aopei Xi

IL-10 directly protects cortical neurons by activating PI-3 kinase and STAT-3 pathways

IL-10 reduces pro-inflammatory responses after ischemic stroke primarily by acting on glia and endothelium, but relatively little is known about the direct effects of IL-10 on cortical neurons, which are often damaged in stroke. We found by PCR and immunohistochemistry that cortical neurons express IL-10 receptor. Treatment of primary cortical neurons in culture with IL-10 increased neuronal survival after exposure to oxygen-glucose deprivation (OGD) or glutamate toxicity. IL-10 also induced phosphorylation of AKT in cortical neurons. Pretreatment with the specific PI-3K inhibitor, wortmannin, attenuated IL-10 mediated neuroprotection against OGD and glutamate. In addition, IL-10 induced STAT-3 phosphorylation. Pre-treatment with a functional blocking antibody to the IL-10 receptor reduced both Stat-3 and AKT phosphorylation and blocked IL-10 mediated protection of cortical neurons. These data suggest that IL-10 provides neuroprotection by acting via IL-10 receptor and PI3K/AKT and STAT-3 signal transduction pathways.

Therapeutic time window and dose response of autologous bone marrow mononuclear cells for ischemic stroke

Although mononuclear cells (MNCs) from bone marrow are being investigated in phase I clinical trials in stroke patients, dose response, therapeutic time window, and biodistribiton have not been well‐characterized in animal stroke models. Long Evans rats underwent common carotid artery/middle cerebral artery occlusion (CCA/MCAo) and 24 hr later were randomized to receive saline IV or a bone marrow aspiration followed by an IV infusion of autologous separated MNCs (1 million, 10 million, or 30 million cells/kg). In another experiment, rats underwent CCAo/MCAo and were randomized at 24 hr, 72 hr, or 7 days after stroke to receive a saline injection or 10 million/kg MNCs. All animals were evaluated on the cylinder and corner tests up to 28 days. MNCs were tracked using Q‐dot nanocrystals to monitor biodistribution. Animals treated with MNCs at 10 million and 30 million cells/kg at 24 hr after stroke had significant reductions in neurological deficits and lesion size compared with saline controls or animals treated with 1 million cells/kg. There was no difference in neurological deficits in the 10 and 30 million cell/kg groups at 28 days. Animals treated with MNCs at 72 hr but not at 7 days showed a significant reduction in neurological deficits by 28 days. Labeled MNCs were found in the brain, spleen, lung, liver, and kidney at 1 hr and exponentially decreased over the ensuing week. In conclusion, we found a maximum reduction in neurological deficits at 10 and 30 million cells/kg and a therapeutic time window up to 72 hr after stroke.

Intra-arterial Delivery Is Not Superior to Intravenous Delivery of Autologous Bone Marrow Mononuclear Cells in Acute Ischemic Stroke

Background and Purpose— Bone marrow–derived mononuclear cells (MNCs) are an investigational autologous cell-based therapy for acute ischemic stroke. Both intravenous (IV) and intra-arterial (IA) administration routes have been used in clinical trials. However, the route of administration to optimize the effect of MNCs is unknown. In this study, we compared the effect of IV versus IA route of administration of MNCs in the rat stroke model. Methods— Long Evans rats were subjected to transient middle cerebral artery occlusion. At 24 hours after stroke, animals were randomly assigned to receive autologous bone marrow–derived MNCs using either the IV or IA delivery route. IV saline served as control. One million cells/kg (low dose) and 30 million cells/kg (high dose) were assessed. Neurological testing, cavity size, serum cytokines, neuroregenerative end points, and MNC biodistribution were evaluated. Results— High-dose MNCs improved functional recovery, reduced lesion size and proinflammatory cytokines, and increased vessel density and neurogenesis markers compared with saline treatment (P<0.05). However, there were no significant differences between IV and IA MNC-treated groups, although IV MNCs reduced serum interleukin-1&bgr; levels compared with IA MNCs (P<0.05). IA MNCs at high dose led to a greater number of cells in the brain at 1 and 6 hours after injection but not in the lungs and spleen. Low-dose MNCs (by IV or IA) did not improve any functional or structural end point compared with saline. Conclusions— At low and high doses of MNCs, we found that IV or IA achieves similar structural and functional outcomes after stroke.

