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+) and glial fibrillary acidic protein immunopositive (GFAP+) 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 concentrations of FGF-2 during aging are associated with decreased numbers of FGF-2+ astrocytes. Quantification of GFAP+ 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 neurogenesis by middle age is likely linked to reduced concentrations 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 during aging is a consequence of age-related impairment in FGF-2 synthesis by astrocytes.

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Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factormediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent.

Aging impairs the competence of the hippocampus for synaptic reorganization after injury. This potentially is due to the inability of the aging hippocampus to up-regulate the critical neurotrophic factors for prolonged periods after injury to levels at which they can stimulate neurite outgrowth and facilitate synaptic reorganization. We hypothesize that the concentrations of neurotrophins in the hippocampus after injury depend on the age at the time of injury. We quantified the concentrations of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) in the hippocampus of young, middle-aged, and aged Fischer 344 rats at 4 days after kainic acid (KA)-induced injury. In comparison with the age-matched intact hippocampus, the KA-lesioned hippocampus exhibited increased levels of BDNF and NGF in all three age groups. In contrast, the NT-3 concentration was unaltered after KA lesion. Notwithstanding similar percentage increases in BDNF after injury, the lesioned middle-aged and aged hippocampus contained 45-52% less BDNF than the lesioned young hippocampus. NGF and NT-3 levels after injury were comparable across the three age groups, however. Furthermore, lower BDNF concentration in the injured aging hippocampus was associated with normal astrocytic response but significantly diminished microglial reaction. Thus, in comparison with the injured young hippocampus, the injured aging hippocampus contains considerably less BDNF but similar levels of NGF and NT-3. Lower BDNF levels in the injured aging hippocampus might underlie the diminished spontaneous healing response observed in the aging hippocampus after injury, particularly in terms of synaptic reorganization and dentate neurogenesis.

A significant upregulation of neurotrophins particularly brainderived neurotrophic factor (BDNF) is believed to be involved in the initiation of epileptogenic changes such as the aberrant axonal sprouting and synaptic reorganization in the injured hippocampus. However, it is unknown which of the neurotrophins are upregulated during the peak period of aberrant mossy fiber sprouting in the chronically injured hippocampus. We measured chronic changes in the levels of BDNF, nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the adult hippocampus using enzyme-linked immunosorbent assay (ELISA) after a unilateral intracerebroventricular administration of kainic acid (KA), a model of temporal lobe epilepsy. For comparison, neurotrophins were also measured from the control intact hippocampus. Further, to see the association between changes in neurotrophin levels and the progression of mossy fiber sprouting, chronic changes in the mossy fiber distribution within the dentate supragranular layer (DSGL) were quantified. In the KA-lesioned hippocampus, the neurotrophins BDNF and NGF were upregulated at 4 days post-lesion, in comparison to their levels in the intact hippocampus. However, the concentration of BDNF reached the baseline level at 45 days post-lesion and dramatically diminished at 120 days post-lesion. In contrast, the upregulation of NGF observed at 4 days post-lesion was sustained at both 45 days and 120 days post-lesion. The concentration of NT-3 was upregulated at 45 days post-lesion but remained comparable to baseline levels at 4 days and 120 days post-lesion. Interestingly, analysis of mossy fiber sprouting revealed that most of the aberrant sprouting in the lesioned hippocampus occurs between 45 days and 120 days post-lesion. Taken together, these results suggest that the period of robust mossy fiber sprouting does not correlate with the phase of post-lesion BDNF upregulation. Rather, it shows a relationship with the time of upregulation of neurotrophins NGF and NT-3.

Grafting of neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise for brain repair after injury or disease, but safety issues have hindered their clinical application. Employing nano-sized extracellular vesicles (EVs) derived from hiPSC-NSCs appears to be a safer alternative because they likely have similar neuroreparative properties as NSCs and are amenable for non-invasive administration as an autologous or allogeneic off-theshelf product. However, reliable methods for isolation, characterization and testing the biological properties of EVs are critically needed for translation. We investigated signatures of miRNAs and proteins and the biological activity of EVs, isolated from hiPSC-NSCs through a combination of anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC). AEC and SEC facilitated the isolation of EVs with intact ultrastructure and expressing CD9, CD63, CD81, ALIX and TSG 101. Small RNA sequencing, proteomic analysis, pathway analysis and validation of select miRNAs and proteins revealed that EVs were enriched with miRNAs and proteins involved in neuroprotective, anti-apoptotic, antioxidant, anti-inflammatory, blood-brain barrier repairing, neurogenic and Aβ reducing activities. Besides, EVs comprised miRNAs and/or proteins capable of promoting synaptogenesis, synaptic plasticity and better cognitive function. Investigations using an in vitro macrophage assay and a mouse model of status epilepticus confirmed the antiinflammatory activity of EVs. Furthermore, the intranasal administration of EVs resulted in the incorporation of EVs by neurons, microglia and astrocytes in virtually all adult rat and mouse brain regions, and enhancement of hippocampal neurogenesis. Thus, biologically active EVs containing miRNAs and proteins relevant to brain repair could be isolated from hiPSC-NSC cultures, making them a suitable biologic for treating neurodegenerative disorders.

Diminished cognitive and mood function are among the most conspicuous symptoms of Gulf War Illness (GWI). Our previous studies in a rat model of GWI have demonstrated that persistent cognitive and mood impairments are associated with substantially declined neurogenesis, chronic low-grade inflammation, increased oxidative stress and mitochondrial dysfunction in the hippocampus. We tested the efficacy of curcumin (CUR) to maintain better cognitive and mood function in a rat model of GWI because of its neurogenic, antiinflammatory, antioxidant, and memory and mood enhancing properties. Male rats were exposed daily to low doses of GWI-related chemicals, pyridostigmine bromide, N,N-diethyl-m-toluamide (DEET) and permethrin, and 5-minutes of restraint stress for 28 days. Animals were next randomly assigned to two groups, which received daily CUR or vehicle treatment for 30 days. Animals also received 5'-bromodeoxyuridine during the last seven days of treatment for analysis of neurogenesis. Behavioral studies through object location, novel object recognition and novelty suppressed feeding tests performed sixty days after treatment revealed better cognitive and mood function in CUR treated GWI rats. These rats also displayed enhanced neurogenesis and diminished inflammation typified by reduced astrocyte hypertrophy and activated microglia in the hippocampus. Additional studies showed that CUR treatment to GWI rats enhanced the expression of antioxidant genes and normalized the expression of multiple genes related to mitochondrial respiration. Thus, CUR therapy is efficacious for maintaining better memory and mood function in a model of GWI. Enhanced neurogenesis, restrained inflammation and oxidative stress with normalized mitochondrial respiration may underlie better memory and mood function mediated by CUR treatment.

Abstract-Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (Յdays) in 52 New Zealand White rabbits undergoing gene transfer (␤-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of Ն4ϫ10 9 plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in ␤-galactosidase expression through 2ϫ10 10 pfu/mL, whereas in in vivo arteries, titers above 4ϫ10 9 pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time-and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1ϫ10 11 pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury. (Circ Res. 1998;82:1253-1262.)