Background and Purpose-We tested the hypothesis that intravenous infusion of bone marrow derived-marrow stromal cells (MSCs) enter the brain and reduce neurological functional deficits after stroke in rats. Methods-Rats (nϭ32) were subjected to 2 hours of middle cerebral artery occlusion (MCAO). Test groups consisted of MCAO alone (group 1, nϭ6); intravenous infusion of 1ϫ10 6 MSCs at 24 hours after MCAO (group 2, nϭ6); or infusion of 3ϫ106 MSCs (group 3, nϭ7). Rats in groups 1 to 3 were euthanized at 14 days after MCAO. Group 4 consisted of MCAO alone (nϭ6) and group 5, intravenous infusion of 3ϫ10 6 MSCs at 7 days after MCAO (nϭ7). Rats in groups 4 and 5 were euthanized at 35 days after MCAO. For cellular identification, MSCs were prelabeled with bromodeoxyuridine. Behavioral tests (rotarod, adhesive-removal, and modified Neurological Severity Score [NSS]) were performed before and at 1, 7, 14, 21, 28, and 35 days after MCAO. Immunohistochemistry was used to identify MSCs or cells derived from MSCs in brain and other organs. Results-Significant recovery of somatosensory behavior and Neurological Severity Score (PϽ0.05) were found in animals infused with 3ϫ10 6 MSCs at 1 day or 7 days compared with control animals. MSCs survive and are localized to the ipsilateral ischemic hemisphere, and a few cells express protein marker phenotypic neural cells. Conclusions-MSCs delivered to ischemic brain tissue through an intravenous route provide therapeutic benefit after stroke. MSCs may provide a powerful autoplastic therapy for stroke.

Background and Purpose-Human umbilical cord blood cells (HUCBC) are rich in stem and progenitor cells. In this study we tested whether intravenously infused HUCBC enter brain, survive, differentiate, and improve neurological functional recovery after stroke in rats. In addition, we tested whether ischemic brain tissue extract selectively induces chemotaxis of HUCBC in vitro. Methods-Adult male Wistar rats were subjected to transient (2-hour) middle cerebral artery occlusion (MCAO).Experimental groups were as follows: group 1, MCAO alone (nϭ5); group 2, 3ϫ10 6 HUCBC injected into tail vein at 24 hours after MCAO (nϭ6) (animals of groups 1 and 2 were killed at 14 days after MCAO); group 3, MCAO alone (nϭ5); group 4, MCAO injected with PBS at 1 day after stroke (nϭ8); and group 5, 3ϫ10 6 HUCBC injected into tail vein at 7 days after MCAO (nϭ5). Rats of groups 3, 4, and 5 were killed at 35 days after MCAO.

Neurologic benefit resulting from hMSC treatment of stroke in rats may derive from the increase of growth factors in the ischemic tissue, the reduction of apoptosis in the penumbral zone of the lesion, and the proliferation of endogenous cells in the subventricular zone.

Abstract-We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1ϫ10 6 ) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (PϽ0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (nϭ12) treated with hMSCs compared with numbers in rats (nϭ12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (PϽ0.05) raised endogenous rat VEGF levels in the IBZ from 10.5Ϯ1.7 ng/mL in the control group to 17.5Ϯ1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (PϽ0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (PϽ0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.

The present study investigates the induction of neurogenesis, reduction of apoptosis, and promotion of basic fibroblast growth factor (bFGF) expression as possible mechanisms by which treatment of stroke with bone marrow stromal cells (MSCs) improves neurological functional recovery. Additionally, for the first time, we treated cerebral ischemia in female rats with intraveneous administration of MSCs. Female rats were subjected to 2 hr of middle cerebral artery occlusion (MCAo), followed by an injection of 3 x 10(6) male (for Y chromosome labeling) rat MSCs or phosphate-buffered saline (PBS) into the tail vein 24 hr after MCAo. All animals received daily injection of bromodeoxyuridine (BrdU; 50 mg/kg, i.p.) for 13 days after treatment for identification of newly synthesized DNA. Animals were sacrificed at 14 days after MCAo. Behavioral tests (rotarod and adhesive-removal tests) were performed. In situ hybridization, immunohistochemistry, and terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) were performed to identify transplanted MSCs (Y chromosome), BrdU, bFGF, and apoptotic cells in the brain. Significant recovery of behavior was found in MSC-treated rats at 7 days in the somatosensory test and at 14 days in the motor test after MCAo compared with control, PBS-treated animals (P<.05). MSCs were found to survive and preferentially localize to the ipsilateral ischemic hemisphere. Significantly more BrdU-positive cells were located in the subventricular zone (P<.05), and significantly fewer apoptotic cells and more bFGF immunoreactive cell were found in the ischemic boundary area (P<.05) of MSC-treated rats than in PBS-treated animals. Here we demonstrate that intravenously administered male MSCs increase bFGF expression, reduce apoptosis, promote endogenous cellular proliferation, and improve functional recovery after stroke in female rats.