General anesthetics protects against cardiac arrest-induced brain injury by inhibiting calcium wave propagation in zebrafish

Dao-Jie Xu, Bin Wang, Xuan Zhao, Yi Zheng, Jiu-Lin Du, Ying-Wei Wang, Dao-Jie Xu, Bin Wang, Xuan Zhao, Yi Zheng, Jiu-Lin Du, Ying-Wei Wang

Abstract

Cardiac arrest is a leading cause of death and disability worldwide. Although many victims are initially resuscitated, they often suffer from serious brain injury, even leading to a "persistent vegetative state". Therefore, it is need to explore therapies which restore and protect brain function after cardiac arrest. In the present study, using Tg (HuC:GCaMP5) zebrafish as a model, we found the zebrafish brain generated a burst of Ca2+ wave after cardiac arrest by in vivo time-lapse confocal imaging. The Ca2+ wave was firstly initiated at hindbrain and then sequentially propagated to midbrain and telencephalon, the neuron displayed Ca2+ overload after Ca2+ wave propagation. Consistent with this, our study further demonstrated neuronal apoptosis was increased in cardiac arrest zebrafish by TUNEL staining. The cardiac arrest-induced Ca2+ wave propagation can be prevented by general anesthetics such as midazolam or ketamine pretreatment. Moreover, midazolam or ketamine pretreatment dramatically decreased the neuronal apoptosis and improved the survival rate in CA zebrafish. Taken together, these findings provide the first in vivo evidence that general anesthetics pretreatment protects against cardiac arrest-induced brain injury by inhibiting calcium wave propagation in zebrafish.

Keywords: Brain injury; Ca2+ wave; Cardiac arrest; General anesthetics; Zebrafish.

Conflict of interest statement

Ethics approval

All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences, and Shanghai Jiaotong University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Cardiac arrest causes a burst of calcium wave in brain. a Schematic showing the experimental scheme. b Schematic showing cardiac arrest (CA) model in zebrafish. A glass micropipette was used to press zebrafish heart (red) until the heart stop beating, neuronal activity was examined with in vivo time-lapse confocal imaging after CA. c Calcium activity of Tg (HuC:GCaMP5) transgenic zebrafish in different brain regions after CA (lateral view). ROI1 (red ellipses), ROI2 (green ellipses) and ROI3 (yellow ellipses) marked neuron in hindbrain, midbrain and telencephalon, respectively. Scale bar: 100 μm. d Calcium activity of three regions of interest in (c) at a higher time resolution. The arrows mark the onset of calcium activities in each region. e Time-lapse confocal images showing the lateral view of the brain in live Tg (HuC:GCaMP5) transgenic zebrafish in control and CA zebrafish at 7 day post-fertilization (dpf). 0 s indicating calcium wave onset. Scale bar: 100 μm. f The change in fluorescence intensity of neuron in midbrain in individual zebrafish during CA. g Occurrence probability of calcium waves in control and CA zebrafish (***P<0.001, chi-square test). h Quantification of the latency of CA to calcium wave generation in CA zebrafish. Numbers in the histograms represent the number of embryos analyzed in each group
Fig. 2
Fig. 2
Midazolam or ketamine pretreatment inhibits cardiac arrest-induced calcium wave propagation. a-d Time-lapse confocal images showing the lateral view of the brain in live Tg (HuC:GCaMP5) transgenic zebrafish in control (a), CA (b), CA + midazolam (0.5 mM)(c) and CA + ketamine (2.5 mM) pretreatment (d) zebrafish at 7 dpf. 0 s indicating calcium wave onset. MDZ, midazolam. Scale bar: 100 μm. e Occurrence probability of calcium waves in each group (***P<0.001, chi-square test). Numbers in the histograms represent the number of embryos analyzed in each group
Fig. 3
Fig. 3
Midazolam or ketamine pretreatment decreases the neuronal apoptosis after cardiac arrest. a-d TUNEL staining of control (a), CA (b), CA + midazolam (0.5 mM)(c) and CA + ketamine (2.5 mM) pretreatment (d) zebrafish at 7 dpf. Red, TUNEL staining, indicating the apoptotic cell; Blue, DAPI-stained nuclei. Scale bar: 10 μm. e Quantification of percentages of TUNEL positive cells in each group (mean ± s.e.m.; ***P<0.001, one-way ANOVA plus Newman–Keuls multiple comparison test). Numbers in the histograms represent the number of sections analyzed in each group
Fig. 4
Fig. 4
Midazolam or ketamine pretreatment improves survival and locomotor function after cardiac arrest. a Survival rate of zebrafish during 5-day follow-up after CA (**P<0.01, ***P<0.001 vs CA group, log-rank test). b Quantification of the spontaneous locomotor distance of 1 day post-CA zebrafish (mean ± s.e.m.; **P<0.01, ***P<0.001, ns, no significance, one-way ANOVA plus Newman–Keuls multiple comparison test). Numbers in the histograms represent the number of embryos analyzed in each group

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