Figure 1.
Markers of oxidative stress accumulate in and around the dentate gyrus of the hippocampus.
(a) Oxidized 8-deoxyguanosine (8OHDG), a marker of oxidized nucleic acid and (c) hydroxynonenal (HNE), a measure of oxidized lipid, both exhibit localized increases in expression throughout the hippocampus. (b, d) Elevated magnification (boxed areas from a, c) indicated that both these markers express highest levels within the SGZ (arrows). Scale bars: 200 µm (a,c), 100 µm (b,d).
Figure 2.
Hippocampal NSCs can be isolated and established as a two-dimensional culture system.
(a–c) Undifferentiated hippocampal NPCs are undifferentiated, expressing insignificant levels of Tuj1 and Doublecortin (DCX). (d–f) Three days following initiation of differentiation, individual progenitor cells become Tuj1 (+) and begin to strongly express DCX. (g, h) Neurogenesis proceeds from focal clusters which expand and contact one another within 7 days of induction of terminal differentiation. (i) Camera lucida of representative of expanding clusters of Tuj1(+) neural progenitors at 12, 24, 48 and 72 hours following differentiation.
Figure 3.
Differentiation of hippocampal NSCs is marked by rapid, transient proliferation and a spike in mitogens.
(a) BrdU incorporation was measured by non-overlapping 24-hr consecutive pulse labeling of differentiating and undifferentiated HPCs (see methods). Upon induction of differentiation, hippocampal NPCs (grey data points) significantly increase their proliferative rate for a period of 72 hours when compared to hippocampal NSCs maintained in medium containing EGF and bFGF (red data points). (b) RT-PCR on NPCs revealed a spike (p*<0.05) in expression of several mitogenic factors, including nerve growth factor β (Ngfb), brain-derived neurotrophic factor (Bdnf) and neurotrophin-3 (Ntf33) three days after differentiation was initiated. (c) Terminal differentiation of hippocampal NPCs is accompanied by decrease in the progenitor marker nestin and increases in the lineage markers GFAP and the early- and forebrain- specific neuronal markers Dcx and calmodulin-dependent kinase alpha (CaMKIIα).
Figure 4.
Hippocampal neurogenesis transiently generates oxidative stress.
(a) Total mitochondria and oxidative load were measured in undifferentiated NPCs, early progenitors and postmitotic neurons. (b, c) Total mitochondria prevalence was measured via Mitotracker biological dye (b) while total oxidative load was measured using Mitosox biological indicator dye (measured as–fold change compared to undifferentiated NSCs). Quantifiable fluorescence emission measurements demonstrate a significant increase in maximal mitochondrial abundance and peak oxidative load occurs during the period enriched for early progenitors. p*<0.0001.
Figure 5.
Identification of a set of oxidative stress-responsive (OR) transcription factors.
(a) A large subset of genes was identified using the IPA database for genes associated with CNS expression and oxidative stress. This list was compared to two publications ([22], [23]) aimed at large-scale identification of genes which exhibit transcriptional response to oxidative stress. Genes appearing in both data sets were cross-referenced to the NCBI Gene Function Database and the Allen Brain Atlas for acknowledged gene function and detectable hippocampal expression, respectively. Genes appearing on this list were selected a priori as potential proxy measures of ambient oxidative stress levels. Gene specific information appears in Table S2. (b) Dentate gyrus was isolated from 500 µm-thick coronal sections using a P300 pipette tip applied to regions shown (dotted line). Sections counterstained with Cresyl Violet.
Figure 6.
ORGs exhibit elevated levels of expression in multiple models of enhanced neurogenesis, and respond more dramatically to antioxidant administration than normal controls.
Infusion of the antioxidant Edaravone (3 mg/kg/day) produces an larger magnitude of decrease in OR genes in CaMKIIα hyper-neurogenic mice compared to controls for both individual genes (16/20; a) and as a group (b). Running (14d) significantly increases the expression of 11/20 ORG members (c), with a significant group increase (d). (e) RT-PCR measures of OR expression. p*<0.05.
Figure 7.
Inhibition of neurogenesis results in the rapid decline of pathophysiological markers of oxidative stress within the SGZ.
The antimitotic agent Ara-C or saline (vehicle) was infused twice daily for 7 days prior to thymidine analog labeling, resulting in a significant reduction in total cell division as measured by thymidine analog incorporation (a–c). Acute Ara-C treatment disrupts the production of oxidized DNA (8OHDG; d, e) and lipid (HNE; f, g) within the SGZ. Note existing 8OHDG and HNE (+) cells are retained in the outer granular layer (arrows; DG defined in d–g with Mayer’s hematoxylin). Scale bars: 50 µm (a, b), 100 µm (d–g). ***p<0.001.