The Akita mutation does not cause RGC death out to 8.5 months of age in DBA/2J mice.

<p>(A, B) To determine if the Akita mutation leads to RGC loss in DBA/2J mice, retinas from 8.5 month old D2.<i>Ins2^Akita/+</i> and D2.<i>Ins2^+/+</i> mice were stained for the RGC marker TUBB3+. (C) TUBB3+ cell counts showed that there was no significant difference in RGCs detected in retinas of D2.<i>Ins2^Akita/+</i> mice compared with D2.<i>Ins2^+/+</i> mice (n = 8 retinas for each genotype; <i>p</i> = 0.138). Scale bar: 30 µm.</p

The Akita mutation does not result in early axon degeneration or axon transport deficiency in DBA/2J mice.

<p>Optic nerve degeneration is a hallmark of glaucoma and optic neuropathies. To determine if the Akita mutation leads to RGC axon loss and optic nerve degeneration in DBA/2J mice, optic nerves from 8.5 month old D2.<i>Ins2^Akita/+</i> and D2.<i>Ins2^+/+</i> mice was assessed using PPD staining (A, B). All of the optic nerves examined from both diabetic and wild-type mice were categorized as having mild damage, which means there was no detectable (or only a low level of damage) damage that is consistent with aged and genetically matched wild-type mice that do not develop glaucoma or diabetic retinopathy. (C) Quantitative analysis of axon counts in 8.5 months old optic nerves showed no differences between D2.<i>Ins2^+/+</i> and D2.<i>Ins2^Akita/+</i> (n = 20 optic nerves for each genotype; p = 0.894). (D–F) Loss of axon transport is a sensitive and early sign of a glaucomatous insult to RGCs and/or RGC dysfunction. To determine if the Akita mutation leads to disruptions in RGC axon transport in DBA/2J mice, intravitreal injections of a fluorescently labeled tracer, CTB-488, were made into 8.5 month old D2.<i>Ins2^Akita/+</i> and D2.<i>Ins2^+/+</i> eyes. (D, E) the superior colliculi of 8.5 months old D2.<i>Ins2^Akita/+</i> mice appeared to fill the same as age-matched D2.<i>Ins2^+/+</i> mice. (F) Quantification of the fluorescent intensity showed there was no significant difference between D2.<i>Ins2^+/+</i> and D2.<i>Ins2^Akita/+</i> mice (n = 8 colliculi for each genotype; <i>p</i> = 0.428). Scale bar: (A, B) 50 µm, (D, E) 150 µm.</p

The Akita mutation increased IOP in DBA/2J mice.

<p>To determine if the Akita mutation caused an increase in IOP in DBA/2J mice, IOP were recorded from D2.<i>Ins2^+/+</i> and D2.<i>Ins2^Akita/+</i> mice at 6 and 8.5 months old. No significant differences in IOP measurements were found between diabetic D2.<i>Ins2^Akita/+</i> and D2.<i>Ins2^+/+</i> at 6 months of age (n = 20 eyes for each genotype; <i>p</i> = 0.106). However by 8.5 months diabetic D2.<i>Ins2^Akita/+</i> showed significant higher IOP levels when compared with wild type D2.<i>Ins2^+/+</i> (n = 38 eyes for each genotype; <i>p</i><0.001).</p

Severe kidney dysfunction and premature death in D2.<i>Ins2^Akita/+</i> mice.

