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

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

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

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