Behavioral Tests in Experiment 1.

<p>MSCs were injected intravenously at 1 h (MSC 1-hour group, <i>n</i> = 7), 1 day (MSC 1-day group, <i>n</i> = 7), or 3 days (MSC 3-day group, <i>n</i> = 7) after MCAO. The control group received normal saline after MCAO (<i>n</i> = 8). Behavioral tests were performed using the rotarod test (A) and by measuring the Longa scores (B) before MCAO and at 1, 4, and 7 days after MCAO. At 7 days after MCAO, the results of the rotarod test showed significant functional recovery in the rats treated with MSCs at 1 h. The Longa scores were also more favorable in the MSC 1-hour group than in the control group (*<i>P</i> < 0.05).</p

MMP-2 and -9 Activities Revealed Using Zymography.

<p>MMP activities in the ischemic hemisphere were measured using gelatin zymography. The MSC 1-hour group showed higher MMP-2 activity than did the control group (<i>P</i> = 0.028). However, MMP-2 activity was not distinct between the MSC 1-day or 3-day group and the control group (A). MMP-9 activity was not different between the MSC-treated groups and the control group (B).</p

Study Design in Experiments 1 and 2.

<p>Experiment 1 was conducted to identify the time point of human mesenchymal stem cell (MSC) transplantation that led to maximal neurological recovery in rats after middle cerebral artery occlusion (MCAO). In Experiment 2, MSCs were administered at 1 h after MCAO, the time point identified in Experiment 1 as being optimal for enabling maximal neurological recovery. Infarction volume, neurological recovery, MMP-2 and MMP-9 activity, and vascular density after MCAO were compared between the MSC 1-hour group and the control group.</p

Neurological Recovery and Infarction Volume.

<p>The surviving animals (MSC 1-hour group, <i>n</i> = 32; control group, <i>n</i> = 27) were subjected to behavioral tests by using the rotarod test and by measuring the modified Neurologic Severity Score (NSS) at baseline and at 1, 4, 7, 10, and 14 days after MCAO. In the rotarod test, the MSC 1-hour group exhibited statistically significant functional recovery at 10 and 14 days (A). The modified NSS at 14 days also revealed greater recovery in the MCS 1-hour group than in the control group. Because the modified NSS scores at 14 days of all 4 rats in control group were 7, the error bar could not be drawn (B). The infarction volume was smaller in the MSC 1-hour group than in the control group at 10 and 14 days after MCAO (C).</p

Vascular Density.

<p>Using the computer-assisted stereological toolbox system, the number and the diameter of vessels were measured based on immunohistochemical staining for collagen type 4 at 10 days after MCAO. The number of the larger capillaries (>10 μm) was significantly higher in the MSC 1-hour group than the control (P = 0.036) (A). After the intracerebrolventricular administration of a selective MMP-2 inhibitor or placebo, vascular density was not significantly different in all ranges of vessel diameter (B).</p

Immunofluorescence staining for MMP-2, NeuN, GFAP, OX-42, and Collagen type 4.

<p>Immunofluorescence staining showed that MMP-2 was not colocalized with neurons (NeuN) or microglia (OX-42), but it was colocalized with astrocytes (GFAP) and and was detected at the outer rim of the capillary basement membrane (collagen type 4), Scale bar = 50 μm.</p

AKAP12-positive cells form a structure to restrict immune cells in the fibrotic scar.

<p>(A) Mouse brains were harvested at serial time points after photothrombotic injury. Brain sections were stained with antibodies for AKAP12 and GS-lectin (a marker for monocytes and macrophages). Nuclei were stained with Hoechst. Scale bar: 20 µm (B) The structure formed by AKAP12-positive cells traps most types of immune cells. Brain sections were stained with antibodies for AKAP12, CD3 (a marker for T cells), MPO (a maker for neutrophils), and IBA1 (a marker for monocytes and macrophages) and GS-lectin (a marker for monocytes, macrophages). Scale bar: 20 µm (C) Mouse brains were harvested at 21 days after photothrombotic injury. AKAP12-positive cells in the fibrotic scar expressed Occludin, E-cadherin and ZO-1 which permit their function as a physical barrier. Scale bar: 40 um. (D) Brain sections were triple-stained with antibodies for AKAP12, GS-lectin and Occludin. Scale bar: 20 µm.</p

AKAP12 is highly expressed in the fibrotic scar surrounding the lesion in the process of CNS repair.

