The persistence of neurogenesis in the forebrain subventricular zone (SVZ) of adult mammals suggests that the mature brain maintains the potential for neuronal replacement after injury. We examined whether focal ischemic injury in adult rat would increase SVZ neurogenesis and direct migration and neuronal differentiation of endogenous precursors in damaged regions. Focal stroke was induced in adult rats by 90-minute right middle cerebral artery occlusion (tMCAO). Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunostaining for cell type-specific markers. Brains examined 10-21 days after stroke showed markedly increased SVZ neurogenesis and chains of neuroblasts extending from the SVZ to the peri-infarct striatum. Many BrdU-labeled cells persisted in the striatum and cortex adjacent to infarcts, but at 35 days after tMCAO only BrdU-labeled cells in the neostriatum expressed neuronal markers. Newly generated cells in the injured neostriatum expressed markers of medium spiny neurons, which characterize most neostriatal neurons lost after tMCAO. These findings indicate that focal ischemic injury increases SVZ neurogenesis and directs neuroblast migration to sites of damage. Moreover, neuroblasts in the injured neostriatum appear to differentiate into a region-appropriate phenotype, which suggests that the mature brain is capable of replacing some neurons lost after ischemic injury.

Inflammation is increasingly recognized as being a critical contributor to both normal development and injury outcome in the immature brain. The focus of this Review is to highlight important differences in innate and adaptive immunity in immature versus adult brain, which support the notion that the consequences of inflammation will be entirely different depending on context and stage of CNS development. Perinatal brain injury can result from neonatal encephalopathy and perinatal arterial ischaemic stroke, usually at term, but also in preterm infants. Inflammation occurs before, during and after brain injury at term, and modulates vulnerability to and development of brain injury. Preterm birth, on the other hand, is often a result of exposure to inflammation at a very early developmental phase, which affects the brain not only during fetal life, but also over a protracted period of postnatal life in a neonatal intensive care setting, influencing critical phases of myelination and cortical plasticity. Neuroinflammation during the perinatal period can increase the risk of neurological and neuropsychiatric disease throughout childhood and adulthood, and is, therefore, of concern to the broader group of physicians who care for these individuals.

Macrophages are viewed as amplifiers of ischemic brain injury, but the origin of injury-producing macrophages is poorly defined. The role of resident brain macrophages—microglial cells—in stroke remains controversial. To determine if microglial cells exert injurious effects after neonatal focal stroke, we selectively depleted these cells with intracerebral injection of liposome-encapsulated clodronate before transient middle cerebral artery occlusion in postnatal day seven rats. Phagocytosis of apoptotic neurons by activated microglia was poor in animals with unmanipulated microglia, and depletion of these cells did not increase the number of apoptotic neurons. Lack of microglia increased the brain levels of several cytokines and chemokines already elevated by ischemia–reperfusion, and also increased the severity and volume of injury, suggesting that microglial cells contribute to endogenous protection during the subacute injury phase. Then, to determine if accumulation of reactive oxygen species in microglia adversely affects phagocytosis of dying neurons and contributes to injury, we delivered reduced glutathione (GSH) into microglia, again using liposomes. Remarkably, pharmacologically increased intracellular GSH concentrations in microglia induced superoxide accumulation in lipid rafts in these cells, further increased the brain levels of macrophage chemoattractants, and exacerbated injury. Taken together, these data show that microglia are part of the endogenous defense mechanisms and that, while antioxidants can protect the injured neonatal brain, high levels of reducing equivalents in activated microglia, GSH, trigger superoxide production, favor the reorganization of lipids, amplify local inflammation and exacerbate injury.

The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood-brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked if the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2–24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70-kDa dextran) and small (3-kDa dextran, Gd-DTPA) tracers remained largely undisturbed 24h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1,266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and MMP-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin and ZO-1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of CINC-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke.

Background and Purpose Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulating repair processes. We investigated whether MSC-treatment improves recovery after neonatal stroke and whether MSC overexpressing brain-derived neurotrophic factor (MSC-BDNF) further enhances recovery. Methods We performed 1.5-hour transient middle cerebral artery occlusion (MCAO) in 10-day-old rats. Three days after reperfusion, pups with evidence of injury by diffusion-weighted MRI were treated intranasally with MSC, MSC-BDNF or vehicle. To determine the effect of MSC-treatment, brain damage, sensorimotor function and cerebral cell proliferation were analysed. Results Intranasal delivery of MSC- and MSC-BDNF significantly reduced infarct size and grey matter loss in comparison to vehicle-treated rats without any significant difference between MSC- and MSC-BDNF-treatment. Treatment with MSC-BDNF significantly reduced white matter loss with no significant difference between MSC- and MSC-BDNF-treatment. Motor deficits were also improved by MSC-treatment when compared to vehicle treated rats. MSC-BDNF-treatment resulted in an additional significant improvement of motor deficits 14 days post-MCAO, but there was no significant difference between MSC or MSC-BDNF 28 days post-MCAO. Furthermore, treatment with either MSC or MSC-BDNF induced long lasting cell proliferation in the ischemic hemisphere. Conclusions Intranasal administration of MSC after neonatal stroke is a promising therapy for treatment of neonatal stroke. In this experimental paradigm MSC and BNDF-hypersecreting-MSC are equally effective in reducing ischemic brain damage.

