Time course and SCS electrode, and the brain region punched out for protein assay.

<p>(A) Scheme showing overall experimental design. (B) Scheme showing experimental design for protein assay. (C) Photograph showing SCS electrode used in this study (diameter: 2 mm; wire length: 60 mm). (D) Scheme showing a rat during stimulation. (E) Brain tissue (diameter: 3 mm showing gray circle), corresponding to the striatum, was punched out from both the lesioned and the intact side.</p

Enhanced endogenous cell proliferation and neuronal differentiation in the SVZ and DG following AFS cell transplantation.

<p>Ki67 (A, E, I, M) and MAP2 (B, F, J, N) staining revealed an apparent increase in the number of positive cells in the SVZ (A-H) and DG (I-P) of the AFS cell-transplanted stroke animals (E-H, M-P) compared to the vehicle-infused stroke animals (A-D, I-L). Ki67 and MAP-2 double-positive cells are shown in panels: D, H, L, and P. Green: Ki67, Red: MAP2, Blue: Hoechst. Scale bar  = 75 µm.</p

TH immunostaining in the substantia nigra pars compacta (SNc), and the ratio to the intact side.

<p>(A) TH immunostaining in the intact SNc. Severe loss of TH-positive neurons was seen in the lesioned side SNc of control group. Preservation of TH-positive neurons was seen at the lesioned side SNc of 50 Hz SCS group. Scale bar: 200 µm. (B) Significant preservation of TH-positive neurons in the lesioned-side SNc of 50 Hz SCS group, compared to those of control group (*p<0.05, n = 10, respectively).</p

Tyrosine hydroxylase (TH) immunostaining in the striatum and the ratio to the intact side.

<p>(A) TH immunostaining in the striatum. Severe loss of TH-positive fibers was seen in the lesioned striatum of control group. Preservation of TH-positive fibers was seen in the lesioned striatum of all SCS groups. Scale bar: 200 µm. (B) The all SCS groups showed significant preservation of TH-positive fibers in the lesioned striatum, compared to those in control group (*p<0.05, n = 10, respectively).</p

Results of ELISA analysis for VEGF and GDNF.

<p>(A, B) In the lesioned striatum, VEGF was significantly increased by SCS at 1 week after 6-OHDA lesion (*p<0.05). At 2 weeks after 6-OHDA lesion, VEGF level in the lesioned striatum also appeared elevated, but did not reach statistical significance. (C, D) GDNF in the striatum of both sides was not significantly increased by SCS at 1 and 2 weeks after 6-OHDA lesion. (C-lesion: control group lesioned side Striatum; C-intact: control group intact side Striatum; S-lesion: 50 Hz SCS group lesioned side Striatum; S-intact: 50 Hz SCS group intact side Striatum, n = 10, respectively).</p

Quantification of cell proliferation and neuronal differentiation in the SVZ and DG.

<p>The number of cells labeled with Ki67 (A) and MAP2 (B) were significantly increased in the SVZ and DG of AFS cell-transplated stroke animals compared to vehicle-infused stroke animals (*p<0.05, **p<0.01, ****p<0.0001) (A, B, D, E). While there was a trend towards increased Ki67/MAP2 double-positive cells in the SVZ (p = 0.0946) (C), the number of cells labeled with both Ki67 and MAP2 were significantly increased in the DG of AFS cell-transplated stroke animals compared to vehicle-infused stroke animals (***p<0.001) (F). Bars represent the mean ± SEM.</p

Experimental design is shown.

<p>Rats were subjected to a 60 minute transient MCAo and received intravenous transplants of AFS cells or vehicle. After behavioral evaluations, all rats were euthanized for immunohistochemical evaluations. EBST: elevated body swing test; MCAo: middle cerebral artery occlusion.</p

Motor deficits were ameliorated in the AFS cell-transplanted stroke animals.

<p>Results revealed that all animals exhibited no detectable bias in swing activity at baseline (p>0.05) (A) and all animals learned to balance on the rotating rod for 60 seconds (p>0.05) (B). EBST revealed a significant biased swing activity on day 2 post-MCAo. EBST conducted on day 60 post-stroke detected a significant decrease in swing bias in the AFS cell-transplanted stroke animals compared to the vehicle-infused stroke animals (*p<0.0001) (A). Rotarod test revealed significant deterioration in motor coordination on day 2 post-MCAo, but on day 60 post-MCAo revealed that the AFS cell-transplanted stroke animals exhibited significantly increased time spent balancing on the rotating rod compared to the vehicle-infused stroke animals (*p<0.0001) (B). Bars represent the mean ± SEM.</p

The results of cylinder test and amphetamine-induced rotation test.

<p>(A) Rats receiving 2 Hz and 50 Hz SCS showed reduction of the contralateral bias at 2 weeks after 6-OHDA lesion, compared to that of rats in control group. (B) The number of amphetamine-induced rotations in all SCS groups decreased, compared to that of control group. There was a significant amelioration in 50 Hz SCS group, compared to control group (*p<0.05, n = 10, respectively).</p

Reference memory, but not spatial navigation, is rescued by AFS cell transplantation.

<p>Results revealed that there were no significant differences in cumulative search error and total distance swam between the two treatment groups (A,B), indicating that spatial navigation was not improved in the AFS cell-transplanted stroke animals compared to the vehicle-infused stroke animals (p>0.05) (A). In contrast, reference memory was significantly improved in AFS cell-transplanted stroke animals compared to vehicle-infused stroke animals (C). Twenty-four hours after training in the MWM, rats were tested in a probe trial in which the platform was missing. Results revealed that AFS cell-transplanted stroke animals spent significantly more time in the target quadrant compared to vehicle-infused stroke animals (*p<0.05). Bars represent the mean ± SEM.</p