Multipotent Adult Progenitor Cells Enhance Recovery After Stroke by Modulating the Immune Response from the Spleen

Stem cell therapy modulates not only the local microenvironment of the brain but also the systemic immune responses. We explored the impact of human multipotent adult progenitor cells (MAPC) modulating splenic activation and peripheral immune responses after ischemic stroke. Hundred twenty‐six Long‐Evans adult male rats underwent middle cerebral artery occlusion. Twenty‐four hours later, they received IV MAPC or saline treatment. At 3 days after infusion, RNA was isolated from the injured cortex and spleen for microarray analysis. Spleen mass, splenocyte phenotype, and releasing cytokines were measured. Serum cytokines, MAPC biodistribution, brain lesion sizes and neurofunctional deficits were compared in rats treated with MAPC or saline with and without spleens. Stroked animals treated with MAPC exhibited genes that more closely resembled animals with sham surgery. Gene categories downregulated by MAPC included leukocyte activation, antigen presentation, and immune effector processing, associated with the signaling pathways regulated by TNF‐α, IL‐1β, IL‐6, and IFN‐γ within the brain. MAPC treatment restored spleen mass reduction caused by stroke, elevated Treg cells within the spleen, increased IL‐10 and decreased IL‐1β released by splenocytes. MAPC reduced IL‐6 and IL‐1β and upregulated IL‐10 serum levels. Compared with saline, MAPC enhance stroke recovery in rats with intact spleens but had no effects in rats without spleens. MAPC restores expression of multiple genes and pathways involved in immune and inflammatory responses after stroke. Immunomodulation of the splenic response by the intravenous administration of MAPC may create a more favorable environment for brain repair after stroke. Stem Cells 2017;35:1290–1302

Ischemic Stroke May Activate Bone Marrow Mononuclear Cells to Enhance Recovery After Stroke

Bone marrow-derived mononuclear cells (MNCs) enhance recovery in rodent stroke models. Since stroke activates the bone marrow, there may be biological differences of autologous MNCs derived poststroke compared with the prestroke setting. We analyzed MNCs harvested from the same Long Evans rats 1 day before and 1 day after ischemic stroke or sham stroke. Stroke was induced by suture occlusion of the middle cerebral artery for 90 min. MNCs were characterized by flow cytometry to identify differences in the percentages of different cell subpopulations. MNCs were also placed in culture and cytokines were measured in the media. In separate experiments, Long Evans rats received 24 h after stroke an intracarotid injection of saline or autologous MNCs, prepared from the same animal, either 1 day before or 1 day after stroke. The rats were then followed on the cylinder and corner tests for 28 days. In poststroke MNCs compared with prestroke MNCs, there was a significant reduction in T and mesenchymal stem cells and a significant increase in CD34+ and natural killer cells. Postsham MNCs showed an elevation in CD11b and CD45R cells compared with presham MNCs. The concentrations of IL-10, IL-6, MCP-1, vascular endothelial growth factor (VEGF), and tumor necrosis factor-α were significantly increased in poststroke MNCs compared with prestroke MNCs. Postsham MNCs showed a decrease in VEGF. Poststroke MNCs in comparison with prestroke MNCs led to a greater recovery on neurological testing and reduced lesion size. Autologous MNCs exert different biological responses when derived from the poststroke setting compared with normal animals.

Nitric oxide facilitates delivery and mediates improved outcome of autologous bone marrow mononuclear cells in a rodent stroke model.

