Zinaida S. Vexler

Rat forebrain neurogenesis and striatal neuron replacement after focal stroke

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.

The role of inflammation in perinatal brain 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.

Microglial Cells Contribute to Endogenous Brain Defenses after Acute Neonatal Focal Stroke

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 whether 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 7 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 whether 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. 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.

Hsp70 Overexpression Sequesters AIF and Reduces Neonatal Hypoxic/Ischemic Brain Injury:

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% O2. 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.

Erythropoietin Improves Functional and Histological Outcome in Neonatal Stroke

Neonatal stroke is a condition that leads to disability in later life, and as yet there is no effective treatment. Recently, erythropoietin (EPO) has been shown to be cytoprotective following brain injury and may promote neurogenesis. However, the effect of EPO on functional outcome and on morphologic changes in neonatal subventricular zone (SVZ) following experimental neonatal stroke has not been described. We used a transient focal model of neonatal stroke in P10 rat. Injury was documented by diffusion weighted MRI during occlusion. Immediately upon reperfusion, either EPO (5U/gm) or vehicle was administered intraperitoneally and animals were allowed to grow for 2 wk. Sensorimotor function was assessed using the cylinder rearing test and then brains were processed for volumetric analysis of the SVZ. Stroke induced SVZ expansion proportional to hemispheric volume loss. EPO treatment markedly preserved hemispheric volume and decreased the expansion of SVZ unilaterally. Furthermore, EPO treatment significantly improved the asymmetry of forelimb use following neonatal stroke. This functional improvement directly correlated with the amount of preserved hemispheric volume. These results suggest EPO may be a candidate in the treatment of neonatal stroke.

Erythropoietin Enhances Long-Term Neuroprotection and Neurogenesis in Neonatal Stroke

Neonatal stroke leads to mortality and severe morbidity, but there is no effective treatment currently available. Erythropoietin (EPO) has been shown to promote cytoprotection and neurogenesis and decrease subventricular zone morphologic changes following brain injury. The long-term cellular response to EPO has not been defined, and local changes in cell fate decision may play a role in functional improvement. We performed middle cerebral artery occlusion in P10 rats. EPO treatment (5 U/g IP) significantly preserved hemispheric brain volume 6 weeks after injury. Furthermore, EPO increased the percentage of newly generated neurons while decreasing newly generated astrocytes following brain injury, without demonstrating long-term differences in the subventricular zone. These results suggest that EPO may neuroprotect and direct cell fate toward neurogenesis and away from gliogenesis in neonatal stroke.

Regulation of hypoxia-inducible factor 1α and induction of vascular endothelial growth factor in a rat neonatal stroke model

Stroke is a devastating condition occurring in at least 1 in 4000 live births in the neonatal period. Since hypoxia-inducible factor (HIF)-1alpha can modulate ischemic injury via induction of target genes that may protect cells against ischemia, and is induced after preconditioning by hypoxia in the neonatal rat brain hypoxia-ischemia model, we evaluated whether HIF-1alpha is induced after focal ischemia-reperfusion, a model for neonatal stroke. We developed an ischemia-reperfusion model in postnatal day 10 (P10) rats by transiently occluding the middle cerebral artery (MCA) for 1.5 h. The MCA territory was reperfused for 0, 4, 8, or 24 h and the expression of HIF-1alpha and its target gene, vascular endothelial growth factor (VEGF), were delineated. HIF-1alpha protein and VEGF protein peaked at 8 h, and declined subsequently at 24 h in injured cortex following 1.5 h of MCA occlusion. Double-immunolabeling indicated that both HIF-1alpha and VEGF are expressed together in neurons with a similar time course of expression. The presence of HIF-1alpha and VEGF after moderate ischemia-reperfusion injury suggests potential avenues to exploit for neuroprotection.

Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia–reperfusion

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-1β and CINC-1 in the systemic circulation but failed to affect the increased levels of IL-1β, 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.

Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat

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 whether 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), gadolinium (III)-diethyltriaminepentaacetic acid tracers remained largely undisturbed 24 h 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 24 h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1266 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 matrix metalloproteinase-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin, and zonula occludens protein 1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of cytokine-induced neutrophil chemoattractant-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.

Macrophages are comprised of resident brain microglia not infiltrating peripheral monocytes acutely after neonatal stroke.

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 post‐reperfusion 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‐β 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 cell‐type 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.