Both Errors (upper) and Latency (lower) measures are presented. Comparing the last day of pre-testing to the last day of testing with concurrent G-CSF administration, mice in both NT groups showed continuing excellent performance irrespective of GCSF treatment. Tg control mice exhibited consistently poor performance for both pre- and during-treatment testing. By sharp contrast, Tg mice treated with GCSF showed significant improvements in both errors and latency during GCSF treatment. Pre-treatment mean ± SEM values are for all animals in that group. *p
Figure 3. G-CSF Treatment Reduces Aβ Deposition…
Figure 3. G-CSF Treatment Reduces Aβ Deposition in Both Hippocampus and Entorhinal Cortex, While Also…
Figure 3. G-CSF Treatment Reduces Aβ Deposition in Both Hippocampus and Entorhinal Cortex, While Also Reducing Soluble Aβ Levels in Hippocampus (A) Quantification of Aβ immunostaining in hippocampus (HC) and entorhinal cortex (EC) in 14.5 and 8.5 month old cohorts revealed very significant reductions in both brain regions for Tg mice treated with G/CSF compared to Tg controls. *p<0.0005 versus Tg controls. (B) Examples of Aβ immunohistochemical staining in HC and EC for G-CSF and saline treated Tg mice from 14.5 month old cohort. The reduction in both size and extent of Aβ deposition is evident in both brain regions. Scale bar = 20 μm. (C) For both cohorts of Tg mice combined, hippocampal levels of soluble Aβ1-40 were significantly reduced in comparison to Tg controls, while the reduction in hippocampal Aβ1-42 levels was nearly significant. *p<0.05 (Kruskal-Wallis test). (D) Also for both cohorts of Tg mice combined, plasma levels of both Aβ1-40 and Aβ1-42 were unaffected by G-CSF treatment.
Figure 4. G-CSF Increases Total Microglial Activity…
Figure 4. G-CSF Increases Total Microglial Activity Selectively in Tg APP+PS1 Mice
(A) Increased microgliosis…
Figure 4. G-CSF Increases Total Microglial Activity Selectively in Tg APP+PS1 Mice (A) Increased microgliosis was evident by quantitative Iba1 immunostaining in both hippocampus and entorhinal cortex of Tg/GCSF mice, but not NT/GCSF mice; * p < 0.00001 versus both NT groups, **p<0.00001 versus Tg group. (B) Saline-treated Tg APP+PS1 mice exhibited clusters of microglia surrounding amyloid, while G-CSF treated Tg mice revealed a significant increase in Iba1 immunoreactivity around and within amyloid deposits in hippocampus (B) and entorhinal cortex (not shown). Scale bar = 50 μm.
Figure 5. Bone Marrow-Derived (GFP+) Cells Contribute…
Figure 5. Bone Marrow-Derived (GFP+) Cells Contribute To Brain Microglial Activity and Are Increased in…
Figure 5. Bone Marrow-Derived (GFP+) Cells Contribute To Brain Microglial Activity and Are Increased in G-CSF Treated Tg Mice (A) GFP+ cells are seen surrounding amyloid plaques (red) in entorhinal cortex (EC) of a saline-treated Tg mouse (left panel) and in a G-CSF-treated Tg mouse (right panel). Scale bar = 20 μm. (B) GFP+ cells surrounding amyloid in hippocampus (HC) of saline-treated Tg mouse (left panel) and in a G-CSF-treated Tg mouse (right panel). In both HC and EC, visual inspection suggested a greater number of GFP+ cells surrounding the Aβ deposits in G-CSF-treated mice. (C) Low power fluorescence photomicrograph of bone marrow-derived (GFP+) cells in a saline-control non-tg mouse (Left panel). GFP+ cells are seen migrating in meningeal vessels (scale bar = 100 μm). An activated GFP+ macrophage, derived from bone marrow of a chimeric Tg mouse is shown engulfing amyloid (red) in a confocal photomicrograph (Zeiss LSM510) with views along X- and Y-planes, (right panel). Scale bar = 10 μm.
