The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity

Abstract

Introduction: Intra-arterial cell infusion is an efficient delivery route with which to target organs such as the ischemic brain. However, adverse events including microembolisms and decreased cerebral blood flow were recently reported after intra-arterial cell delivery in rodent models, raising safety concerns. We tested the hypothesis that cell dose, infusion volume, and velocity would be related to the severity of complications after intra-arterial cell delivery.

Methods: In this study, 38 rats were subjected to a sham middle cerebral artery occlusion (sham-MCAO) procedure before being infused with allogeneic bone-marrow mesenchymal stem cells at different cell doses (0 to 1.0 × 10(6)), infusion volumes (0.5 to 1.0 ml), and infusion times (3 to 6 minutes). An additional group (n = 4) was infused with 1.0 × 10(6) cells labeled with iron oxide for in vivo tracking of cells. Cells were infused through the external carotid artery under laser Doppler flowmetry monitoring 48 hours after sham-MCAO. Magnetic resonance imaging (MRI) was performed 24 hours after cell infusion to reveal cerebral embolisms or hemorrhage. Limb placing, cylinder, and open field tests were conducted to assess sensorimotor functions before the rats were perfused for histology.

Results: A cell dose-related reduction in cerebral blood flow was noted, as well as an increase in embolic events and concomitant lesion size, and sensorimotor impairment. In addition, a low infusion velocity (0.5 ml/6 minutes) was associated with high rate of complications. Lesions on MRI were confirmed with histology and corresponded to necrotic cell loss and blood-brain barrier leakage.

Conclusions: Particularly cell dose but also infusion velocity contribute to complications encountered after intra-arterial cell transplantation. This should be considered before planning efficacy studies in rats and, potentially, in patients with stroke.

Figure 1

Study design. Cells were transplanted 48 hours after the sham-operation, and laser Doppler flowmetry (LDF) was used to monitor the cerebral blood flow during the infusion. MRI was performed on postoperative day 3, followed by the limb-placing test, the cylinder test, and the open field test. At the end, rats were perfused for histology.

Figure 2

Changes of cerebral blood flow (CBF) monitored by laser Doppler flowmetry (LDF). (A) Changes in CBF in rats infused with different doses of cells. CBF is reduced as the cell dose is increased. *P <0.05 compared to PBS group. (B) Changes in CBF in rats infused with different volumes and in different time spans. ^# P <0.05 compared with the 0.5 ml/3 minute group.

Figure 3

MRI images. (A) Typical microembolisms in rats receiving different cell doses. (B) Typical micro-occlusions in groups with different infusion volumes delivered over different time spans. Scale bar, 2 mm.

Figure 4

Open field test in rats infused with different cell doses. Velocity (A), total distance moved (B), frequency in center zone (C), and moving duration (D) 24 hour after cell infusion were chosen to assess general motor function. Although no significant difference was found between the groups, the motor function after cell transplantation seemed to decrease as the cell dose increased, which was confirmed with Spearman correlation analyses.

Figure 5

Histology after intra-arterial cell infusion in rats. (A) T₂-weighted coronal slices from a rat infused with 0.5 × 10⁶ cells in 0.5 ml within 3 minutes. The white boxed region reveals a typical microembolism. Nissl staining (B), silver staining (C), and IgG staining (D) show the pathologic changes in the boxed region. Scale bar in (A) is 2 mm, and for (B)-(D) is 250 μm.

Figure 6

Tracking of infused cells. (A) T₂-weighted image of a rat infused with 1.0 × 10⁶ iron-labeled cells in 0.5 ml within 3 minutes. (B) T_2* image at the same level. Cell infusion evokes hypointense spots on T_2* sequence primarily located in the ipsilateral hemisphere within and around the lesions seen in T₂. Prussian blue staining reveals cells inside the microvessel and attached to the vascular wall (C). It is also evidence that cells had migrated across the vascular wall and disaggregated (D). Scale bar for (A, B) is 2 mm, and for (C, D) is 20 μm.