had significantly higher Fulton’s Condition Factor than controls at 2 weeks after HE (P = 0.005 and P = 0.003, respectively). No significant differences occurred across different time points after HE (P = 0.78) (Figure 5C).

3.3 Time to emerge from start chamber

Latency for emerging from the start (dark) chamber was not significantly different when comparing larval groups at the same time following HE (P = 0.99). No significant effect in emergence time from the start chamber occurred as a result of acute severe hypoxic exposure and severe hypoxic exposure + p38 MAPK inhibition when compared from the control group (P = 0.96, and P = 0.80, respectively).

However, the time to emerge from the start chamber was significantly shorter in larvae 2 weeks after HE, regardless of experimental group. Time to emerge from the start chamber was significantly shorter in zebrafish larvae at 2 weeks after HE (P = 0.03) (Figure 6). Controls at 1 week after HE took 96% longer (P = 0.004) to emerge than controls at 2 weeks after HE. Similarly, the HE group took 96% longer to emerge from the start chamber at 1 week after HE, compared to larvae at 2 weeks after HE (P = 0.024). Larvae with HE + p38 MAPK inhibition had a 117% longer emergence time at 1 week after HE than 2 weeks after HE (P = 0.03).

Effects of acute severe hypoxic exposure and p38 MAPK inhibition on time to emerge from the start chamber in zebrafish larvae. N values are at the bottom of each bar. No significant differences occurred among groups within the same week of severe hypoxic exposure (P = 0.99). However, there were significant differences between week 1 and week 2 following hypoxic exposure (P = 0.03).

3.4 Time spent in arena zones

Time of emergence from the start chamber was used as a covariant to analyze its effect in the time spent in each of the zones of the arena. p38 MAPK inhibitor had a significant effect on the time spent in the thigmotaxis zone (P = 0.02), while the week after HE had no significant effect (P = 0.09). Larvae with HE + p38 MAPK inhibition spent significantly more time in the thigmotaxis zone (17% based on estimated marginal means) compared to the HE group with no p38 MAPK inhibition, when considering emergence time as a covariant at 1 week after HE (P = 0.04) (Figure 7A). Time spent in the thigmotaxis zone by the control, HE, and HE + p38 MAPK inhibition groups did not differ between 1 and 2 weeks following HE (P = 0.25, P = 0.2, and P = 0.91, respectively). Time to emerge from the start chamber had a significant effect on the time spent in the thigmotaxis zone (P = < 0.001).

Effects of severe hypoxic exposure and p38 MAPK inhibition on arena zone distribution in zebrafish larvae. N values are at the bottom of each bar. Time of emergence from start chamber was treated as a covariant with the time spent on each zone, except for zone 3 (see text for further information). (A) Thigmotaxis zone observed means. (B) Thigmotaxis zone estimated marginal means. (C) Zone 1 (outer intermedium) observed means. (D) Zone 1 estimated marginal means. (E) Zone 2 (internal intermedium) observed means. (F) Zone 2 estimated marginal means. (G) Zone 3 (center zone, novel object zone), observed means. Error bars are plotted as ± S. E. M. *p < 0.05. Different letters indicate significant differences between groups.

Time spent in zone 1 was not significantly different between groups (P = 0.92) at any point in time, nor across time after HE (P = 0.19) (Figures 7C,D). The Treatment Factor was not significantly different in the time spent in zone 2 (Figures 7E,F) and zone 3 (novel object zone) (Figure 7G) (P = 0.11, and P = 0.94, respectively), nor was the Age Factor after acute severe hypoxic exposure (P = 0.41 and P = 0.97, respectively). However, the HE group spent significantly less time in the novel object zone, when compared to controls at 2 weeks after HE, when time of emergence from start chamber serves as a covariant (P = 0.04). Controls spent significantly longer time in the novel object zone (zone 3) at 2 weeks after HE then controls at 1 week after HE, when emergence time is considered as a covariant (P = 0.02). However, the covariant did not have a significant effect (P = 0.65), thus, observed means are plotted instead (Figure 7G). Report of the two-way ANOVAs with covariant for the zones 0–3 are included in Table 1.

Behavioral metricInterceptTreatmentAgeTreatment vs. AgeEmergence timeThigmotaxis*F = 798.60
P < 0.001F = 3.90
P = 0.02F = 2.89
P = 0.09F = 2.98
P = 0.05F = 598.57
P = < 0.001Zone 1*F = 116.98
P < 0.001F = 0.08
P = 0.92F = 1.77
P = 0.18F = 1.60
P = 0.20F = 21.57
P = < 0.001Zone 2*F = 120.91
P < 0.001F = 2.24
P = 0.11F = 0.69
P = 0.41F = 0.34
P = 0.71F = 7.42
P = 0.007Zone 3*F = 336.07
P < 0.001F = 0.06
P = 0.94F = 0.002
P = 0.96F = 1.86
P = 0.16F = 0.20
P = 0.65Distance to novel object*F = 10.07
P = 0.002F = 0.07
P = 0.94F = 0.86
P = 0.36F = 0.08
P = 0.92F = 8.25
P = 0.004

Results from the general linear model used to analyze the time spent in the different arena zones in zebrafish during acute severe hypoxic exposure assessed with Two-way ANOVA.

*Indicates rank transformation. Statistical significance was considered with α ≤ 0.05.

3.5 Average distance to the novel object—neophilia

No significant differences occurred among groups at the same point in time after HE (P = 0.94), and across weeks after HE (P = 0.36) (Figure 8A). Statistics for the two-way ANOVA with covariant for the average distance to the novel object are included in Table 1.

Effects of severe hypoxic exposure and p38 MAPK inhibition on locomotion in larval zebrafish. (A) Effects of severe hypoxic exposure and p38 MAPK inhibition on the average distance from the novel object in zebrafish larvae. N values are at the bottom of each bar. No significant differences in distance from novel object occurred between treatments (P = 0.936) or with age (P = 0.356). (B) Effect of severe hypoxic exposure and p38 MAPK inhibition in mean swimming velocity (cm/s) in zebrafish larvae. (C) Effect of severe hypoxic exposure and p38 MAPK inhibitio