Experimental setup of the hypoxia-reoxygenation brain damage model in zebrafish through oxygen deprivation. (A) A simulation diagram of the hypoxia device designed for zebrafish. (B) The dissolved oxygen levels in the hypoxic aquarium gradually declined as nitrogen perfusion increased, ultimately reaching the lowest level of 0.145 mg/L at the 16th minute. (C) qRT-PCR analysis demonstrated a significant up-regulation in mRNA levels of hif-1αa and hif-1αb following a 6-h H/R (H/R-6 h) treatment. (D) Western blotting revealed a notable elevation in Hif-1α protein level after 6-h H/R treatment. H/R-3 h: hypoxia followed by 3 h of reoxygenation; H/R-6 h: hypoxia followed by 6 h of reoxygenation. Six to seven brains were analyzed per group. Data are shown as mean ± S.E.M. Statistical significance is denoted as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 compared to the control.

Establishment of a sevoflurane postconditioning model for zebrafish and cerebral infarction detected by TTC staining. (A) The sevoflurane inhalation device for zebrafish. (B) TTC-stained brain sections of zebrafish in the control, H/R, H/R+Sevo, and H/R+TDZD-8 groups. The white regions represent the infarct area, whereas the red regions represent the non-infarct area. Scale bar = 1 mm. (C) Statistical analysis of the infarct area/total cerebral area in the four groups. Six brains in each group were analyzed. Data are shown as mean ± S.E.M. The significance level was *** p< 0.001, with analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.

Apoptosis in zebrafish brains. (A) Representative images of horizontal sections of zebrafish brain in control, H/R, H/R + Sevo, and H/R + TDZD-8 groups. From the left to the right: TUNEL-positive images, DAPI-positive images, and the merged images. (B) Statistical analysis of TUNEL-positive regions in zebrafish brains, with four brains included in each group. Data are expressed as mean ± S.E.M. Scale bar =100 μm. The significance levels were ** p < 0.01 and *** p < 0.001, with analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.

Ultrastructural morphology of mitochondria and oxidative stress in zebrafish brain. (A) Representative transmission electron microscopy images of mitochondria in zebrafish brains from the control, H/R, H/R + Sevo, and H/R + TDZD-8 groups. Green arrows indicate healthy mitochondria, red arrows indicate injured mitochondria, and yellow arrows indicate partially restored mitochondria in terms of morphology. Scale bar = 200 nm. (B-C) The quantification of mitochondrial number and the percentage of injured mitochondria in the zebrafish brains conducted using electron microscopy, with a sample size of 15 neurons in five zebrafish per group. (D-F) Levels of glutathione (GSH), oxidized glutathione (GSSG), and total GSH (T-GSH) in zebrafish brain measured under hypoxia, sevoflurane postconditioning, and TDZD-8 pretreatment, with six brains included in each group. Data are expressed as mean ± S.E.M. The significance levels were * p < 0.05, **p < 0.01, and ***p < 0.001, with analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.

Sevoflurane postconditioning and TDZD-8 treatment effectively facilitated phosphorylation of Akt and GSK-3β. (A) Representative western blot images depicting protein levels in the control, H/R, H/R + Sevo, and H/R + TDZD-8 groups. Band intensity analysis showed the ratios of p-Akt to Akt (B) and p-GSK-3β to GSK-3β (C). Six brains were included in each group. Data are expressed as mean ± S.E.M. The significance levels were * p < 0.05 and ***p < 0.001, with analyzed by one-way ANOVA followed by Tukey’s multiple comparison test.

Sevoflurane postconditioning promoted MAP2 expression and zebrafish visual neurobehavioral recovery. (A) Representative western blotting images showing MAP2 protein levels in the control, H/R, H/R + Sevo, and H/R + Sevo + Nocodazole groups. (B) Band intensity analysis showing the ratio of MAP2 to β-actin. (C) Representative images of MAP2 immunofluorescence in the zebrafish midbrain. (D) Relative intensity analysis of MAP2-positive cells in the control, H/R, H/R + Sevo, and H/R + Sevo + Nocodazole groups. (E) The schematic diagram of the experimental setup used for investigating optokinetic response behavior in adult zebrafish. (F) Statistical analysis of the frequency of optokinetic response of zebrafish in the control, H/R, H/R + Sevo, and H/R + Sevo + Nocodazole groups. Six animals in each group were used in western blotting and immunofluorescence staining. Eight animals in each group were used to in optokinetic response behavior. Scale bar = 500 μm. Data are expressed as mean ± S.E.M. The significance levels were ** p < 0.01 and ***p < 0.001, with analyzed by one-way ANOVA followed by Tukey’s multiple comparison test

The overall illustration of the protective effects of sevoflurane postconditioning against H/R-induced brain injury in zebrafish. Sevoflurane postconditioning enhances the phosphorylation of Akt and GSK-3β, increases the expression of MAP2, and subsequently promotes the polymerization of MAP2 and microtubules. This process effectively inhibits cerebral infarction and neuron apoptosis, improves mitochondrial morphology integrity, and ultimately facilitates the recovery of neurobehavioral function of H/R zebrafish.