Michael Chopp

Therapeutic Benefit of Intravenous Administration of Bone Marrow Stromal Cells After Cerebral Ischemia in Rats

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 (n=32) were subjected to 2 hours of middle cerebral artery occlusion (MCAO). Test groups consisted of MCAO alone (group 1, n=6); intravenous infusion of 1×106 MSCs at 24 hours after MCAO (group 2, n=6); or infusion of 3×106 MSCs (group 3, n=7). Rats in groups 1 to 3 were euthanized at 14 days after MCAO. Group 4 consisted of MCAO alone (n=6) and group 5, intravenous infusion of 3×106 MSCs at 7 days after MCAO (n=7). 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 (P <0.05) were found in animals infused with 3×106 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.

VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain

VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF(165) (rhVEGF(165)) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF(165) to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF(165) to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF(165) to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

Human marrow stromal cell therapy for stroke in rat: Neurotrophins and functional recovery

ObjectiveTo test the effect of IV-injected human bone marrow stromal cells (hMSC) on neurologic functional deficits after stroke in rats. Methods Rats were subjected to transient middle cerebral artery occlusion and IV injected with 3 × 106 hMSC 1 day after stroke. Functional outcome was measured before and 1, 7, and 14 days after stroke. Mixed lymphocyte reaction and the development of cytotoxic T lymphocytes measured the immune rejection of hMSC. A monoclonal antibody specific to human cellular nuclei (mAb1281) was used to identify hMSC and to measure neural phenotype. ELISA analyzed neurotrophin levels in cerebral tissue from hMSC-treated or nontreated rats. Bromodeoxyuridine injections were used to identify newly formed cells. Results Significant recovery of function was found in rats treated with hMSC at 14 days compared with control rats with ischemia. Few (1 to 5%) hMSC expressed proteins phenotypic of brain parenchymal cells. Brain-derived neurotrophic factor and nerve growth factor significantly increased, and apoptotic cells significantly decreased in the ischemic boundary zone; significantly more bromodeoxyuridine-reactive cells were detected in the subventricular zone of the ischemic hemisphere of rats treated with hMSC. hMSC induced proliferation of lymphocytes without the induction of cytotoxic T lymphocytes. ConclusionNeurologic 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.

Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats.

Background and Purpose— Erythropoietin (EPO) promotes proliferation and differentiation of erythroid progenitors and the survival of maturing erythroid cells. Here, we investigated the role of EPO in brain repair after stroke. Methods— Rats were treated with recombinant human EPO (rhEPO) at 24 hours after the onset of embolic stroke. An array of behavior tests was performed. Rats were euthanized 28 days after stroke for measurements of infarct volume, angiogenesis, and neurogenesis. In vitro, neurospheres derived from the subventricular zone (SVZ) of the rat and cerebral endothelial cells derived from the mouse were treated with rhEPO. Capillary-like tube formation and neuronal differentiation were measured. Results— Treatment with rhEPO significantly improved functional recovery, along with increases in density of cerebral microvessels at the stroke boundary and numbers of BrdU, doublecortin, and nestin immunoreactive cells in the SVZ. rhEPO treatment significantly increased brain levels of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). In vitro, rhEPO enhanced capillary tube formation of cerebral endothelial cells, which was inhibited by a specific VEGF receptor 2 antagonist (SU1498). Incubation of neurospheres derived from stroke SVZ with anti-EPO neutralizing antibody inhibited neurogenesis, whereas incubation of stroke-derived neurospheres with rhEPO enhanced neurogenesis. Conclusion— Our data suggest that EPO-increased VEGF and BDNF may be involved in angiogenesis and neurogenesis, which could contribute to functional recovery.

Nitric Oxide Measured by a Porphyrinic Micro Sensor in Rat Brain after Transient Middle Cerebral Artery Occlusion

We measured, in vivo, the local concentration of nitric oxide (NO) in cerebral tissue, during and after transient middle cerebral artery occlusion in the rat (n = 8). Baseline concentration of NO was <10−8 M; upon initiation of ischemia, NO concentration increased to ∼10−6 M and then declined. Reperfusion likewise stimulated an increase in NO concentration to above baseline level. Administration of N-nitro-l-arginine methyl ester (n = 4), an inhibitor of nitric oxide synthase, before onset of ischemia, maintained NO at basal levels. Our data indicate that large increases in NO occur at onset of ischemia, which may affect tissue response to an ischemic insult.

