Krystal Schaar

Functional assessments in the rodent stroke model

Stroke is a common cause of permanent disability accompanied by devastating impairments for which there is a pressing need for effective treatment. Motor, sensory and cognitive deficits are common following stroke, yet treatment is limited. Along with histological measures, functional outcome in animal models has provided valuable insight to the biological basis and potential rehabilitation efforts of experimental stroke. Developing and using tests that have the ability to identify behavioral deficits is essential to expanding the development of translational therapies. The present aim of this paper is to review many of the current behavioral tests that assess functional outcome after stoke in rodent models. While there is no perfect test, there are many assessments that are sensitive to detecting the array of impairments, from global to modality specific, 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.

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.