Morphological Analysis of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish

Abstract

As the number of elderly individuals is increasing in modern society, the need for a relevant gerontology model is higher than ever before. Aging can be defined by specific cellular hallmarks, described by López-Otín and colleagues, who provided a map which can be used to scavenge the aging tissue environment. As revealing the presence of individual hallmarks does not necessarily indicate aging, here we provide different (immuno)histochemical approaches that can be used to investigate several aging hallmarks—namely, genomic damage, mitochondrial dysfunction/oxidative stress, cellular senescence, stem cell exhaustion, and altered intercellular communication—in the killifish retina, optic tectum, and/or telencephalon at a morphological level. In combination with molecular and biochemical analysis of these aging hallmarks, this protocol offers the opportunity to fully characterize the aged killifish central nervous system.

Age-related dysregulation of the retinal transcriptome in African turquoise killifish

“…Fish were euthanized as described above. Intracardial perfusion with phosphate buffered saline (NaHPO 4 .2H 2 O, 8mM; KH 2 PO 4 , 2 mM; NaCl, 0.15M; KCl, 3mM; pH 7.4) and 4% paraformaldehyde (PFA, in PBS, Merck-Aldrich) was executed, all as described (Bergmans et al, 2023c; Mariën et al, 2022). Eyes were collected and post-fixed overnight in 4% PFA, followed by three rinses in PBS.…”

“…Next, eyes were incubated until saturation using an increasing series of sucrose concentrations (10, 20, 30% weight by volume in PBS) and embedded in 1.25% agarose and 30% sucrose in PBS for cryosectioning. For each eye, 10 µm sagittal cryosections were serially collected on eight SuperFrost Plus Adhesion Slides (Epredia) and stored at −20°C until further use (Bergmans et al, 2023c).…”

The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment

“…17. Carry out perfusion and sectioning as described in the indicated Steps from Protocol: Morphological analysis of the aging hallmarks in the central nervous system of the fast-aging African turquoise killifish<prot107827> (Bergmans, 2022b). i.…”

“…30. Define the central optic tecti sections based on the killifish brain atlas published by D'Angelo, i.e., sections comparable to those in Figure 2 panel m -q of (D'Angelo, 2013) and transfer the selected sections from Step 43 in Protocol: Morphological analysis of the aging hallmarks in the central nervous system of the fast-aging African turquoise killifish<prot107827> (Bergmans et al, 2022b) to a net insert in a 12-well plate filled with 1X PBS using a paint brush. These net inserts are tiny baskets with a net-based bottom that fit within the well of 12-well plate.…”

“…Step 34 in Protocol: Morphological analysis of the aging hallmarks in the central nervous system of the fast-aging African turquoise killifish<prot107827> (Bergmans et al, 2022b), remove the cryosections of brains that were post-fixed for 1 h from the freezer.…”