Figure 1.
Analysis of infarct severity and extent on PET/CT imaging in representative sham and MCAO brains.
A. Coronal view of a sham-operated animal showing normal symmetrical glucose uptake. B. One day post-MCAO, there is decreased uptake on the side of the infarct. C. Axial and coronal images showing placement of 2 mm thick ovoid regions of interest (ROIs) [symmetrical across the midline, arbitrary colors] across the cerebral hemispheres. D. Mean standardized uptake value (SUV) ratios (infarcted to contralateral side ROI) are plotted against slice position comparing infarcted and sham-operated control rat shown in A–C. The severity of the infarct is reflected both by the number of slices affected, and the degree to which activity is suppressed (relative to normal) in each slice. Over 4 weeks, there was a modest spontaneous improvement in FDG uptake, which correlated with the improvement seen in mNSS scores from 6 to 4 (not shown). Imaging was repeated for 3 sham-lesioned and 3 MCAO lesioned rats.
Figure 2.
Spontaneous recovery of behavioral deficits after MCAO.
Panel A represents sensorimotor loss as measured by the mNSS score. mNSS scores are normalized to a maximum of 18 on the mNSS scale (100% deficit). Note that following MCAO there was substantial spontaneous recovery in the mNSS score. N = 4–6, *p<0.01 and **p<0.001 compared to scores at 1 d post-MCAO. Panel B represents the rats' ability to climb the sidewall of a cylinder using the affected limb after MCAO. All rats were tested prior to MCAO and at 1, 2, 3 and 4 weeks after MCAO and the asymmetry index calculated (See Methods). Note that the decline in forelimb climbing remained even at 4 weeks post-MCAO. N = 4–6, ** p<0.001. Panel C represents the ability of the test animal to turn toward the unimpaired right side when placed in a corner. All rats are tested prior to MCAO, turning 50% of the time to the right and 50% of the time to the left. Values represent % of trials in which the rat turned toward the unimpaired side after MCAO. Note that following MCAO, this ability declined dramatically. ** p<0.001. In all 3 panels, the higher the value, the greater the deficit. (N = 4–8 ± SEM).
Figure 3.
Whole body and brain images following IV injection of In-111 oxine–labeled BMSCs.
(A) Images show persistent activity in the lungs, with relative activity in the liver (arrows) and spleen (arrowheads) increasing over time. Bladder activity is also seen. (B) Brain images at various times after injection. Larger magnification through a pinhole collimator with increasing proximity to the forebrain in later images (20 hrs, 44 hrs, 70 hrs) showed higher activity on the side of the infarct. Approximate position of left eye is marked in each image. (N = 4).
Figure 4.
Alkaline phosphatase labeled rBMSCs in vitro and after IV administration into MCAO rats.
Panel A.: Lung sections 3 days after transplantation; BMSCs show intense activity in the small airways. Note distinct green punctate Alk Phos histochemical staining. Inset: Human alk phos in BMSCs in culture. B. Brain sections from the same animal showing activity in the peri-infarcted tissue. C. Brain at 8 days after transplantation shows some activity at the edge of the infarct zone (“I” = infarct, with no intact tissue). (N = 3) Scale bars in A,C = 100 um; B = 50 um.
Figure 5.
Analysis of blood composition before and after MCAO and rat BMSC treatment.
Blood was collected from the tail vein before and 24 hr after MCAO and 6 and 24 hours after IV injection of BMSCs and quantitatively analyzed for counts of white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells (LUC) by Bioreliance (Rockville, MD). (N = 3) *p<0.05 and **p<0.01.
Figure 6.
Immunocytochemical analysis of reactive glia and activated microglia in the region of the infarct 4 weeks after BMSC treatment in MCAO rats.
Panel A. Representative section through the brain of MCAO rat (center) showing area of infarction (red). Area in rectangle on ipsilateral (left) or contralateral (right) sides of MCAO shown enlarged after staining with GFAP (reactive astrocytes) and CD11 (activated microglia) or merged images in side panels. Panel B. Area labeled “B” in rectangle of Panel A is shown at higher power after staining for GFAP or CD11; Insets: magnified small rectangles of B. Bars in A and B = 100 µm. (N = 4).
Figure 7.
Correlation of BMSC treatment with a significant improvement in behavior over time.
All behavioral assessments as described in Fig. legend 2. Panel A. Sensorimotor behavior as assessed on the mNSS scale 1 or 4 weeks after PBS or BMSC treatment (compared to mNSS score at 1 day post-MCAO set to 100%). Panel B. Climbing ability as assessed on the forelimb asymmetry test at 1, 2, 3 and 4 weeks after PBS or BMSC treatment. When compared to pre-MCAO performance on day 0, PBS groups wer significantly different at all treatment times while those treated with BMSCs differed at 1 and 2 weeks post-treatment but normalized towards control at later time points. Importantly, forelimb asymmetry was significantly different (p<0.001) between BMSC and PBS treatment groups at all 4 treatment times. Panel C. Corner test evaluated at 1 day, 1 and 4 weeks after PBS or BMSC treatment (compared to pre-MCAO performance on day 0). N = 6, *p<0.01 and **p<0.001.