Stereological Analysis of Microglia in Aged Male and Female Fischer 344 Rats in Socially Relevant Brain Regions

Abstract

Aging is associated with a substantial decline in the expression of social behavior as well as increased neuroinflammation. Since immune activation and subsequent increased expression of cytokines can suppress social behavior in young rodents, we examined age and sex differences in microglia within brain regions critical to social behavior regulation (PVN, BNST, and MEA) as well as in the hippocampus. Adult (3-month) and aged (18-month) male and female F344 (N = 26, n = 5-8/group) rats were perfused and Iba-1 immunopositive microglia were assessed using unbiased stereology and optical density. For stereology, microglia were classified based on the following criteria: (1) thin ramified processes, (2) thick long processes, (3) stout processes, or (4) round/ameboid shape. Among the structures examined, the highest density of microglia was evident in the BNST and MEA. Aged rats of both sexes displayed increased total number of microglia number exclusively in the MEA. Sex differences also emerged, whereby aged females (but not males) displayed greater total number of microglia in the BNST relative to their young adult counterparts. When morphological features of microglia were assessed, aged rats exhibited increased soma size in the BNST, MEA, and CA3. Together, these findings provide a comprehensive characterization of microglia number and morphology under ambient conditions in CNS sites critical for the normal expression of social processes. To the extent that microglia morphology is predictive of reactivity and subsequent cytokine release, these data suggest that the expression of social behavior in late aging may be adversely influenced by heightened inflammation.

Keywords: Fischer 344; aging; limbic system; microglia; rat; social behavior.

Figure 1

(A) Regions of interest for stereological analysis of microglia (adapted from Paxinos & Watson, 1986). (B) Representative photomicrographs (scale bar = 500 µm) showing areas for stereological analysis of microglia (BNST, PVN, CA1, CA3, DG, and MEA). (C) Optical fractionator in Stereo Investigator. Microglia that were within the counting frame (or touching the green line) were included, whereas those that touched the red line were excluded. This prevents duplicate counting of microglia. Arrows point to example Iba-1 positive microglia. (D) Representative photomicrographs (scale bar = 25 µm) of Type 1 (thin ramified processes), Type 2 (thick long processes), Type 3 (stout processes), and Type 4 (round/ameboid shape).

Figure 2

(A) Regional differences in microglia density. Significant differences between ROIs indicated with (*). Density of Iba1+ microglia in BNST (B), PVN (C), MEA (D), CA1 (E), CA3 (F), and DG (G). Significant main effects of Sex are indicated by (#). For significant interactions, different letters indicate groups that differ significantly from one another.

Figure 3

Estimated total number of Type 1/2 and Type 3/4 Iba1+ microglia in BNST (A), PVN (B), MEA (C), CA1 (D), CA3 (E), and DG (F). Aged rats had significantly more microglia in the MEA, regardless of microglia type, whereas aged females (but not males) had more microglia in the BNST and DG. Significant differences in microglia type are indicated by (^) and significant differences between adult and aged rats are indicated by (*). For significant interactions, different letters indicate groups that differ significantly from one another.

Figure 4

Average estimated soma area (µm²) of microglia in the (A) BNST, (B) PVN, (C) MEA, (D) CA1, (E) CA3, and (F) DG. Microglia soma were significantly larger in 18-month-old rats in BNST, MEA, and CA3. In the PVN, 3-month-old females had significantly lower soma areas, relative to all other experimental groups. significant differences between adult and aged rats are indicated by (*). For significant interactions, different letters indicate groups that differ significantly from one another.