Abstract
Purpose :
Neuroinflammation is an early and persistent feature of retinal pathophysiology. Understanding the interaction of retinal neurons and resident glia cells (microglia, astrocytes, and Müller glia) is important for identifying disease mechanisms and drug targets. Attributing neuroinflammatory events to resident or infiltrating immune cells is non-trivial since simple tools and models to separate these do not exist. Using a retinal explant model, where axotomy and maintenance of the retina ex vivo drives rapid retinal ganglion cell neurodegeneration, we assessed resident neuroinflammation
Methods :
Retina from C57BL/6J mice were dissected and maintained as retinal explants for 0 (control) or 24 hrs. LPS (100 ng / mL) or Valproic acid (1 mM) were dissolved in the culture media. Retinal neuroinflammation was quantified by morphological and protein analysis. Fixed retinas were immunofluorescently labeled for RBPMS (retinal ganglion cells), IBA1 (microglia), and GFAP (astrocytes), imaged on a confocal microscope, and 3D morphology reconstructed using Imaris. Dissociated retinas were assessed for cytokine and chemokine expression by protein array (109 proteins)
Results :
Twenty-four hours after axotomy, retinas displayed significantly altered microglia and astrocyte morphologies. The morphology of microglia processes was, on average, more contracted and voluminous, consistent with a shift towards inflammatory phenotypes. Astrocyte networks increased in filament density and connectivity. Key inflammatory cytokines were significantly upregulated (Ccl5, Cxcl10, Ccl2, Il-1α, Cxcl1, Icam-1). This is consistent with in vivo retinal ganglion cell injury. As a putative positive control, LPS (a known inflammatory activator), further retracted microglial morphology and further increased Ccl5, Cxcl10, and Il-12, but did not affect astrocyte morphology. Valproic acid provided neuroprotection, significantly reduced morphological changes of both microglia and astrocytes, and maintained Ccl5, Cxcl10, Ccl22, Il-12, Ccl2, and Cxcl1 at uninjured control levels
Conclusions :
We demonstrate that the retinal explant model allows for the isolated study of resident glial neuroinflammation without the influence of the circulatory system. This model allows for rapid testing of compounds which may modify neuroinflammation and identifies Valproic acid as a drug which limits retinal neuroinflammation
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.