Abstract
Hypoxic ischaemic injury (HIE) in the neonatal brain has significant consequences on neurodevelopment and increases the occurrence of neurological deficits in infants. HIE is also a leading cause of neonatal seizures. Therapeutic options for the treatment of HIE are very limited. Hypoxia-ischemia directly damages brain tissue in a primary-wave of injury which activates a cascade of events triggering local and systemic inflammatory responses, driven by the innate immune system, which contribute to a significant secondary-wave of injury taking place as early as 6 hours post-hypoxia-ischaemia. Levels of the well documented inflammatory microRNA, miR-155 are elevated in rodent seizure and epilepsy models. Here, we assessed the impact of, miR-155 deletion in myeloid cells, on regulating inflammation and seizure severity in a preclinical model of neonatal hypoxia-induced seizures (Hypoxia-Sz). Wildtype miR-155 (miR-155+/+LysMCre) mice were compared to a mouse line in which miR-155 was deleted in myeloid cells (miR-155fl/fl LysMCre). We demonstrate significant upregulation of miR-155 target genes, brain-derived neurotrophic factor (bdnf), arginase-2 (arg-2), ship-1 and socs-1 in miR-155fl/fl LysMCre mice compared to controls at various time points following Hypoxia-Sz. Conversely, we report decreased mRNA levels of pro-inflammatory cytokines IL-1β and IL-6 and lower protein levels of IL-1β in miR-155fl/fl LysMCre mice as compared to WTs. Myeloid miR-155 deletion significantly reduced behavioural seizure severity score, reduced electrographically (EEG) measured seizure frequency and seizure burden as compared to mice to wildtypes, suggesting miR-155 regulation of seizure occurrence in this model. Behavioural tests for motor functions at 5 weeks post Hypoxia-Sz demonstrated differences between genotypes. Excitingly this work highlights that inhibition of miR-155, specifically in myeloid cells, may hold therapeutic benefit for both seizures and comorbidities associated with hypoxic brain injury.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
This preprint has been revised to include the Acknowledgement of EU funding as follows. Acknowledgments This project has received funding from the European Unions Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 897880. This is the only revision to the manuscript.
Abbreviations
- ANOVA
- – Analysis of Variance
- Arg
- - Arginase
- ATP
- – Adenosine Triphosphate
- BBB
- – Blood Brain Barrier
- Bcl6
- – B-cell lymphoma 6 protein
- BDNF
- – Brain Derived Neurotrophic Factor
- Bic gene
- – B-cell integration cluster gene
- C/EBP
- – CCAAT/enhancer-binding proteins
- CBA
- – Cytometric Bead Array
- CNS
- – Central Nervous System
- CP
- – Cerebral Palsy
- DC
- – Dendritic Cell
- dMCAO
- – distal Middle Cerebral Artery Occlusion
- DMEM
- – Dulbecco’s Modified Eagle Medium
- DNA
- – Deoxyribonucleic Acid
- DSS
- – Dextran Sulphate Sodium
- EEG
- – Electroencephalograph
- ELISA
- – Enzyme Linked Immunosorbent Assay
- GC
- – Glucocorticoids
- HIE
- – Hypoxic Ischaemic Encephalopathy
- HIF
- – Hypoxia Inducible Factor
- HRI
- – Hypoxia Reperfusion Injury
- Hypoxia-Sz/Hypoxic-Sz
- – Hypoxia induced Seizures
- IFN
- – Interferon
- IL
- – Interleukin
- IP
- – Intraperitoneal
- IRF
- – Interferon Regulatory Factor
- IRI
- – Ischaemia Reperfusion Injury
- ISRE
- – Interferon-Sensitive Response Elements
- KSRP
- - KH-type Splicing Regulatory Protein
- LSLB
- – Low Stringency Lysis Buffer
- Maf-b
- – Musculoaponeurotic fibrosarcoma-oncogene homolog-bD
- MCP
- – Monocyte Chemoattractant Protein
- MMP
- – Matrix Metalloproteinase
- MRTF
- – Microsurgical Research and Training Facility
- NCS
- – Non-Convulsive Seizures
- NF-kB
- – Nuclear Factor kappa-light-chain-enhancer of activated B cells
- OGD/R
- – Oxygen Glucose Deprivation/Reoxygenation
- PBS
- – Phosphate Buffered Saline
- PI3K-AKT
- - Phosphatidylinositol-3-Kinase - Protein kinase B
- PRR
- – Pattern Recognition Receptors
- RCLB
- – Red Cell Lysis Buffer
- RISC
- – RNA Induced Silencing Complex
- RNA
- – Ribonucleic Acid
- ROS
- – Reactive Oxidative Species
- RT-PCR
- – Real Time quantitative Polymerase Chain Reaction
- SDS-PAGE
- – Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis
- SHIP-1
- - Src homology 2 (SH2) domain containing inositol polyphosphate 5-phosphatase 1
- SOCS-1
- – Suppressor of Cytokine Signalling-1
- TBST
- - Tris-buffered Saline-Tween
- TF
- – Transcription Factor
- TGF
- – Transforming Growth Factor
- TH
- – Therapeutic Hypothermia
- TLE
- – Temporal Lobe Epilepsy
- TLR
- – Toll Like Receptor
- TNF
- – Tumour Necrosis Factor
- UTR
- – Untranslated Region
- WB
- – Western Blot
- WHO
- – World Health Organisation
- WT
- – Wildtype