GFAP deficiency increases age-related loss of retinal ganglion cell axons in mice

Abstract

Purpose : Glial fibrillary acid protein (GFAP) is a major intermediate filament component of astrocytes and contributes significantly to their biological functions. In human and experimental glaucoma, astrocytes show signs of reactivity, distinct morphological changes and an increased immunoreactivity for GFAP. Several studies in animal models of glaucoma reported on an association of the reactive phenotype with severe axon loss. On the other hand, inhibition of astrocyte reactivity increased axon loss in a murine glaucoma model. In this study we aimed to investigate how a complete deficiency of GFAP influences retinal ganglion cell (RGC) survival with increasing age.

Methods : 12-week-old and 12-month-old GFAP deficient (GFAP-/-) mice and their wildtype (WT) littermates were analyzed in this study. IOP was measured prior to sacrifice. Whole retinae were stained against RBMPS and RGC somata were quantified. ON axons were quantified on PPD-stained semithin sections. For immunofluorescence sagittal sections of 12-month-old animals were stained against Glutamine Synthetase (GS) and Connexin 43 (Cx43) to visualize changes in astrocyte and Müller Cell reactivity.

Results : We found no difference in IOP between 12-week-old WT and GFAP-/- mice (WT:15.68 ± 2.89 mmHg, GFAP-/-: 16.03 ± 2.06 mmHg, n = 16, p = 0.73). Also, axon numbers were not affected by GFAP deficiency (WT: 49270 ± 4158, GFAP-/-: 47242 ± 6025, n = 6, p = 0.51). IOP in 12-month-old GFAP-/- mice was significantly higher than in WT mice of the same age (WT:12.24 ± 2.97 mmHg, GFAP-/-: 15.66 ± 2.62 mmHg, n = 22, p = 0.04). Absolute numbers of ON axons in GFAP-/- mice were 17% lower than in WT animals (WT: 46550 ± 2536, GFAP-/-: 38690 ± 3247, n = 6, p = 0.001). Optic nerve diameter in the myelinated region was not altered by GFAP deficiency (WT: 108632 ± 5013µm², GFAP-/-: 96538 ± 15321µm², n = 6, p = 0.1). RGC numbers were 17% lower in GFAP deficient than in WT mice (WT: 2473 ± 193 RGC/mm², GFAP-/-: 2054 ± 281 RGC/mm², n = 8, p = 0.004). Immunofluorescence staining against GS and Cx43 indicated a lower macroglial reactivity in GFAP deficient mice.

Conclusions : We conclude that GFAP deficiency increases age dependent RGC loss in mice, probably by altering astrocyte and/or Müller cell homeostatic functions.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.