Postpartum Plasma-Derived Exosomes Confer Mitochondrial Stabilization and Neuroprotection against Ischemic Stroke

The molecular mechanisms governing adaptive neuroprotection during the postpartum period remain unknown. We hypothesized that circulating exosomes contain bioactive cargo (such as Hsp20) that confer neuroprotection against ischemic injury during the postpartum period.

Exosomes were isolated from plasma of postpartum female mice (ppExos) and control female mice, and from serial blood samples obtained from healthy human volunteers during pregnancy (3rd trimester) and again on postpartum day 2. Exosomal size and protein markers were confirmed via nanoparticle tracking analysis and Western blotting. Neuroprotection with exosome treatment was assessed in vitro using mouse neuronal and astrocyte cultures and human retinal pigment cell line subjected to simulated ischemia. In vivo neuroprotection was assessed using transient middle cerebral artery occlusion (MCAO) in young adult and aged mice. Mitochondrial integrity and reactive oxygen species (ROS) production were evaluated by live cell imaging. In vivo neuroprotection was evaluated by assessing infarct volume and neurobehavioral scores. Changes in mitochondrial dynamics were measured by immunofluorescence and Western blot. Human exosomes were sent for proteomic assessment (SomaScan^™) followed by differential expression analysis (R software v4). Protein quantification was validated by immunoblot.

ppExos significantly reduced infarct volumes and improved neurological deficits post-MCAO in both young and aged female mice. In vitro, ppExos reduced ROS generation in all cell types after simulated ischemia and reduced mitochondrial fragmentation in astrocytes. Mitochondrial fusion proteins Mfn2 and Opa1 proteins were elevated in maternal postpartum brains, and preserved in both astrocyte and neuronal cell cultures after in vitro ischemia with ppExo treatment. Proteomics revealed significant upregulation of heat shock protein 20 (Hsp20) in human ppExos, while Western blot validated elevated Hsp20 in both human and mouse ppExos. Simulated ischemia significantly reduced Hsp20 in astrocyte and neuronal cultures which was reversed by treatment with ppExos. In conclusion, ppExos represent a previously unrecognized, naturally optimized neuroprotective agent that enhances mitochondrial resilience and antioxidant defenses associated with enhanced Hsp20 expression. These findings establish a novel platform for sex-informed, cell-free therapies in ischemic cerebrovascular accidents.