T lymphocytes invade into TKO brain.

<p>(A, B) The WT (A) and TKO (B) mice at 7 weeks of age were prepared for brain sections, which were immunostained with anti-CD3 antibody to show T cell infiltration. (C) The brain section from the TKO mouse at age of 10 month old was stained with hematoxylin and eosin (H&E). Scale bars, 50 µm. (D) Brain diagram shows the regions of figures A–C. (E, F) Flow cytometric analysis of infiltrated TCRαβ-positive cells in TKO brains. Cell preparation and flow cytometry procedures were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064812#s2" target="_blank">Methods and Materials</a>. There are increased TCRαβ-positive cells in the TKO brain (E, 5.6% of leukocytes) than the WT brain (F, 0.8% of leukocytes). This is one representative for each genotype, n = 3.</p

TKO mice produce increased proinflammatory cytokines.

<p>(A) TNF-α level in the WT and TKO mouse serum were measured by Ready-set-go ELISA kits (eBiosciences). Data are shown as means±SD, n = 9, p = 0.0002. (B) Thioglycollate-induced peritoneal macrophages (MФ) were treated with LPS for 2, 3, 4 and 5 hrs. TNF-α released into medium was measured as above. Data are shown as means ±SD for five wells per group in a single experiment and are representative of those in three experiments. N = 3, **P<0.001. (C) Real-time qPCR quantification of IL-1β and IL-6 mRNA in the lymph nodes. The total RNA was extracted by TRIzol and reversely transcribed using transcribed using qScript™ cDNA Supermix kit (Quanta Biosciences, MD). Real time qPCR was performed to measure the relative mRNA level of IL-1β and IL-6 genes in the WT (open) and TKO (solid) L.N. Data are shown as means ± SD, n = 3, *P<0.05, **P<0.002. Statistics was performed by the one-way ANOVA test using ProStat Ver 5.5.</p

Autoantibodies are deposited on the microvessels of the TKO brain.

<p>Both WT (A) and TKO (B) mice at age of 8 weeks were deep anesthetized by 2.5% Avertin and the blood vessels were flushed through transcardial perfusion using hepatinized 1×PBS for 5 min followed by 8 min of 4% PFA perfusion fixation. After dissection, the brain tissues were postfixed in 4% PFA at 4°C for overnight. Coronal sections at the middle of cortex were cut on cryostat and stained with FITC labeled rabbit-anti-mouse IgG. The images were taken on fluorescent microscope. Bars, 200 µm. (C) Serum antibody isotyping was performed on the sera isolated from WT, TKO and <i>Axl^−/−Mertk^−/−</i> (AM), using mouse monoclonal antibody isotyping reagents following manufacturer’s instruction (Sigma-Aldrich). The data is expressed as mean±SD, n = 6. The statistics was performed by ANOVA Tukey’s multiple comparison tests using Prostat ver5.5 program **<i>P</i><0.001.</p

The TKO hippocampal CA3 regions show altered mossy fiber projection and sprouting.

<p>Timm staining of the Zn²⁺-containing mossy fiber terminals was performed on the hippocampus of WT and TKO mice at ages of 8–10 months. (A and A-a) Mossy fibers in the WT brains were terminated in the stratum lucidum, above the CA3 pyramidal cells layer (arrowheads), and below and within the pyramidal cell layer in the proximal portion of CA3 (half-open arrowhead). (B and B-b) Mossy fiber sprouting in the suprapyramidal bundle (arrows), and a fuzzy distal boundary of the suprapyramidal bundle spreading across the pyramidal cell layer into CA2 (asterisk) were shown on the TKO brain sections. Triangle in (B) shows the lightly stained and diffused intra and infrapyramidal bundles. Scale bars, 100 µm in (A and B), and 50 µm in (A-a and B-b).</p

TKO brains develop ubiquitinated protein-aggregates and lipofuscin deposition.

<p>(A, B) Ubiquitinated protein aggregates were detected by anti-ubiquitin antibody. The brain cryosections from TKO (n = 5) or WT (n = 3) mice at ages of 6–8 months were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064812#s2" target="_blank">Methods and Materials</a>. 20 micron of cryostat sections was cut and immunostained with anti-ubiquitin (red, 1∶100, RnD) and -GFAP (green, 1∶200, Santa Cruz) antibodies. Nuclei were stained blue with DAPI. Fluorescent images were obtained with a confocal microscope (LSM510, Zeiss). (C–E) Lipofuscin autofluorescence was accumulated in the TKO brains. The brain sections were prepared from the WT (C) and TKO (D and E) mice at age of 2 months old, and stained with DAPI for cell nuclei; the sections were observed and photographed under UV laser light. The lipofuscins were shown as red aggregates at one side of blue-stained nuclei. Scale Bars are 50 µm in (A and B), and 100 µm in (C–E).</p

The BBB integrity is altered in TKO mice.

<p>(A–D) Mice at 6–8 months of age were <i>i.v</i>. injected with Evans blue for 30 min. Histological and image processes follow procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064812#s2" target="_blank">Methods and Materials</a>. Extravasation of Evans blue into parenchyma can be clearly detected in the TKO brains (A and B), in contrast to barely-detected leakage in the WT brain (C and D). (E, F) Mice at 6–8 months of age were <i>i.v</i>. injected with FITC-dextran (70 kDa) for 20 min prior to systemic transcardial perfusion of 1×PBS supplemented with 1 unit/mL of heparin. Brains were harvested, cryosectioned at 30 µm in thickness and stained with DAPI. Fluorescent images in (A–F) were observed using a Zeiss microscope equipped with Apotome and obtained with a deep-cooled CCD imaging system, and analyzed using Carl Zeiss imaging systems software version 4.8.2. Scale bars in (A–F) are 50 µm. (G) For fluorescent measurement of Evens blue retained in brains, the brain tissues obtained from WT and TKO mice that had been perfused with hepatinized PBS were weighed and homogenized in formamide, followed by incubation at 56°C for 30 min, and centrifugation for 15 min at maximal speed in a Beckman Coulter microfuge-18 centrifuge. The Evans blue retained in the supernatant was measured by absorbance at the optical density (OD) of 600 nm on a fluorescent spectrophotometer (Molecular Devices, CA). Absorbance was normalized to the sample weight. Data is expressed as mean ±SD, n = 5, p = 0.005. (H) The integrity of the brain blood vessel endothelial cells was measured using a transwell permeability assay system. The brain endothelial cells were isolated from WT, TKO, AM and TA mice at age of postnatal day 12 and cultured for 7 days in medium 131 supplemented with microvascular growth supplements (Life Technologies). Cell permeability assay was described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064812#s2" target="_blank">Methods and Materials</a>. The value at each time point in each genotype was the mean of three independent cultures, and each culture was pooled endothelial cells from at least four pups with same genotype.</p

TKO brains show increased apoptosis and cell death.

<p>Mice at 6–8 weeks of age were deeply anesthetized by 2.5% Avertin prior to transcardial perfusion with 1 unit/mL of heparin dissolved in 1×PBS. Brains were harvested, cryosectioned at 20 micron. (A–D) TUNEL labeling showed increased apoptosis in the TKO brains (B and D) as compared to the negative labeling in the WT brains (A and C). (E and F) Purkinje cell loss was shown in the TKO cerebellum (F and F-f) but not in the WT brains (E and E-e). Purkinje cells were immunostained with antibody for calbindin D28K and cell nuclei were stained with DAPI. Fluorescent images were obtained with a confocal microscope (LSM510, Zeiss). Scale Bars in (A–F), 100 µm; and in (E-e and F-f), 50 µm.</p