Efficacy and dose-dependent safety of intra-arterial delivery of mesenchymal stem cells in a rodent stroke model

Abstract

Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) for acute ischemic stroke is attractive for clinical translation. However, studies using rat model of stroke have demonstrated that IA MSCs delivery can decrease middle cerebral artery (MCA) flow, which may limit its clinical translation. The goal of this study is to identify a dose of IA MSCs (maximum tolerated dose; MTD) that does not compromise MCA flow and evaluate its efficacy and optimal timing in a rat model of reversible middle cerebral artery occlusion (rMCAo). We sought to determine if there is a difference in efficacy of acute (1 h) versus sub-acute (24 h) IA MSCs treatment after rMCAo. Adult female Sprague-Dawley rats underwent rMCAo (90 min) and an hour later a single dose of MSCs (at de-escalating doses 1 × 10(6), 5 × 10(5), 2 × 10(5), 1 × 10(5) and 5 × 10(4)) was given using IA route. MSCs were suspended in phosphate buffered saline (PBS) and PBS alone was used for control experiments. We measured the percent change in mean laser Doppler flow signal over the ipsilateral MCA in de-escalating doses groups to determine MTD. The results demonstrated that the lowering of IA MSC dose to 1 × 10(5) and below did not compromise MCA flow and hence an IA MSC dose of 1 × 10(5) considered as MTD. Subsequently, 1 h and 24 h after rMCAo, rats were treated with IA MSCs or PBS. The 24 h delivery of IA MSCs significantly improved neurodeficit score and reduced the mean infarct volume at one month as compared to control, but not the 1 h delivery. Overall, this study suggests that the IA delivery of MSCs can be performed safely and efficaciously at the MTD of 1 × 10(5) delivered at 24 hours in rodent model of stroke.

Figure 1. Lower doses of IA MSCs mitigate adverse effect IA injection on MCA blood flow.

(A) Experimental timeline showing rMCAo for 90 minutes followed by withdrawal of the suture to allow reperfusion. At 60 minutes of reperfusion, IA MSC or vehicle only injection was given, followed by LDF monitoring for 60 min. (B) Comparison of relative LDFS worsening from baseline to final recording, among de-escalating dose groups (C) The 1×10⁵ dose and placebo has significantly less maximum LDFS worsening as compared to the 1×10⁶ dose. The comparisons in B and C were done using general linear modeling (GLM) to compare mean differences among groups. LDFS =  Laser Doppler Flow Signal.

Figure 2. Comparison of LDFS changes over time and microvascular occlusion between dose-groups.

(A) IA MSC dose of 1×10⁵ and IA placebo have a transient and less MCA LDF worsening during 60 minutes post injection as compared to 5×10⁵ dose using mixed model. LDFS =  Laser Doppler Flow Signal. (B) On comparing the total number of microvessels with complete occlusion among the two dose groups, there were a significantly lower number of complete occlusions in the lower dose group. Mean ±SD, * P<0.05, ANOVA. (C) Representative brain sections from IA MSC 1×10⁵ dose group showing GFP+ MSCs identified by 3, 3″-diaminobenzidine (DAB), showing localized complete filling of microvessels at 3–5 days post-injection, as well as MSCs just outside the vessel wall in brain parenchyma and (D) high power field of representative brain sections from IA 5×10⁵ dose group showing single MSC partly inside and partly outside microvessel wall.

Figure 3. Functional neurologic outcomes are superior in the IA MSC_24 h group.

(A) Experimental timeline of the efficacy study showing 90 min rMCAo followed by 24 hour reperfusion except in group receiving IA MSCs at 1 hour reperfusion followed by ND score assessment at 1,7,14,21 & 28 days. (B) On day 1 post rMCAo, the ND scores were not significantly different among groups. The ND score of the 1×10⁵ group progressively decreased over time and at 28 days was significantly lower than the other groups. C, The day 28 ND score was significantly lower in the IA MSC_24 h group as compared to all the remaining groups. ND =  Neurodeficit

Figure 4. Comparison of infarct volume among treatment groups.

(A) Geometric mean infarct volumes are compared among groups after log transformation to achieve normal distribution. Only the IA MSC_24 h group shows significantly reduced infarct volume as compared to the IA PBS_24 h. B, Frequency infarct map statistically comparing the location of the mean infarct volume in the IA MSC_24 h and IA PBS_24 h groups using color- coded representation of the percent of rats showing infarction in each brain region using “Fisher's exact test” with a color-coded representation of the “p-value”, the color bar in “1-p” format. Displays are in “Coronal presentation”; middle sections are selected. The IA MSC_24 h group shows a much reduced infarction frequency, particularly surrounding the core as quantized by the Fisher test.