Stem/Progenitor Cell Proliferation Factors FGF-2, IGF-1, and VEGF Exhibit Early Decline During the Course of Aging in the Hippocampus: Role of Astrocytes ASHOK K. SHETTY, 1,2 * BHARATHI HATTIANGADY, 1,2 AND GEETHA A. SHETTY 3 1 Department of Surgery (Neurosurgery), Duke University Medical Center, Durham, North Carolina 2 Department of Medical Research Service, Veterans Affairs Medical Center, Durham, North Carolina 3 Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina KEY WORDS astrocytes; dentate neurogenesis; ELISA; fibroblast growth factor-2; insulin-like growth factor-1; stem/progenitor cell dif- ferentiation; vascular endothelial growth factor ABSTRACT Dentate neurogenesis, important for learning and memory, declines dramatically by middle age. Although studies have shown that this age-related decrease can be reversed to some extent by exogenous applications of mitogenic factors, it is unclear whether one or more of these factors exhibits decline by middle age. We hypothesize that multiple stem/progenitor cell proliferation factors exhibit early decline during the course of aging in the hippocampus, and some of these declines are linked to age-related alterations in hippocampal astrocytes. We measured the concentrations of fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF) in the hippocampus of young, middle-aged, and aged F344 rats, using enzyme-linked immunosorbent assay (ELISA). In addition, we quantified the total number of FGF-2 immunopositive (FGF-2 þ ) and glial fibrillary acidic protein immunopositive (GFAP þ ) cells in the dentate gyrus and the entire hippocampus. Our results provide new evidence that the concentrations of FGF-2, IGF-1, and VEGF decline considerably by middle age but remain steady between middle age and old age. Further, decreased concentra- tions of FGF-2 during aging are associated with decreased numbers of FGF-2 þ astrocytes. Quantification of GFAP þ cells, and GFAP and FGF-2 dual immunostaining analyses, reveal that aging does not decrease the total number of astrocytes but fractions of astrocytes that express FGF-2 decline considerably by middle age. Thus, dramatically decreased dentate neuro- genesis by middle age is likely linked to reduced concentra- tions of FGF-2, IGF-1, and VEGF in the hippocampus, as each of these factors can individually influence the proliferation of stem/progenitor cells in the dentate gyrus. Additionally, the results demonstrate that decreased FGF-2 concentration dur- ing aging is a consequence of age-related impairment in FGF-2 synthesis by astrocytes. V V C 2005 Wiley-Liss, Inc. INTRODUCTION Hippocampus is one of the brain regions most suscepti- ble to aging (Geinisman et al., 1995; Shetty and Turner, 1999a,b; Driscoll et al., 2003). One of the conspicuous age-related changes in the hippocampus includes greatly diminished neurogenesis in the dentate gyrus (DG) as early as middle age (Kuhn et al., 1996; Nacher et al., 2003; Rao et al., 2005), which may contribute to age- related learning and memory impairments (Drapeau et al., 2003). Although the age-related declines in dentate neurogenesis have been correlated with decreased prolif- eration of stem/progenitor cells in the DG (Kuhn et al., 1996), the precise reasons for age-related decreases in neurogenesis are unknown as the extent of production of new neurons in the mature DG depends on multiple fac- tors (Limke and Rao, 2003). These include the concentra- tions of stem/progenitor cell proliferation factors (Son- ntag et al., 1999; Lichtenwalner et al., 2001; Jin et al., 2002, 2003; Kim et al., 2003; Newton et al., 2003), the presence of cell death in the dentate granule cell layer (Gould and Tanapat, 1997), the concentration of stress hormones and the neurotransmitter serotonin (Gould et al., 1998; Gould, 1999), and the state of vascular niche (Palmer et al., 2000; Monje et al., 2002). It is plausible that decreased dentate neurogenesis during aging is due to the overall age-related alterations in all or most of the above factors. However, decreased levels of stem/progenitor cell proliferation factors, such as fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF), in the microenvironment of the subgra- nular zone (SGZ) are likely to be one of the major causes of age-related decreases in dentate neurogenesis (Hall- bergson et al., 2003). This is because multiple studies have shown that dentate neurogenesis is considerably influenced by the levels of FGF-2, IGF-1, or VEGF in the hippocampus. For example, subcutaneous injections of FGF-2 enhance dentate neurogenesis in both neonatal and adult brain (Wagner et al., 1999; Cheng et al., 2002) *Correspondence to: Ashok K. Shetty, Division of Neurosurgery, DUMC Box 3807, Duke University Medical Center, Durham, NC.27710. E-mail: Ashok.Shetty@duke.edu Grant sponsor: National Institutes of Health (National Institute for Aging); Grant number: RO1AG20924; Grant sponsor: Department of Veterans Affairs. Received 5 September 2004; Accepted 7 January 2005 DOI 10.1002/glia.20187 Published online 30 March 2005 in Wiley InterScience (www.interscience.wiley. com). V V C 2005 Wiley-Liss, Inc. GLIA 51:173–186 (2005)