Figure 6. G- CSF Treatment Enhances Synaptophysin…
Figure 6. G- CSF Treatment Enhances Synaptophysin Immunostaining in Hippocampus for Both NT and Tg…
Figure 6. G- CSF Treatment Enhances Synaptophysin Immunostaining in Hippocampus for Both NT and Tg Mice (A and B) Increased staining was evident in both CA1 and CA3 regions of the hippocampus of mice given G-CSF treatment in both 14.5 month and 8.5 month old cohorts. Interestingly, increased immunostaining was evident between NT and Tg controls due to aberrant synaptic terminal staining of Tg mice, particularly on the periphery of Aβ deposits. (C) Photomicrographic examples depicting G-CSF’s enhancement of synaptophysin immunostaining in the CA1 regions of hippocampus are presented for both NT and Tg mice. Scale bar = 50 μm.
Figure 7
( A ) Neural stem/progenitor…
Figure 7
( A ) Neural stem/progenitor cells (NSCs) co-express G-CSF receptor and nestin. Polyclonal…
Figure 7 (A) Neural stem/progenitor cells (NSCs) co-express G-CSF receptor and nestin. Polyclonal rabbit anti-G-CSF-R 1:500 and goat anti-nestin antibody 1/200 (both fromSanta Cruz Biotechnology; Santa Cruz, CA) were incubated for 24 hrs at 4 C, washed with PBS, then incubated with secondary antibodies for 1 hr (anti-rabbit-rhodamine and anti-goat FITC). Image taken with a Zeiss LSM510 confocal microscope. Red= G-CSF receptor; green= nestin. Scale= 10 μm. (B) Addition of G-CSF to hippocampal NSC in basal media containing DMEM+10% FBS for 48 hrs, (in absence of EGF and bFGF), resulted in a dose-dependent increase in 3H-thymidine uptake expressed as percent change from control conditions (DMEM+FCS). * p <0.05 compared to control uptake.
Figure 8. G-CSF Treatment Increased Neurogenesis in…
Figure 8. G-CSF Treatment Increased Neurogenesis in Dentate Gyrus
(A) Mean number of calretinin+ cells…
Figure 8. G-CSF Treatment Increased Neurogenesis in Dentate Gyrus (A) Mean number of calretinin+ cells was estimated in NT and Tg mice from cohort 2 mice (8.5 months of age) treated with G-CSF or saline. G-CSF significantly increased numbers of calretinin+ cells in Tg mice by 36.5%. (*p < 0.05; two-tailed t-test). Mean number of calretinin+ cells in the Tg mice was slightly lower than in NT mice, but this result did not reach statistical significance. (B) Immunofluorescent images of calretinin+ cells in dentate gyrus of hippocampus. Upper panels illustrate calretinin+ cells in a G-CSF treated Tg mouse and lower panels show cells from a saline-treated Tg mice. (Green = Calretinin-immunoreactivity; Blue = DAPI). Panels on the right are magnifications of the indicated regions, showing immature neurons expressing calretinin in the subgranular zone of the dentate gyrus (scale bar = 100 μm in left panels and 10 μm in right panels).
Figure 9. Diagrams depicting proposed effects of…
Figure 9. Diagrams depicting proposed effects of G-CSF treatment on the dynamic equilibrium between soluble…
Figure 9. Diagrams depicting proposed effects of G-CSF treatment on the dynamic equilibrium between soluble and deposited Aβ in the brain (Unmodulated) Newly produced Aβ, resulting from the actions of both β- and γ secretase on APP, enters an equilibrium between soluble and deposited (insoluble) Aβ in the brain. Some bone marrow-derived microglia surround Aβ plaques (green), continually engulfing deposited Aβ. Transport of soluble Aβ from brain to blood plasma is concentration-dependent on levels of soluble Aβ in the brain. (G-CSF Treatment) A relatively short course of G-CSF treatment increases the penetration of bone marrow-derived microglia (green) into the brain and increases activation of reside brain microglia, thus enhancing Aβ engulfment (removal) from plaques. This causes a shift “to the right” in the equilibrium between soluble Aβ ⇔ deposited Aβ, decreasing soluble levels of brain Aβ and resulting in less soluble Aβ transport into the blood.