Annexin A6 in Eye-Lavage Extracellular Vesicles—a Candidate Biomarker for Sex-Specific Differences in Mild Hypoxic–Ischaemic Brain Damage—Findings from an Explorative Neonatal Mouse Model Study

Extracellular vesicles (EVs) play important roles in cell communication, including via transfer of cargo proteins, and are promising biomarkers for pathobiological processes. Reliable biomarkers for mild neonatal hypoxia ischaemia (HI) are scarce. Wrongly determined HI severity often leads to adverse long-term neurological outcomes, with more detrimental effects in males. Therefore, identification of sex-specific markers for HI is of great interest. We recently reported the potential of EVs from eye lavage as novel non-invasive biomarkers of neonatal HI brain injury, differing in proteomic cargoes between mild and severe HI brain insults. In continuation, the current study therefore focused on assessing sex-specific differences in the same mouse model of mild HI with respect to Annexin A6 (ANXA6) EV protein cargo, at different time points (0 h, 24 h and 48 h) post HI insult. Using direct stochastic optical reconstruction microscopy (dSTORM) quantifying and co-localising ANXA6 to EV subtypes, our results indicate more marked changes in males compared with females, with a peak of positive ANXA6 detection in EVs at 24 h post HI in males. PanEV+TT+ EVs contained most ANXA6, followed by the PanEV+-labelled EVs. In the females, less ANXA6-positive staining was observed in EVs of all three time points in the HI groups compared with the males. In the sham and naïve control animals, ANXA6 detection suggested developmental reduction of ANXA6 in both sexes. However, in the male HI groups that developmental reduction was eliminated and ANXA6 remained at an increased level at all three time points. In addition, EV subtype labelling showed some differences between time points within each sex group and between sexes. PanEV+ single labelled EVs were more abundant in the females compared with the males in the 0 h and 24 h post-HI groups and PanEV+TT+-labelled EVs were more abundant in females than males at the 0 h post-HI time point. Our pilot findings indicate that increased ANXA6 labelling in eye-lavage EVs is associated with the mild HI model with more marked changes in males, where ANXA6 positivity peaked at 24 h post HI. Outcomes of this study contribute to EV-based biomarker development for HI diagnostics, with potential clinical value, including sex-specific differences. Future studies should include validation in further biological replicates in larger cohort sizes and assessment in human neonatal clinical samples.