Background Bone marrow mononuclear cells (MNC) represent an investigational treatment for stroke. The objective of this study was to determine the relevance of vasoactive mediators, generated in response to MNC injection, as factors regulating cerebral perfusion (CP), the biodistribution of MNC, and outcome in stroke. Methods Long Evans rats underwent transient middle cerebral artery occlusion. MNC were extracted from the bone marrow at 22 hrs and injected via the internal carotid artery or the femoral vein 2 hours later. CP was measured with MRI or continuous laser Doppler flowmetry. Serum samples were collected to measure vasoactive mediators. Animals were treated with the Nitric Oxide (NO) inhibitor, L-NAME, to establish the relevance of NO-signaling to the effect of MNC. Lesion size, MNC biodistribution, and neurological deficits were assessed. Results CP transiently increased in the peri-infarct region within 30 min after injecting MNC compared to saline or fibroblast control. This CP increase corresponded temporarily to serum NO elevation and was abolished by L-NAME. Pre-treatment with L-NAME reduced brain penetration of MNC and prevented MNC from reducing infarct lesion size and neurological deficits. Conclusions NO generation in response to MNC may represent a mechanism underlying how MNC enter the brain, reduce lesion size, and improve outcome in ischemic stroke.

Autologous Bone Marrow Mononuclear Cells Exert Broad Effects on Short- and Long-Term Biological and Functional Outcomes in Rodents with Intracerebral Hemorrhage

Autologous bone marrow-derived mononuclear cells (MNCs) are a potential therapy for ischemic stroke. However, the effect of MNCs in intracerebral hemorrhage (ICH) has not been fully studied. In this study, we investigated the effects of autologous MNCs in experimental ICH. ICH was induced by infusion of autologous blood into the left striatum in young and aged male Long Evans rats. Twenty-four hours after ICH, rats were randomized to receive an intravenous administration of autologous MNCs (1 × 10(7) cells/kg) or saline. We examined brain water content, various markers related to the integrity of the neurovascular unit and inflammation, neurological deficit, neuroregeneration, and brain atrophy. We found that MNC-treated young rats showed a reduction in the neurotrophil infiltration, the number of inducible nitric oxide synthase-positive cells, and the expression of inflammatory-related signalings such as the high-mobility group protein box-1, S100 calcium binding protein B, matrix metalloproteinase-9, and aquaporin 4. Ultimately, MNCs reduced brain edema in the perihematomal area compared with saline-treated animals at 3 days after ICH. Moreover, MNCs increased vessel density and migration of doublecortin-positive cells, improved motor functional recovery, spatial learning, and memory impairment, and reduced brain atrophy compared with saline-treated animals at 28 days after ICH. We also found that MNCs reduced brain edema and brain atrophy and improved spatial learning and memory in aged rats after ICH. We conclude that autologous MNCs can be safely harvested and intravenously reinfused in rodent ICH and may improve long-term structural and functional recovery after ICH. The results of this study may be applicable when considering future clinical trials testing MNCs for ICH.

Cryopreservation of Bone Marrow Mononuclear Cells Alters Their Viability and Subpopulation Composition but Not Their Treatment Effects in a Rodent Stroke Model

The systemic administration of autologous bone marrow (BM) derived mononuclear cells (MNCs) is under investigation as a novel therapeutic modality for the treatment of ischemic stroke. Autologous applications raise the possibility that MNCs could potentially be stored as a banked source. There have been no studies that investigate the effects of cryopreservation of BM-MNCs on their functional abilities in stroke models. In the present study, C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAo) for 60 minutes and then divided into two treatment groups: fresh MNCs versus cryopreserved MNCs. BM-MNCs were collected at 22 hours after MCAo and were stored in liquid nitrogen for 12 months in cryopreserved MNCs group. BM-MNCs cellular viability, composition, and phenotype of the various subpopulations of mice BM-MNCs were evaluated by flow cytometry, and the behavioral recovery of stroke animals was tested with freshly harvested MNCs versus cryopreserved MNCs by corner test and ladder rung test. We found that long-term cryopreservation negatively impacts the cellular viability of bone marrow MNCs. Cryopreservation also alters the cellular composition of various subpopulations within the MNCs. However, despite the changes observed in cryopreserved cells, both fresh and frozen MNCs have similar beneficial effect on behavioral and histological outcomes.