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. 2011 Oct;42(10):2923-31.
doi: 10.1161/STROKEAHA.110.606368. Epub 2011 Aug 11.

The CCR2/CCL2 interaction mediates the transendothelial recruitment of intravascularly delivered neural stem cells to the ischemic brain

Robert H Andres  1 , Raymond Choi, Arjun V Pendharkar, Xavier Gaeta, Nancy Wang, Jaya K Nathan, Joshua Y Chua, Star W Lee, Theo D Palmer, Gary K Steinberg, Raphael Guzman
Affiliations

The CCR2/CCL2 interaction mediates the transendothelial recruitment of intravascularly delivered neural stem cells to the ischemic brain

Robert H Andres et al. Stroke. 2011 Oct.

Abstract

Background and purpose: The inflammatory response is a critical component of ischemic stroke. In addition to its physiological role, the mechanisms behind transendothelial recruitment of immune cells also offer a unique therapeutic opportunity for translational stem cell therapies. Recent reports have demonstrated homing of neural stem cells (NSC) into the injured brain areas after intravascular delivery. However, the mechanisms underlying the process of transendothelial recruitment remain largely unknown. Here we describe the critical role of the chemokine CCL2 and its receptor CCR2 in targeted homing of NSC after ischemia.

Methods: Twenty-four hours after induction of stroke using the hypoxia-ischemia model in mice CCR2+/+ and CCR2-/- reporter NSC were intra-arterially delivered. Histology and bioluminescence imaging were used to investigate NSC homing to the ischemic brain. Functional outcome was assessed with the horizontal ladder test.

Results: Using NSC isolated from CCR2+/+ and CCR2-/- mice, we show that receptor deficiency significantly impaired transendothelial diapedesis specifically in response to CCL2. Accordingly, wild-type NSC injected into CCL2-/- mice exhibited significantly decreased homing. Bioluminescence imaging showed robust recruitment of CCR2+/+ cells within 6 hours after transplantation in contrast to CCR2-/- cells. Mice receiving CCR2+/+ grafts after ischemic injury showed a significantly improved recovery of neurological deficits as compared to animals with transplantation of CCR2-/- NSC.

Conclusions: The CCL2/CCR2 interaction is critical for transendothelial recruitment of intravascularly delivered NSC in response to ischemic injury. This finding could have significant implications in advancing minimally invasive intravascular therapeutics for regenerative medicine or cell-based drug delivery systems for central nervous system diseases.

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Figures

Figure 1
Figure 1
Migration of CCR2+/+ and CCR2−/− neural stem cells (NSC) in response to CCL2 and CXCL12a in vitro (A). CCR2+/+, but not CCR2−/−, NSC showed a dose-dependent increase in transmigration in response to CCL2 after 1 hour (B) or 2 hours (C). No significant differences in the number of transmigrated CCR2+/+ and CCR2−/− cells were found after stimulation with CXCL12a for 1 hour (D). Results are representative of 2 independent experiments. Data are mean±standard error of the mean (n=8). *P<0.05.
Figure 2
Figure 2
Experimental setup (A). Distribution of neural stem cells (NSC) in the postischemic brain at 3 days after intracarotid delivery (B). In the CCR2+/+ group, most NSC were found in the ischemic penumbra of the cortex and striatum (B). Significantly fewer cells were detected in the CCR2−/− group (C). Representative photomicrographs of NSC in the ipsilateral hemisphere (D). Data are mean±standard error of the mean (n=8 per group, 2 independent experiments). *P<0.05. Scale bar: 100 μm.
Figure 3
Figure 3
Bioluminescence imaging demonstrating the recruitment dynamics of CCR2+/+ and CCR2−/− neural stem cells (NSC) to the postischemic brain at 6 (A), 24 (B), and 48 hours (C) after intra-arterial delivery. Representative images of transplanted animals with wild-type (CCR2+/+) and knockout (CCR2−/−) NSC, photon flux measurements (photon flux), and calculated left-right (L/R) distribution ratios (left/right ratio). At all time points, luciferase activity was significantly higher in mice transplanted with CCR2+/+ NSC (A—C). In both groups, the differences in the L/R ratio decreased during the given time period. Pseudocolor scale bars represent luminescence photon output in photons/s/cm2/sr. Data are mean±standard error of the mean (n=3 per group). *P<0.05. n.s., not significant.
Figure 4
Figure 4
Number of recruited cells and differentiation of intra-arterially delivered neural stem cells (NSC) in the postischemic brain at 14 days after transplantation. Experimental set-up (A). Significantly higher numbers of cells were found in the ipsilateral hemisphere in the CCR2+/+ group (B). Colocalization studies with the neuronal (NeuN, neuronal class III b-tubulin (TuJ1)) and astrocytic (GFAP) markers did not show any significant differences in NSC differentiation in vivo between the CCR2+/+ and the CCR2−/− group (C). Representative confocal photomicrographs from the ipsilateral and contralateral hemispheres demonstrating colocalization of NeuN (red, upper pannel) and GFAP (red, lower panel) with green fluorescent protein (GFP)-positive (green) NSC (D). Data are mean±standard error of the mean (n=12 per group, 2 independent experiments). *P<0.05, **P<0.01. n.s. indicates not significant. Scale bars: 200 μm (split color panel), 20 μm (high-magnification orthogonal images).
Figure 5
Figure 5
Compared to baseline (B) performance, stroke resulted in a marked locomotor impairment in mice grafted with CCR2+/+ and CCR2−/− neural stem cells (NSC) in the horizontal ladder test at 1 day (1D) after transplantation. Mice transplanted with CCR2+/+ NSC showed a significantly better recovery than CCR2−/− grafted animals as soon as after 3 days (3D) after transplantation, which was sustained after 4 weeks (4W) (A). Time-course analysis of GFP-positive cells demonstrating a marked decrease in the number of surviving cells between 3 days and 2 weeks after transplantation in both the CCR2+/+ and the CCR2−/− group. There was no marked change between 2 weeks and 4 weeks, and there remained a statistically significant difference between animals transplanted with CCR2+/+ as compared to CCR2−/− NSC (B). Analysis of activated microglia (CD68) and Fluoro-Jade (FJ)–positive degenerating neurons revealed lower CD68-positive cell numbers (C) and lower FJ-positive cells (D) in animals transplanted with CCR2+/+ as compared to CCR2−/− NSC. Data are mean±standard error of the mean (n=6 per group). *P<0.05. Representative fluorescence images for CD68 (C) and FJ (D). Low-magnification image for FJ fluorescence showing the typical extent of stroke (D, inset). Scale bars (C, D): 100 μm.
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