Intravenous Administration of Human Bone Marrow Stromal Cells Induces Angiogenesis in the Ischemic Boundary Zone After Stroke in Rats

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 (1×106) 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 (P <0.05) 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 (n=12) treated with hMSCs compared with numbers in rats (n=12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (P <0.05) raised endogenous rat VEGF levels in the IBZ from 10.5±1.7 ng/mL in the control group to 17.5±1.6 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 (P <0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (P <0.01) 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.

TREATMENT OF NEURAL INJURY WITH MARROW STROMAL CELLS

We describe our preclinical studies on the use of bone-marrow stromal cells (MSC; an uncharacterised mixed population of plastic-adherent cells) in the treatment of neural injury. These cells obtained from donor rats or human beings have been directly transplanted into brain or administered intra-arterially or intravenously. MSC selectively target injured tissue and promote functional recovery. Signals that target inflammatory cells to injured tissue probably direct MSC to injury sites. Although some MSC express proteins typical of neural cells, the possibility that benefit is derived by replacement of infarcted tissue with differentiated MSC is highly unlikely. MSC activate endogenous restorative responses in injured brain, which include angiogenesis, neurogenesis, and synaptogenesis. Given the robust therapeutic benefit of these cells in the treatment of experimental neural injury, and the fact that MSC have been used in the treatment of other human disease, there is justification for further preclinical studies leading to clinical trials for the treatment of neural injury such as stroke.

Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia

Progenitor cells in the subventricular zone of the lateral ventricle and in the dentate gyrus of the hippocampus can proliferate throughout the life of the animal. To examine the proliferation and fate of progenitor cells in the subventricular zone and dentate gyrus after focal cerebral ischemia, we measured the temporal and spatial profiles of proliferation of cells and the phenotypic fate of proliferating cells in ischemic brain in a model of embolic middle cerebral artery occlusion in the adult rat. Proliferating cells were labeled by injection of bromodeoxyuridine (BrdU) in a pulse or a cumulative protocol. To determine the temporal profile of proliferating cells, ischemic rats were injected with BrdU every 4 h for 12 h on the day preceding death. Rats were killed 2-14 days after ischemia. We observed significant increases in numbers of proliferating cells in the ipsilateral cortex and subventricular zone 2-14 days with a peak at 7 days after ischemia compared with the control group. To maximize labeling of proliferating cells, a single daily injection of BrdU was administered over a 14-day period starting the day after ischemia. Rats were killed either 2 h or 28 days after the last injection of BrdU. A significant increase in numbers of BrdU immunoreactive cells in the subventricular zone was coincident with a significant increase in numbers of BrdU immunoreactive cells in the olfactory bulb 14 days after ischemia and numbers of BrdU immunoreactive cells did not significantly increase in the dentate gyrus. However, 28 days after the last labeling, the number of BrdU labeled cells decreased by 90% compared with number at 14 days. Clusters of BrdU labeled cells were present in the cortex distal to the infarction. Numerous cells immunostained for the polysialylated form of the neuronal cell adhesion molecule were detected in the ipsilateral subventricular zone. Only 6% of BrdU labeled cells exhibited glial fibrillary acidic protein immunoreactivity in the cortex and subcortex and no BrdU labeled cells expressed neuronal protein markers (neural nuclear protein and microtubule associated protein-2). From these data we suggest that focal cerebral ischemia induces transient and regional specific increases in cell proliferation in the ipsilateral hemisphere and that proliferating progenitor cells may exist in the adult cortex.

Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation.

We tested the hypothesis that transplantation of bone marrow stromal cells (MSCs) into the spinal cord after a contusion injury promotes functional outcome. Rats (n = 31) were subjected to a weight driven implant injury. MSCs or phosphate buffered saline was injected into the spinal cord I week after injury. Sections of tissue were analyzed by double-labeled immunohistochemistry for MSC identification. Functional outcome measurements using the Basso-Beattie-Bresnehan score were performed weekly to 5 weeks post-injury. The data indicate significant improvement in functional outcome in animals treated with MSC transplantation compared to control animals. Scattered cells derived from MSCs expressed neural protein markers. These data suggest that transplantation of MSCs may have a therapeutic role after spinal cord injury.

Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat

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 × 106 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.