Anesthesia-Induced Hypothermia Attenuates Early-Phase Blood-Brain Barrier Disruption but Not Infarct Volume following Cerebral Ischemia.

Blood-brain barrier (BBB) disruption is thought to facilitate the development of cerebral infarction after a stroke. In a typical stroke model (such as the one used in this study), the early phase of BBB disruption reaches a peak 6 h post-ischemia and largely recovers after 8-24 h, whereas the late phase of BBB disruption begins 48-58 h post-ischemia. Because cerebral infarct develops within 24 h after the onset of ischemia, and several therapeutic agents have been shown to reduce the infarct volume when administered at 6 h post-ischemia, we hypothesized that attenuating BBB disruption at its peak (6 h post-ischemia) can also decrease the infarct volume measured at 24 h. We used a mouse stroke model obtained by combining 120 min of distal middle cerebral arterial occlusion (dMCAo) with ipsilateral common carotid arterial occlusion (CCAo). This model produced the most reliable BBB disruption and cerebral infarction compared to other models characterized by a shorter duration of ischemia or obtained with dMCAO or CCAo alone. The BBB permeability was measured by quantifying Evans blue dye (EBD) extravasation, as this tracer has been shown to be more sensitive for the detection of early-phase BBB disruption compared to other intravascular tracers that are more appropriate for detecting late-phase BBB disruption. We showed that a 1 h-long treatment with isoflurane-anesthesia induced marked hypothermia and attenuated the peak of BBB disruption when administered 6 h after the onset of dMCAo/CCAo-induced ischemia. We also demonstrated that the inhibitory effect of isoflurane was hypothermia-dependent because the same treatment had no effect on ischemic BBB disruption when the mouse body temperature was maintained at 37°C. Importantly, inhibiting the peak of BBB disruption by hypothermia had no effect on the volume of brain infarct 24 h post-ischemia. In conclusion, inhibiting the peak of BBB disruption is not an effective neuroprotective strategy, especially in comparison to the inhibitors of the neuronal death signaling cascade; these, in fact, can attenuate the infarct volume measured at 24 h post-ischemia when administered at 6 h in our same stroke model.

Mouse-induced pluripotent stem cells generated under hypoxic conditions in the absence of viral infection and oncogenic factors and used for ischemic stroke therapy.

Induced pluripotent stem (iPS) cells are considered as having the greatest potential for use in cell-based therapies. However, at least two hurdles remain: integrating viral transgenes and introducing the c-Myc and Klf4 oncogenes. In a previous study, fibroblasts were incapable of generating iPS cells in the absence of both oncogenes and viral infection. For the present study, we tested our hypothesis that iPS cells can be generated without oncogenes and viral infection under hypoxic conditions and used for cell therapies. By avoiding oncogenic factors and virus integration, this strategy would decrease the potential for cancer formation. According to our observations, the repeated transfection of two expression plasmids (Oct4 and Sox2) into mouse embryonic fibroblasts (MEFs) and combined hypoxic condition resulted in the generation of a novel iPS cell. At 6 h post-transfection, MEFs were subjected to hypoxic conditions (3% O2) for 24 h; this procedure was repeated four times. The resulting MEFs were seeded on feeder cells on day 9; iPS cell clones were observed 12 days post-seeding and designated as iPS-OSH. Data for cell morphology, stem cell marker staining, gene expression profiles, and embryonic body, teratoma, and chimeric mouse formation indicated iPS-OSH pluripotent capability. Neural precursor cells differentiated from iPS-OSH cells were used to treat an ischemic stroke mouse model; results from a behavior analysis indicate that the therapeutic group surpassed the control group. Further, iPS-OSH-derived neural precursor cells differentiated into neurons and astrocytes in mouse stroke brains. In conclusion, we generated a novel iPS-OSH in the absence of viral infection and oncogenic factors and could use it for ischemic stroke therapy.