Authors :
Presenting Author: Shivani Sonawane, MSc – CEITEC, Masaryk University and 1st Department of Neurology, Faculty of Medicine, Masaryk University and St. Anne´s University Hospital, Brno, Czech Republic – member of ERN EpiCARE
Radim Jaroušek, MSc – Faculty of Medicine, Masaryk University, Czech Republic
Dagmar Al Tukmachi, MSc – Central European Institute of Technology, Masaryk University, Czech Republic
Nicolas Blavet, PhD – Central European Institute of Technology, Masaryk University, Czech Republic
Milan Brázdil, MD, PhD – 1st Department of Neurology, Faculty of Medicine, Masaryk University and St. Anne´s University Hospital, Brno, Czech Republic – member of ERN EpiCARE
Hana Kubova, PhD – Institute of Physiology, Academy of Science, Czech Republic
Rationale:
Neonatal seizures (NS) are the most common neurological events affecting newborns. They often cause irreversible brain damage, leading to the development of neuropsychiatric impairments, and in some cases initiate epilepsy, but the whole mechanism underlying these alterations remains unknown. Using a model of flurothyl-induced recurrent seizures in neonatal rats we aimed to study changes in the dynamic of microRNA (miRNA) and mRNA expression profiles in both sexes. Our data may provide a novel insight into the NS-related pathophysiology.
Methods:
Starting at postnatal day 6 (P6), Wistar rats of both sexes were exposed to flurothyl-induced generalized tonic-clonic seizures (5 seizures/day) for five consecutive days.
Littermates of both sexes were used as controls (n=36). Hippocampal tissue was collected 24 hours, 7 days, and 3 months after the last seizure. Total RNA was isolated and subjected to miRNA and mRNA library preparation followed by sequencing (Illumina). The expression of each differentially expressed (DE) miRNA and genes was validated using RT-qPCR.:
The miRNA sequencing analysis revealed sex- and interval-related regulation patterns following flurothyl-induced seizures. At 24 hours after the last seizure, 6 miRNAs were DE in females and 2 in males, with miR-129-2-3p and -132-3p common in both sexes. At 7 day intervals, 3 miRNAs were DE in males (miR-204-5p, -34c-5p, -1298) and 2 in females (miR-24-2-5p,-449a-5p) but no shared miRNA was observed. By 3 months, no miRNAs were dysregulated in males while 8 miRNAs were DE in females including miR-214-3p, -448-3p,-451-5p,-484. Approximately 90% of the dysregulated miRNAs were shown to differ between the sexes.
The mRNA sequencing analysis revealed significant differences in gene expression between the sexes. We identified 364 DE genes at 24 hours, 22 in 7 days, and 48 mRNAs in 3 months post-last seizures. Whereas, 91, 29, and 17 DE mRNAs in males. About 8-10% of DE genes overlapped between the sexes at the first two time points (24h;7d). Pathway analysis showed involvement of various biological processes such as inflammatory responses (Ptgs2, Il1b, Il6, Itgb1, Il1r1, Stat3), neuroprotection (Gfap, Bdnf, Bag3, Gadd45g), ion transport (Wnk3, Slc13a4, Slc36a1), calcium signaling (Hbegf, Pval, Ryr1), neurotransmitter receptor activity (P2x7r, Grin2c, Chrna5) and stress response (Hspb1, Crym, Anxa4, Ptx3). Our correlation studies highlighted a negative and positive association between some DE miRs and mRNAs.
Conclusions:
The miRNA and mRNA sequencing revealed sex-specific differences in gene expression and regulatory responses following flurothyl-induced seizures. Females exhibited a more robust response while males showed limited and transient response. Our finding highlighted the importance of considering the sex differences in molecular responses to seizures.
Funding: Supported by project nr. LX22NPO5107 (MEYS): Financed by European Union – Next Generation EU.