<p>(A) High and sustained blood glucose levels were found in males D2.<i>Ins2^Akita/+</i> mice compared to their respective D2.<i>Ins2^+/+</i> littermates during the time of the study (n = 10 mice for each genotype and age; <i>p</i><0.001 for each age). (B) Severe loss of weight and wasting were found in D2.<i>Ins2^Akita/+</i> compared with D2.<i>Ins2^+/+</i> mice (n = 10 for each genotype and age; <i>p</i><0.001 each age). (C) Sudden and early death occurred in the diabetic D2.<i>Ins2^Akita/+</i> before 9 months (n>40 mice). (D) Kidney weight/body weight ratio was significantly higher in D2.<i>Ins2^Akita/+</i> mice versus D2.<i>Ins2^+/+</i> (n = 10 kidneys). (E–F) Pathological changes associated with diabetic kidney disease at 6.5 months. (E) Decreased immunoreactivity of nestin was evident in D2.<i>Ins2^Akita/+</i> glomerulus when compared with D2.<i>Ins2^+/+</i>. No differences in CD31 immunoreactivity in the glomerular capillary loop were found between diabetic and wild-type mice. (F) MECA79 immunoreactivity was observed in the glomeruli of D2.<i>Ins2^Akita/+</i> mice, but not in D2.<i>Ins2^+/+</i> glomeruli. The MECA79 immunoreactivity was coincident with CD45 positive leukocytes (arrows) in these affected glomeruli. Non-specific green fluorescence in kidney tubules was observed. Scale bars: (E) 10 µm, (F) 30 µm.</p

Vascular leakage and astrocytes reactivity in D2.<i>Ins2^Akita/+</i> retinas.

<p>(A, B) To determine if the Akita mutation caused an increase in vascular permeability in DBA/2J mice, systemic injections of FITC-dextran amine 70 kDa were made into 6 month old D2.<i>Ins2^+/+</i> and D2.<i>Ins2^Akita/+</i> mice. Astrocytes were labeled with GFAP (purple). While FITC fluorescence signal was restricted to blood vessels in D2.<i>Ins2^+/+</i> retinas (n = 10), there was clear leaked fluorescence signal in the majority of D2.<i>Ins2^Akita/+</i> retinas (8 out of 10 retinas examined). (C) GFAP fluorescence intensity (purple) was significantly increased in D2.<i>Ins2^Akita/+</i> retinal astrocytes when compared with D2.<i>Ins2^+/+</i> retinas (<i>p<</i>0.05, n = 10). Scale bar: 50 µm.</p

Astrocytic AQP4 is decreased in aged cortical astrocytes.

<p>(A) AQP4 immunoreactivity (blue) and protein levels (B) are significantly decreased in the aged cortex. (C) Astrocyte reactivity is increased in the cortex of aged mice determined by increased immunoreactivity of astrocytic GFAP (green) when compared with young mice. (D) Electron micrographs showing examples of astrocyte endfeet (As, white region surrounding the vessels) abnormalities such as swelling and big vacuoles (*) in aged mice. Astrocyte endfeet abnormalities were not observed in all cases in aged mice and never seen in young mice. Values in (B) are relative mean <u>+</u> SEM to the young values, <i>n</i> = 4 per group. **<i>p</i> < 0.005 (B) by unpaired <i>t</i> test. Scale Bars: 50 μm (A and C) and 2 μm (D). DP = degenerated pericyte, EC = endothelial cell and BV = blood vessel. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

<i>Apoe</i> expression is reduced in the parietal cortex and CA1 of aged mice.

<p>(A) Representative sections of the FPC from young and aged brains hybridized with an <i>Apoe</i> riboprobe. (B) Quantification of <i>Apoe</i> hybridized area in the cortex shows a significant decrease of <i>Apoe</i> expression in the aged mice. (C) Representative sections of the hippocampal CA1 region from young and aged brains hybridized with an <i>Apoe</i> riboprobe. (D) <i>Apoe</i> hybridized area in the CA1 shows a significant decrease of <i>Apoe</i> expression in the aged mice. (E) Representative sections of the CC from young and aged brains hybridized with <i>Apoe</i> riboprobe. (F) A significant increase of <i>Apoe</i> hybridized area is measured on the aged CC. (G) Representative images of <i>Apoe</i> in situ hybridization in the cortex and CC showing decreased expression of <i>Apoe</i> in the cortex and increased expression in the CC in aged mice compared with young mice. (H) No differences in APOE protein levels were found by western blotting of whole brains from young and aged mice. Values in (B, D, and F) are relative mean <u>+</u> SEM to the young values, <i>n</i> = 6 per group. In (D) **<i>p</i> < 0.001 and in (F) *<i>p</i> = 0.0364 by unpaired <i>t</i> test. Scale Bars: 600 μm (A), 50 μm (C), 100 μm (E and G). The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

Schematic illustration of age-related changes in the neurovascular unit that are prevented by exercise.

