Extracellular Vesicle Signatures from Eye Lavage as Novel Non-invasive Biomarkers for Hypoxic Ischaemic Insult – Findings from a Neonatal Mouse Model

Runci Li¹Sarah R Needham^2,3Igor Kraev⁴Mariya Hristova^1*Sigrun Lange^5*

• ¹University College London Elizabeth Garrett Anderson Institute for Women's Health, London, United Kingdom
• ²Rutherford Appleton Laboratory, Didcot, United Kingdom
• ³Science & Technology Facilities Council Central Laser Facility, Didcot, United Kingdom
• ⁴The Open University Faculty of Science Technology Engineering and Mathematics, Milton Keynes, United Kingdom
• ⁵University of Westminster School of Life Sciences, London, United Kingdom

Neonatal hypoxia ischaemia (HI) affects 1-3 per 1000 live births, is a major cause of infant mortality and morbidity and leads to adverse long term neurological outcomes, while reliable biomarkers are scarce. Extracellular vesicles (EVs) are small membrane vesicles released from cells and play key roles in cellular communication through transfer of diverse cargoes, including proteins, and can be isolated from various body fluids. Here we developed a new non-invasive method of biofluid-EV profiling, isolating EVs from eye lavage. Our data demonstrate that in a neonatal HI mouse model of mild and severe insult, significant differences are found in EV signatures of eye lavage. We identified increased EV numbers alongside modification in EV size profiles and EV's proteomic cargo signatures, in eye lavage from HI animals, compared to controls. Protein-protein interaction network analysis of EV proteome cargoes identified enrichment in Gene ontology and KEGG pathways in the HI groups associated with various homeostatic and disease related pathways. Specific changes for the mild HI group included pathways for Ribosome biogenesis, Translation, RNA processing, Gene expression, Blood coagulation, Innate immunity, Antioxidant activity, Phospholipid binding, Post-synapse, Cell cortex, and HIF-1 signalling. Enriched pathways associated only with the EV proteome of the severe HI group associated with Cytoskeleton organisation, peptide cross-linking, Monosaccharide biosynthesis, Peroxidase activity, Extrinsic component of plasma membrane, GAIT complex, Mast cell granulation, Ruffle, and Sealing of nuclear envelope by ESCRT-III. Here, we report a new non-invasive method using eye lavage EV signatures to identify changes in response to HI. Our results highlight eye lavage EVs as potential clinical biomarkers for predicting changes occurring in the brain and eye due to neonatal HI, including due to different severity of injury.