Macrophages can be both beneficial and detrimental after CNS injury. We previously showed rapid accumulation of macrophages in injured immature brain acutely after ischemia-reperfusion. To determine whether these macrophages are microglia or invading monocytes, we subjected post-natal day 7 (P7) rats to transient 3 h middle cerebral artery (MCA) occlusion and used flow cytometry at 24 and 48 h postreperfusion to distinguish invading monocytes (CD45high/ CD11b+) from microglia (CD45low/medium/CD11b+). Inflammatory cytokines and chemokines were determined in plasma, injured and contralateral tissue 1-24 h post-reperfusion using ELISA-based cytokine multiplex assays. At 24 h, the number of CD45+/CD11b+ cells increased 3-fold in injured compared to uninjured brain tissue and CD45 expression shifted from low to medium with less than 10% of the population expressing CD45high. MCA occlusion induced rapid and transient asynchronous increases in the pro-inflammatory cytokine IL-b and chemokines cytokine-induced neutrophil chemoattractant protein 1 (CINC-1) and monocyte-chemoattractant protein 1 (MCP-1), first in systemic circulation and then in injured brain. Double immunofluorescence with celltype specific markers showed that multiple cell types in the injured brain produce MCP-1. Our findings show that despite profound increases in MCP-1 in injured regions, monocyte infiltration is low and the majority of macrophages in acutely injured regions are microglia. Keywords: cytokines, flow cytometry, inflammation, macrophage, microglia, neonatal stroke. Inflammation is a significant contributing factor to neurodegenerative diseases. Depending on injury setting, the relative involvement of systemic and local inflammation, and communication between the two compartments can vary (Carson and Sutcliffe 1999;Baggiolini 2001;Perry 2004). It is well known that stroke in adult triggers a robust inflammatory reaction, largely involving an influx of peripheral leukocytes into the brain parenchyma (for reviews, see Feuerstein et al. 1997;Dirnagl et al. 1999;Han and Yenari 2003) and disruption of the blood-brain barrier (BBB) (Gidday et al. 2005). In the adult, neutrophils are typically the first leukocyte type to infiltrate (Barone et al. 1991;Garcia et al. 1994;Matsuo et al. 1995) and are followed by macrophages and lymphocytes (Barone et al. 1991;Garcia et al. 1994). The damaging role of infiltrating neutrophils in ischemia was shown by studies in which neutrophil depletion (Garcia et al. 1994) or administration of anti-adhesion molecules (Kishimoto and Rothlein 1994;Fassbender et al. 1999) reversed the reduced local tissue perfusion, BBB disruption, and the release of free radicals, proteinases, and other cytotoxins seen after stroke. The key role of macrophages in cerebral ischemic injury has also been firmly established (Feuerstein et al. 1997;Dirnagl et al. 1999;Han and Yenari 2003). Macrophage populations in the injured brain, however, are diverse (Carson et al. 1998;Dalmau et al. 2003) Abbreviations used: BBB, blood-bra...

The incidence of neonatal stroke is high and currently there are no strategies to protect the neonatal brain from stroke or reduce the sequelae. Agents capable of modifying inflammatory processes hold promise. We set out to determine whether delayed administration of one such agent, minocycline, protects the immature brain in a model of transient middle cerebral artery (MCA) occlusion in 7-day-old rat pups. Injury volume in minocycline (45 mg/kg/dose, beginning at 2 h after MCA occlusion) and vehicle-treated pups was determined 24 h and 7 days after onset of reperfusion. Accumulation of activated microglia/macrophages, phosphorylation of mitogen-activated protein kinase (MAPK) p38 in the brain, and concentrations of inflammatory mediators in plasma and brain were determined at 24 h. Minocycline significantly reduced the volume of injury at 24 h but not 7 days after transient MCA occlusion. The beneficial effect of minocycline acutely after reperfusion was not associated with changed ED1 phenotype, nor was the pattern of MAPK p38 phosphorylation altered. Minocycline reduced accumulation of IL-1beta and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1beta, IL-18, MCP-1 or CINC-1 in the injured brain tissue. Therefore, minocycline provides early but transient protection, which is largely independent of microglial activation or activation of the MAPK p38 pathway.

Apoptosis is implicated in neonatal hypoxic/ischemic (H/I) brain injury among various forms of cell death. Here we investigate whether overexpression of heat shock protein (Hsp) 70, an antiapoptotic protein, protects the neonatal brain from H/I injury and the pathways involved in the protection. Postnatal day 7 (P7) transgenic mice overexpressing rat Hsp70 (Tg) and their wild-type littermates (Wt) underwent unilateral common carotid artery ligation followed by 30 mins exposure to 8% O(2). Significant neuroprotection was observed in Tg versus Wt mice on both P12 and P21, correlating with a high level of constitutive but not inducible Hsp70 in the Tg. More prominent injury was observed in Wt and Tg mice on P21, suggesting its continuous evolution after P12. Western blot analysis showed that translocation of cytochrome c, but not the second mitochondria-derived activator of caspase (Smac)/DIABLO and apoptosis-inducing factor (AIF), from mitochondria into cytosol was significantly reduced in Tg 24 h after H/I compared with Wt mice. Coimmunoprecipitation detected more Hsp70 bound to AIF in Tg than Wt mice 24 h after H/I, inversely correlating with the amount of nuclear, but not cytosolic, AIF translocation. Our results suggest that interaction between Hsp70 and AIF might have reduced downstream events leading to cell death, including the reduction of nuclear AIF translocation in the neonatal brains of Hsp70 Tg mice after H/I.