<p>In the aged cortex of sedentary mice, neurovascular dysfunction is evident by decreased numbers of pericytes, decline in BM coverage, and increased transcytosis on endothelial cells. Expression of AQP4 in astrocyte endfeet and down-regulation of <i>Apoe</i> (purple) are also found as part of the age-related dysfunction of the neurovascular unit. In addition, decrease in synaptic proteins such as synaptophysin (SYN) is found in aged neurons. The number of proinflammatory IBA1⁺ microglia/monocytes expressing high levels of <i>C1qa</i> RNA is also increased in the aged cortex and HP indicating age-related neuroinflammation in aged mice. These age-related changes were successfully prevented by 6 months of voluntary running during aging, indicating the important contribution of physical activity on preservation of cerebrovascular function during aging.</p

Transcriptional profiling predicts age-related neurovascular dysfunction.

<p>(A) The number of DE genes in three brain regions (FPC/CC; HP; RB) comparing 21-month-old mice to 4-month-old control mice. (B) Pathways overrepresented in the DE genes from the FPC/CC region. For further details and the overrepresented pathways from other regions, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s016" target="_blank">S4 Table</a>. (C) Genes relevant to the neurovascular unit were generally down-regulated. (D) DE genes from the FPC/CC region in the ECM-receptor interaction pathway were down-regulated (green). (E–F) Fibrin intra- and extravascular deposits (red) were significantly increased in aged cortex compared to young cortex. In (<b>F</b>) values are relative mean <u>+</u> Standard Error of the Mean (SEM) to the young values, <i>n</i> = 6 mice per group, **<i>p</i> = 0.0073 by unpaired <i>t</i> test. Scale Bars: 50 μm. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p

Exercise reduces <i>C1qa</i>⁺ microglia/monocytes in the aged cortex.

<p>(A) Representative sections from an aged sedentary and aged runner mouse immunostained with PDGFRβ (magenta) for pericytes and IBA1 (green) for microglia/monocyte cells. (B) Quantification of IBA1⁺ cells in aging, aged sedentary (Sd), and aged runner (Rn) mice shows a decrease in microglia/monocyte density in the aged runners versus the aged sedentary mice. (C) Correlation between PDGFRβ⁺ cells and IBA1⁺ cells (R² = 0.49, <i>p</i> = 0.01). Decreased number of pericytes correlates with increasing number of microglia/monocyte cells. (D) Representative sections of the cortex hybridized with <i>C1qa</i> riboprobe and coimmunostained with IBA1 (green) in young, aged Sd and aged Rn mice. (E) Quantification of <i>C1qa</i>/IBA1⁺ (magenta bars) and IBA1⁺ (green bars) cells in the cortex of young, aged Sd, and aged Rn mice showing a significant increase in <i>C1qa</i>⁺/IBA1 positive cells in aged Sd mice and a significant decline of these cells in the aged runner mice. A new quantification of IBA1⁺ cells (different from B) was performed for the analysis of <i>C1qa</i>⁺/IBA1 cells. (F) Representative sections of the hippocampal CA1 hybridized with <i>C1qa</i> riboprobe and coimmunostained with IBA1 (green) in young, aged Sd, and aged Rn mice. (G) <i>C1q</i>/IBA1⁺ (magenta bars) and IBA1⁺ (green bars) cells in the hippocampal CA1 are significantly increased in aged Sd mice and significantly reduced in aged Rn mice. In panels (B, E, and G) values are mean <u>+</u> SEM, <i>n =</i> 4 per group. In (B) **<i>p</i> = 0.0032 and *<i>p</i> = 0.0148, in (E) **<i>p =</i> 0.0065 and 0.0044, ***<i>p =</i> 0.0006 and *<i>p</i> = 0.0194 and in (G) ***<i>p <</i> 0.0006,***<i>p</i> = 0.0003, **<i>p =</i> 0.0025 and *<i>p</i> = 0.0302 by ANOVA followed by Tukey’s posthoc tests. Scale Bars: 50 μm. The data used to make this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002279#pbio.1002279.s001" target="_blank">S1 Dataset</a>.</p