Tracking transplanted bone marrow stem cells and their effects in the rat MCAO stroke model.

In this study, rat bone marrow stromal stem cells (BMSCs) were tracked after IV administration to rats with experimental stroke caused by middle cerebral artery occlusion (MCAO). In addition, the effects of BMSC treatment on blood cell composition, brain glia and sensorimotor behavior was studied and compared to that which occurred spontaneously during the normal recovery process after stroke. We found that the vast majority of radiolabeled or fluorescently labeled BMSCs traveled to and remained in peripheral organs (lungs, spleen, liver) 3 days after IV injection in the MCAO rat. Once in the circulation, BMSCs also produced rapid alterations in host blood cell composition, increasing both neutrophil and total white blood cell count by 6 hours post-injection. In contrast, few injected BMSCs traveled to the brain and almost none endured there long term. Nonetheless, BMSC treatment produced dramatic changes in the number and activation of brain astroglia and microglia, particularly in the region of the infarct. These cellular changes were correlated with a marked improvement in performance on tests of sensory and motor function as compared to the partial recovery of function seen in PBS-injected control rats. We conclude that the notable recovery in function observed after systemic administration of BMSCs to MCAO rats is likely due to the cellular changes in blood and/or brain cell number, activation state and their cytokine/growth factor products

Systematic interaction network filtering identifies CRMP1 as a novel suppressor of huntingtin misfolding and neurotoxicity

Assemblies of huntingtin (HTT) fragments with expanded polyglutamine (polyQ) tracts are a pathological hallmark of Huntington's disease (HD). The molecular mechanisms by which these structures are formed and cause neuronal dysfunction and toxicity are poorly understood. Here, we utilized available gene expression data sets of selected brain regions of HD patients and controls for systematic interaction network filtering in order to predict disease-relevant, brain region-specific HTT interaction partners. Starting from a large protein-protein interaction (PPI) data set, a step-by-step computational filtering strategy facilitated the generation of a focused PPI network that directly or indirectly connects 13 proteins potentially dysregulated in HD with the disease protein HTT. This network enabled the discovery of the neuron-specific protein CRMP1 that targets aggregation-prone, N-terminal HTT fragments and suppresses their spontaneous self-assembly into proteotoxic structures in various models of HD. Experimental validation indicates that our network filtering procedure provides a simple but powerful strategy to identify disease-relevant proteins that influence misfolding and aggregation of polyQ disease proteins.DFG [SFB740, 740/2-11, SFB618, 618/3-09, SFB/TRR43 A7]; BMBF(NGFN-Plus) [01GS08169-73, 01GS08150, 01GS08108]; HDSA Coalition for the Cure; EU (EuroSpin) [Health-F2-2009-241498, HEALTH-F2-2009-242167]; Helmholtz Association (MSBN, HelMA) [HA-215]; FCT [IF/00881/2013]info:eu-repo/semantics/publishedVersio

Analysis of blood composition before and after MCAO and rat BMSC treatment.

<p>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.</p

Correlation of BMSC treatment with a significant improvement in behavior over time.

<p>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.</p

Analysis of infarct severity and extent on PET/CT imaging in representative sham and MCAO brains.

<p>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.</p

Whole body and brain images following IV injection of In-111 oxine–labeled BMSCs.

<p>(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).</p

Alkaline phosphatase labeled rBMSCs in vitro and after IV administration into MCAO rats.

<p>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.</p

Spontaneous recovery of behavioral deficits after MCAO.

<p>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).</p