Abstract
Purpose :
The role and mechanisms of efferocytosis (phagocytosis of apoptotic cells) in traumatic optic neuropathy (TON) is poorly understood. Mouse retinas subjected to TON show upregulation of the cluster of differentiation 47 (CD47) protein, a “don’t eat me” signal which inhibits phagocytosis when it binds to its ligand, signal regulatory protein α (SIRPα) on myeloid cells. Therefore, we hypothesize disrupting CD47 interaction with SIRPα on myeloid cells may enhance efferocytosis, thus providing neuronal protection.
Methods :
C57BL/6J male mice were subjected to TON model known as optic nerve crush (ONC). Crush was three seconds long using self-closing forceps and contralateral eye was utilized as non-injury control. On day four post ONC, injured retinas were treated with 1ul (0.66 µg) intravitreal injection of anti-CD47 (Bio X Cell) or anti-IgG (Bio X Cell) for injured control. Retinas were perfused fixed and collected at days 4-14 post ONC. Potential mechanisms of CD47 regulation were explored using western blot and efferocytic efficiency was assessed using immunohistochemistry (IHC) on flatmounts and quantification of colocalized IBA-1 (myeloid marker) positive cells with TUNEL (apoptosis marker) positive cells. Neuroprotection was assessed using optical coherence tomography (OCT) and IHC ImageJ analysis of NeuN (neuronal marker) positive cells.
Results :
Lysates of mouse retinas subjected to TON injury show concurrent upregulation of tumor necrosis factor-α (TNF-α) with CD47 at days 4-10 after ONC. Blockade of CD47 using CD47 neutralizing antibodies reduced total number of retinal apoptotic cells (p=0.03) and increased the co-localization of myeloid cells with apoptotic (TUNEL positive) cells (p=0.04) compared to injured control retinas at day 5. Optical coherence tomography revealed anti-CD47 treatment rescues degeneration of retinal ganglion cell layer (P=0.02) which was confirmed using IHC NeuN staining (p=0.05) at day 14.
Conclusions :
CD47 is upregulated in mice subjected to ONC, a TON mouse model, and is potentially regulated via TNF-α. Our results are consistent with our hypothesis that intravitreal treatment using anti-CD47 post ONC enhances efferocytosis, reduces total apoptotic cells, and has shown to be neuroprotective. The CD47/ SIRPα axis is a promising target in TON.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.