Region specific neuron loss in the aged canine hippocampus is reduced by enrichment
Introduction
The hippocampus and entorhinal cortex are brain regions essential for intact cognitive abilities and appear to be particularly vulnerable to the aging process. In normal human aging, stereological studies have revealed neuron loss in the hilus and subiculum of the hippocampus (Simic et al., 1997, West, 1993, West et al., 2000), and within islands in layer II of the entorhinal cortex (Heinsen et al., 1994, Simic et al., 2005). Neuron loss also occurs in the hilus of the hippocampus of aged rats (Azcoitia et al., 2005, Cadiacio et al., 2003, Shetty and Turner, 1999), but not in any area or layer of the entorhinal cortex, even when cognitive status is considered (Merrill et al., 2001). By contrast, neuron loss has not been reported in any subregion of the hippocampus (Keuker et al., 2003) or any layer of the entorhinal cortex of non-human primates (Gazzaley et al., 1997, Merrill et al., 2000).
Neuron number and function may be modulated by environmental enrichment. Environmental enrichment involves rearing animals in a socially and physically stimulus-rich environment (Varty et al., 2000). This typically involves housing animals in groups to increase social contact, and adding novel objects to the immediate environment to stimulate exploratory behavior. Environmental enrichment in mice increases the number of hippocampal dentate gyrus neurons and neurogenesis (Kempermann et al., 1997).
Neuron number and function may also be compromised by oxidative damage. Increasing oxidative damage to proteins, lipids and nucleotides may contribute to neuron dysfunction in normal and pathological aging in humans (Ames et al., 1993, Harman, 1992). Mitochondria are a source of damaging free radicals and they are in turn, particularly vulnerable to free radicals. Oxidative damage produces mutations in mitochondrial DNA, alters membrane fluidity and phospholipid composition, and leads to dysfunctional mitochondrial proteins (Shigenaga et al., 1994). The result is a loss of biochemical and physiological function of mitochondria in the cell, which impairs normal cellular activities. In support of the hypothesis that oxidative damage may lead to neuron death, providing rats with Vitamin E increases neurogenesis (Cecchini et al., 2003, Ciaroni et al., 1999, Cuppini et al., 2002) and improves cell survival in the dentate gyrus (Ciaroni et al., 2002, Cuppini et al., 2002, Ferri et al., 2003).
The aging dog exhibits many features of normal brain aging that develop in humans. Aged dogs show declines in cognitive function (Head et al., 1995, Milgram et al., 1999, Tapp and Siwak, 2006, Tapp et al., 2003a, Tapp et al., 2003b, Tapp et al., 2004b), decreased brain volume (Su et al., 1998, Su et al., 2005, Tapp et al., 2004a, Tapp et al., 2006), behavioral alterations (Head et al., 1997, Siwak et al., 2001, Siwak et al., 2002, Siwak et al., 2003), and neuropathology including the accumulation of human-type beta-amyloid (Head et al., 1998, Russell et al., 1996, Wisniewski et al., 1970), increased oxidative damage (Head et al., 2002, Papaioannou et al., 2001, Rofina et al., 2004, Skoumalova et al., 2003) and apoptosis (Anderson et al., 2000, Kiatipattanasakul et al., 1996). Previous studies that examined neuron number in the canine brain report significant decreases with age in the cingulate gyrus, superior colliculus and claustrum (Ball et al., 1983, Morys et al., 1994). Calbindin-positive GABAergic neurons in the prefrontal cortex of the canine brain are also vulnerable to aging (Pugliese et al., 2004). No evidence of neuron loss, however, has been reported in other brain regions including the hippocampus in the aged dog.
The goal of the present study was to examine the effect of age on neuron number in the canine hippocampus and entorhinal cortex using the optical fractionator in the left hemisphere. We hypothesized that neuron loss in the canine hippocampus, like that in humans, will be region specific and may also occur in the entorhinal cortex. We further tested the hypothesis that an antioxidant fortified food or behavioral enrichment or both would reduce age-related loss of neurons or increase the number of neurons relative to untreated control animals. We further examined the relationship between neuron number and cognitive function to determine whether changes in neuron number could account for the cognitive enhancing effects of the behavioral enrichment and antioxidant fortified food.
Section snippets
Aging study animals
The brains from 5 aged (13.0–15.0 years old) and 5 young (3.4–4.5 years old) Beagles from the Lovelace Respiratory Research Institute (LRRI, Albuquerque, NM) breeding colony were used to examine age-related differences in neuron number in the hippocampus and entorhinal cortex. Unilateral neuron numbers were acquired for each subregion of the left hippocampus separately. Unilateral neuron numbers were determined for the left entorhinal cortex and in layer II specifically.
Treatment study animals
Twenty-four
Aging study
Table 2, Table 3 show a significant and selective age-related loss of neurons in the hilus of the hippocampus [F(1, 8) = 10.28, p = .012]. There were no significant differences in neuron number between the young and aged dogs in the granule cell layer of the dentate gyrus (p = .546), area pCA3 (p = .476), area dCA3 (p = .662), total area CA3 (p = .869), area CA2 (p = .809), area CA1 (p = .915), the subiculum (p = .280) of the hippocampus, or within the entorhinal cortex (p = .561) or layer II of the entorhinal
Discussion
This study examined neuron number as a function of age in the canine hippocampus and entorhinal cortex of the left hemisphere using the optical fractionator. The present results reveal a significant subregion specific age-related loss of neurons in the hilus of the canine hippocampus that is similar to observations in normal aged humans. Further, hilar neuron number in aged animals could be modified by providing animals with a program of behavioral enrichment but not with an antioxidant
Conclusions
The aged canine hippocampus is vulnerable to neuron loss in the hilus. Age-related losses of hilar neurons in the canine may be reduced by behavioral enrichment but do not appear to be significantly affected by antioxidant treatment. These results extend previous studies in rodents to a higher mammal with consistent results. Although both treatment conditions improved cognition in aged canines, the changes in neuronal function seem to be mediated through independent molecular pathways.
Acknowledgements
This research was supported by the National Institute on Aging (NIA AG12694 and AG17066), and by the United States Army Medical Research and Material Command under Contract No. DAMD17-98-1-8622. The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as an official Department of the Army position, policy, or decision unless so designated by other documentation. Additional support was provided by the Natural Sciences and Engineering
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