<p>(A) Brain sections were stained with antibody against AKAP12. AKAP12 was highly expressed in the normal meninges, and the number of AKAP12-positive cells in the scar tissue increased over time. Yellow boxes were magnified in right panels. Scale bar: 500 µm (large panels), 50 µm (magnified panels) (B) The AKAP12-positive area of three representative sections per mouse was analyzed using Image J. (Mean ± S.D.; n = 4 mice per each time; P*<0.05, P#<0.001) (C) Mouse brains were harvested at 21 days after photothrombotic injury. Brain sections were stained with antibody against AKAP12, GFAP (a marker for the glial scar) and fibronectin (a marker for the fibrotic scar). Yellow box was magnified in lower panels. Scale bar: 500 µm (largest panel), 200 µm (magnified panels) (D) AKAP12 gene KO was confirmed through genomic PCR and tissue western blotting. PCR analysis of mouse brain DNA showing the AKAP12 WT and KO alleles in the WT or AKAP12 KO mice and immunoblotting analysis of the brain lysates from the WT and KO mice using two different AKAP12 antibodies. (E) Mouse brains were harvested at 21 days after photothrombotic injury. The specificity of the AKAP12 signal was supported by lack of AKAP12 staining in the fibrotic scar in the AKAP12 KO mice. Scale bar: 200 um [LC: lesion core, FS: fibrotic scar, GS: glial scar].</p

The expression of AKAP12 is very low in the endothelial cells and astrocytes of the adult mouse brain.

<p>The mouse brain was extracted on day 21 after injury, and brain sections were stained with antibodies for AKAP12, GFAP (the marker for astrocyte) and CD31 (the marker for endothelial cell). Nuclei were counterstained with Hoechst 33342. AKAP12 was little expressed in the endothelial cells of vessels in normal meninges (panel a) and cortex (panel b). Endothelial cells of vessels in the fibrotic scar also hardly expressed AKAP12 (panel c). AKAP12 level was very low in normal astrocytes wrapping vessels (panel d). The expression of AKAP12 seems to be slightly increased in reactive astrocytes forming the glial scar. However, it was insignificant compared to that of the meningeal cells (panel e). Scale bar: 50 µm (leftmost panels), 20 µm (panel a, b, d and e have same magnification except panel c).</p

AKAP12 KO mice showed larger demyelinated and infarcted tissue compared to WT from the extended infiltration of immune cells.

<p>(A) The areas infiltrated by CD45-positive leukocytes increased in the AKAP12 KO mice on day 21 after injury. Brain sections were co-stained with antibodies against fibronectin and CD45 (the marker for pan leukocytes). Scale bar: 500 µm (left panel), 200 µm (magnified panel) (FS: fibrotic scar) (B) The AKAP12 KO mice showed more severe inflammation compared to the WT mice. The areas infiltrated by the IBA1-positive immune cells and the number of IBA1-positive cells per unit area near the lesion site increased in the AKAP12 KO mice on day 21 after injury (mean ± S.D.; n = 6 mice per WT and KO and 3 positions per mouse; P#<0.001). Scale bar: 200 µm (C) The AKAP12 KO mice showed increased demyelination compared to the WT. Luxol fast blue (LFB) staining was performed to evaluate demyelination (the asterisk region) in the injured brain (mean ± S.D.; n = 4 mice per WT and KO; P**<0.01). Scale bar: 2 mm (D) The AKAP12 KO mice showed increased tissue damage compared to the WT. Triphenyltetrazolium chloride (TTC) staining to evaluate the infarct volume (Mean ± S.D.; n = 4 mice per WT and KO; P#<0.001). Scale bar: 2 mm.</p