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Aging-related changes in staining optical density of Insulin Receptor α, Insulin-like Growth Factor 1b Receptor and Leptin Receptor in male murine brain

Introduction: Hypothalamic nuclei and hippocampus are shown to be implicated in feeding behaviour and energy homeostasis regulation. Aging seems to influence the metabolic status by inducing changes in those brain areas. Therefore, it is of crucial importance to understand the different patterns of changes that take place in these regions, in order to better understand the impact of aging on the energy homeostasis. Materials & Methods: Male C57BL/6 mice (n=5) were sacrificed at five time points: 3 months old (3MO), 6 months old (6MO), 12 months old (12MO), 16 months old (16MO) and 20 months old (20MO).The animals were anesthetized with isoflurane, and perfused with 4% paraformaldehyde in phosphate buffer saline (pH = 7, 4). Brains were isolated, perfused, cryoprotected using 30% saccharose, and stored at -80 °C. The specimens were cut on cryostat (Leica CM3050S) in 35 microns thick slices. The following regions of interest were identified: arcuate hypothalamic nucleus (Arc), periventricular hypothalamic nucleus (PeV) and dentate gyrus of the hippocampus (DG). The regions were immunohistochemically stained for Insulin Receptor α (IRa), Insulin-like Growth Factor 1b Receptor (IGF1-Rb) and Leptin Receptor (ObR). Slices were photographed using Zeiss Axioskop 2 MOT microscope, and quantification of staining optical density was performed using ImageJ software. Results: Immunohistochemical analysis showed that IRa tends to yield denser signal in older animals in comparison with younger ones in Arc and PeV, but not in DG, where its optical density decreased as the animal was aging (3MO 6, 15%, 6MO 6, 71%, 12MO 2, 56%, 16MO 2, 37% and 20MO 1, 04%). ObR signal does not seem to change significantly in aging, with an exception of marked depletion in Arc of 20MO mice when compared to the younger animals (16MO 46, 2% vs. 20MO 4, 19%). IGF1-Rb showed a steady trend of increase in signal density as the mice age, except for PeV where optical density does not change much until an animal reaches 20 months (average optical density for 3MO-16MO being 3, 36% vs. 20MO reaching 18, 41%). Conclusion: Results showed that Arc, PeV and DG in male mice might undergo different patterns of aging- related changes in IRa, ObR and IGF1-Rb expression. Further study should involve reward regions due to close connection with satiety centers. Funding: The study has been funded by Croatian Science Foundation under project number IP-09-2014- 2324. Ethical Committee Approval: Ministry for Agriculture, Committee for Animal Research: registration number HR- POK-005.

aging ; insulin ; leptin ; hypothalamus ; hippocampus

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