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Neuroprotection of NSC Therapy is Superior to Glibenclamide in Cardiac Arrest-Induced Brain Injury via Neuroinflammation Regulation

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Abstract

Cardiac arrest (CA) is common and devastating, and neuroprotective therapies for brain injury after CA remain limited. Neuroinflammation has been a target for two promising but underdeveloped post-CA therapies: neural stem cell (NSC) engrafting and glibenclamide (GBC). It is critical to understand whether one therapy has superior efficacy over the other and to further understand their immunomodulatory mechanisms. In this study, we aimed to evaluate and compare the therapeutic effects of NSC and GBC therapies post-CA. In in vitro studies, BV2 cells underwent oxygen–glucose deprivation (OGD) for three hours and were then treated with GBC or co-cultured with human NSCs (hNSCs). Microglial polarization phenotype and TLR4/NLRP3 inflammatory pathway proteins were detected by immunofluorescence staining. Twenty-four Wistar rats were randomly assigned to three groups (control, GBC, and hNSCs, N = 8/group). After 8 min of asphyxial CA, GBC was injected intraperitoneally or hNSCs were administered intranasally in the treatment groups. Neurological-deficit scores (NDSs) were assessed at 24, 48, and 72 h after return of spontaneous circulation (ROSC). Immunofluorescence was used to track hNSCs and quantitatively evaluate microglial activation subtype and polarization. The expression of TLR4/NLRP3 pathway-related proteins was quantified via Western blot. The in vitro studies showed the highest proportion of activated BV2 cells with an increased expression of TLR4/NLRP3 signaling proteins were found in the OGD group compared to OGD + GBC and OGD + hNSCs groups. NDS showed significant improvement after CA in hNSC and GBC groups compared to controls, and hNSC treatment was superior to GBC treatment. The hNSC group had more inactive morphology and anti-inflammatory phenotype of microglia. The quantified expression of TLR4/NLRP3 pathway-related proteins was significantly suppressed by both treatments, and the suppression was more significant in the hNSC group compared to the GBC group. hNSC and GBC therapy regulate microglial activation and the neuroinflammatory response in the brain after CA through TLR4/NLRP3 signaling and exert multiple neuroprotective effects, including improved neurological function and shortened time of severe neurological deficit. In addition, hNSCs displayed superior inflammatory regulation over GBC.

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Data Availability

All data supporting the conclusions of this manuscript are provided in the text and figures. Please contact the author for data requests.

Abbreviations

NSC:

Neural stem cell

CA:

Cardiac arrest

GBC:

Glibenclamide

OGD:

Oxygen–glucose deprivation

OGD/R:

Oxygen-glucose deprivation and re-oxygenation

hNSCs:

Human NSCs

TLR4:

Toll-like receptor 4

NLRP3:

NLR pyrin domain containing 3

NDS:

Neurological-deficit scores

ROSC:

Return of spontaneous circulation

AD:

Alzheimer's disease

DMEM:

Dulbecco's modified Eagle medium

bFGF:

Basic fibroblast growth factor

EGF:

Epidermal growth factor

FBS:

Fetal bovine serum

CPR:

Cardiopulmonary resuscitation

MAP:

Mean arterial pressure

ABG:

Arterial blood gas

ECG:

Electrocardiogram

EEG:

Electroencephalogram

DMSO:

Dimethyl sulfoxide

qEEG-IQ:

Quantitative EEG-information quantity

lac:

Blood lactic acid

SND:

Severe neurological deficit

PFA:

Paraformaldehyde

HDS:

Histopathological damage scoring

BSA:

Bovine serum albumin

RIPA:

Radioimmunoprecipitation assay

PMSF:

Phenylmethanesulfonyl fluoride

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene fluoride membrane

ECL:

Enhanced chemiluminescence

A. U.:

Arbitrary units

TTM:

Targeted temperature management

ICUs:

Intensive care units

IntDen:

Integrated density

LOS:

Length of stay

ROS:

Reactive oxygen species

ROSC:

Return of spontaneous circulation

RNS:

Reactive nitrogen species

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Acknowledgements

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Funding

This work was partially supported by R01HL118084, R01NS110387, RO1NS125232 from the United States National Institute of Health (all to Xiaofeng Jia). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Zhuoran Wang, Shuai Zhang, Jian Du, Songyu Chen & Xiaofeng Jia

  2. Program in Trauma, Department of Neurology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Brittany Bolduc Lachance

  3. Anesthesiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Brian M. Polster

  4. Orthopedics, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Xiaofeng Jia

  5. Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Xiaofeng Jia

  6. Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Xiaofeng Jia

  7. Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA

    Xiaofeng Jia

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  1. Zhuoran Wang
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Contributions

Zhuoran Wang performed the in vivo study and related in vitro study, analyzed the data and wrote the manuscript; Shuai Zhang performed and analyzed the cellular study; Jian Du prepared neural stem cells; Songyu Chen assisted with in vitro studies; Brian Polster provided critical appraisal in the cellular model; Zhuoran Wang, Brittany Bolduc Lachance, and Xiaofeng Jia revised the manuscript; Xiaofeng Jia conceived the original idea, designed the experiments, and finalized the manuscript. The authors read and approved the final manuscript.

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Correspondence to Xiaofeng Jia.

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All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Maryland, Baltimore.

Human and Animal Ethics

All rats were maintained following NIH guidelines for the humane care of animals.

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Presented, in part, at the Society of Critical Care Medicine’s 50th Critical Care Congress with Star Research Achievement Award in Jan 2021.

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Wang, Z., Zhang, S., Du, J. et al. Neuroprotection of NSC Therapy is Superior to Glibenclamide in Cardiac Arrest-Induced Brain Injury via Neuroinflammation Regulation. Transl. Stroke Res. 14, 723–739 (2023). https://doi.org/10.1007/s12975-022-01047-y

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