Background-Blood-brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models. Methods and Results-Primary astrocytes, ECs, or cocultures were prepared with or without added microglia. Primary ECs were more resistant to oxygen-glucose deprivation/reperfusion than astrocytes. ECs plus astrocytes showed intermediate vulnerability. Microglia added to cocultures nearly doubled cell death. This increase was prevented by minocycline and apocynin. In vivo, minocycline reduced infarct volume and neurological deficits and markedly reduced BBB disruption and hemorrhage in mice after experimental stroke. Conclusions-Inhibition of microglial activation may protect the brain after ischemic stroke by improving BBB viability and integrity. Microglial inhibitors may prove to be an important treatment adjunct to fibrinolysis.

SUMMARY Chaperones, especially the stress inducible Hsp70, have been studied for their potential to protect the brain from ischemic injury. While they protect from both global and focal ischemia in vivo and cell culture models of ischemia/reperfusion injury in vitro, the mechanism of protection is not well understood. Protein aggregation is part of the etiology of chronic neurodegenerative diseases such as Huntington's and Alzheimer's, and recent data demonstrate protein aggregates in animal models of stroke. We now demonstrate that overexpression of Hsp70 in hippocampal CA1 neurons reduces evidence of protein aggregation under conditions where neuronal survival is increased. We have also demonstrated protection by the cochaperone Hdj-2 in vitro and demonstrated that this is associated with reduced protein aggregation identified by ubiquitin immunostaining. Hdj-2 can prevent protein aggregate formation by itself, but can only facilitate protein folding in conjunction with Hsp70. Pharmacological induction of Hsp70 was found to reduce both apoptotic and necrotic astrocyte death induced by glucose deprivation or oxygen glucose deprivation. Protection from ischemia and ischemia-like injury by chaperones thus involves at least anti-apoptotic,anti-necrotic and anti-protein aggregation mechanisms.

Mild hypothermia is neuroprotective, but the reasons are not well known. Inflammation contributes to ischemic damage; therefore, we examined whether the protection by hypothermia may be attributable to alterations in the inflammation. We examined whether hypothermia might alter the inflammatory cell-associated inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) and peroxynitrite generation in experimental stroke and inflammation. Rats underwent 2 hr of middle cerebral artery occlusion (MCAO). Brain inflammation was modeled by intravenous lipopolysaccharide (LPS) (2 mg/kg) injection. Temperature was maintained at 33 degrees C for 2 hr immediately after MCAO and LPS injection, delayed 2 hr after MCAO or maintained at 38 degrees C. Cultured microglia were activated with LPS and then incubated at 33 or 37 degrees C. Both intraischemic and delayed mild hypothermia attenuated infarct size by 40% (p < 0.05). Immunohistochemistry was performed to identify cell type, iNOS, and peroxynitrite. The majority of iNOS- and peroxynitrite-positive cells were activated microglia-macrophages, and mild hypothermia significantly decreased the numbers of immunoreactive cells at 72 hr by >50% (p < 0.05). After ischemia, mild hypothermia decreased NO production by 40%. Similarly, hypothermia attenuated NO and iNOS in LPS-injected rats, as well as in cultured microglia. Aminoguanidine, an iNOS inhibitor, also attenuated infarct size and NO in ischemic and inflammation models. We conclude that mild hypothermia significantly inhibits the inflammatory response by affecting microglial iNOS-NO generation. Therapies directed against microglia or their activation may be useful in treating stroke.