# Extracellular vesicle signatures from eye lavage as novel non-invasive biomarkers for hypoxic ischaemic insult—findings from a neonatal mouse model

## TL;DR

This study introduces a non-invasive method using eye lavage to detect biomarkers for neonatal hypoxia-ischaemia, offering potential for early diagnosis and monitoring.

## Contribution

A novel non-invasive method using eye lavage extracellular vesicles to detect biomarkers for neonatal hypoxia-ischaemia is introduced.

## Key findings

- EVs from eye lavage show distinct signatures in neonatal hypoxia-ischaemia mouse models.
- Mild and severe HI groups exhibit different proteomic pathway enrichments in EV cargo.
- Eye lavage EVs reflect brain and eye changes caused by varying severities of HI injury.

## Abstract

Neonatal hypoxia ischaemia (HI) affects 1–3 per 1,000 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 the 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 insults, significant differences are found in EV eye lavage signatures. We identified increased EV numbers and modifications in EV size profiles and EV's proteomic cargo signatures in eye lavage from HI animals compared to controls. A protein–protein interaction network analysis of the EV proteome cargoes identified enrichment in Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways in the HI groups associated with various homeostatic and disease-related pathways. The specific changes in 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. The enriched pathways only associated with the EV proteome of the severe HI group included cytoskeleton organisation, peptide cross-linking, monosaccharide biosynthesis, peroxidase activity, extrinsic component of plasma membrane, the GAIT complex, mast cell granulation, ruffle, and sealing of the nuclear envelope by the endosomal sorting complex required for transport 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 that occur in the brain and eye due to different neonatal HI injury severities.