More mitochondria are present in the corneal epithelium following trephine injury.

Axonal mitochondria engulfed by corneal epithelial cells after trephine injury retain metabolic function for up to 6 h.

Corneal epithelial cells upregulate macropinocytosis within minutes following corneal trephine injury.

Corneal epithelial cell macropinocytosis also mediates the in vitro engulfment of isolated and vesicle-bound mitochondria.

Gap junction inhibitors do not reduce engulfment of mitochondria by corneal epithelial cells in vitro.

To determine the mechanisms used to internalize mitochondria by corneal epithelial cells after in vivo corneal trephine injury and in vitro in corneal epithelial cells.

Male and female mice were subjected to trephine injury and euthanized immediately, 6, and 24 h after injury. Macropinocytosis was quantified in vivo using 70 kD fluorescent dextran. Mitochondrial content was assessed by immunofluorescence and metabolic activity quantified by Seahorse assay immediately and 6 h after injury. In vitro experiments using human corneal and limbal epithelial (HCLE) cells and isolated mitochondria were performed to assess mitochondrial transfer in the presence of the gap junction inhibitor 18α-glycyrrhetinc acid and the macropincytosis inhibitor ethylisopropylamiloride.

Mitochondria accumulate within apical epithelial cell layers within minutes of trephine injury. Macropinocytosis also increases within minutes of trephine injury. Oxygen Consumption Rates increase in the corneal epithelium 6 h after trephine injury in males and females. Inhibiting gap junctions increases mitochondrial engulfment while inhibiting macropinocytosis prevents engulfment of mitochondria by corneal epithelial cells in vitro.

Molecules released by injured cells and severed axons induce macropinocytosis in corneal epithelial cells within minutes of trephine injury. An increase in oxygen consumption rate in the corneal epithelium after trephine injury indicates that axonal mitochondria can evade lysosomal degradation for at least 6 h. In vitro studies using isolated labeled and unlabeled mitochondria and control and mechanically stressed human corneal epithelial cells confirm the involvement of macropinocytosis in the engulfment of free and vesicle bound mitochondria by corneal epithelial cells.

Cornea

Epithelium

Axons

Macropinocytosis

Mitochondria

© 2025 The Authors. Published by Elsevier Inc.