Age-related neuronal loss in the rat brain starts at the end of adolescence

Abstract

Aging-related changes in the brain have been mostly studied through the comparison of young adult and very old animals. However, aging must be considered a lifelong process of cumulative changes that ultimately become evident at old age. To determine when this process of decline begins, we studied how the cellular composition of the rat brain changes from infancy to adolescence, early adulthood, and old age. Using the isotropic fractionator to determine total numbers of neuronal and non-neuronal cells in different brain areas, we find that a major increase in number of neurons occurs during adolescence, between 1 and 2-3 months of age, followed by a significant trend of widespread and progressive neuronal loss that begins as early as 3 months of age, when neuronal numbers are maximal in all structures, until decreases in numbers of neurons become evident at 12 or 22 months of age. Our findings indicate that age-related decline in the brain begins as soon as the end of adolescence, a novel finding has important clinical and social implications for public health and welfare.

Keywords: aging; atrophy; brain size; neuronal loss; number of neurons.

Figure 1

Appearance of dissociated nuclei and detection of NeuN immunocytochemistry. Images are representative of the appearance of free cell nuclei of cerebral cortex, cerebellum, olfactory bulb and rest of brain prepared with the isotropic fractionator. Same field is shown on the left (Dapi) and right (NeuN immunocytochemistry). NeuN-positive nuclei are indicated by vertical arrows in cerebral cortex, olfactory bulb, and rest of brain; NeuN-negative labeled nuclei are indicated by oblique arrows in cerebellum (a small group of unlabeled nuclei in the bottom right of the image is not indicated). Notice that isolated nuclei are well separated, intact, and easy to identify; immunocytochemistry is virtually free of background staining; and Dapi-stained nuclei are thus easily scored as NeuN-labeled or unlabeled. All images shown at same magnification. Scale bar, 50 μm.

Figure 2

Changes in body and brain mass with age. Body (A) and brain mass (B) increase gradually between ages 1 and 22 months, while total numbers of brain neurons (C) increase between ages 1 and 2–3 months then decline progressively with age, and total numbers of other brain cells (D) increase between ages 1 and 2–3 months, but then do not change significantly with age. Each point represents one individual. Closed circles, males; open circles, females. Large horizontal line, average value for that age; small horizontal lines, one standard deviation from the mean. “Brain” corresponds to the sum of cerebral cortex, hippocampus, cerebellum, and rest of brain, excluding the olfactory bulb for consistency with other studies reported by our group (Herculano-Houzel et al., 2011). ANOVA shows an effect of age for all parameters (body mass, F-ratio 33.0177, p < 0.0001; brain mass, F-ratio 9.0818, p < 0.0001; brain neurons, F-ratio 13.6292, p < 0.0001; other cells, F-ratio 2.8235, p = 0.0356).

Figure 3

Progressive increase in brain structure mass with age. Structure mass increases progressively with age in the cerebral cortex (A,Cx), hippocampus (B,Hp), cerebellum (C,Cb), olfactory bulb (D,OB) and rest of brain (E,RoB). Each point represents one individual. Closed circles, males; open circles, females. Large horizontal line, average value for that age; small horizontal lines, one standard deviation from the mean. ANOVA shows an effect of age for cerebral cortex (F-ratio 7.4735, p = 0.0001), cerebellum (F-ratio 15.1961, p < 0.0001) and rest of brain (F-ratio 9.8994, p < 0.0001), but not for hippocampus (F-ratio 2.3212, p = 0.0642) and olfactory bulb (F-ratio 1.8248, p = 0.1266).

Figure 4

Numbers of neurons increase until the end of adolescence, then decrease with age. Numbers of neurons increase between 1 and 2–3 months and then decrease progressively with age in the cerebral cortex (A,Cx), hippocampus (B,Hp), cerebellum (C,Cb), olfactory bulb (D,OB) and rest of brain (E,RoB). Each point represents one individual. Closed circles, males; open circles, females. Large horizontal line, average value for that age; small horizontal lines, one standard deviation from the mean. ANOVA shows an effect of age for all structures (cerebral cortex, F-ratio 5.8245, p = 0.0007; hippocampus, F-ratio 5.2071, p = 0.0016; cerebellum, F-ratio 11.1325, p < 0.0001; olfactory bulb, F-ratio 7.1240, p < 0.0001; rest of brain, F-ratio 5.1337, p = 0.0011).

Figure 5

Numbers of other cells increase until the end of adolescence. Numbers of other (non-neuronal) cells increase between 1 and 2–3 months and then decrease progressively with age only in the olfactory bulb (D,OB) and rest of brain (E,RoB), but not in the cerebral cortex (A,Cx), hippocampus (B,Hp), and cerebellum (C,Cb). Each point represents one individual. Closed circles, males; open circles, females. Large horizontal line, average value for that age; small horizontal lines, one standard deviation from the mean. ANOVA shows an effect of age for hippocampus (F-ratio 2.6538, p = 0.0418), olfactory bulb (F-ratio 7.2727, p < 0.0001) and rest of brain (F-ratio 5.8304, p = 0.0004), but not for cerebral cortex (F-ratio 0.2965, p = 0.9326) and cerebellum (F-ratio 1.4832, p = 0.2200).

Figure 6

Numbers of neurons change concertedly across structures. Correlated variations in numbers of neurons across brain structures in rats aged between 3 and 22 months. Each point represents one individual. Closed circles, males; open circles, females. Spearman correlation p-values are listed; where p < 0.05, the correlation coefficient is also given. Significant correlations are highlighted.