Huaxin Sheng

Oxidants, antioxidants and the ischemic brain

SUMMARY Despite numerous defenses, the brain is vulnerable to oxidative stress resulting from ischemia/reperfusion. Excitotoxic stimulation of superoxide and nitric oxide production leads to formation of highly reactive products, including peroxynitrite and hydroxyl radical, which are capable of damaging lipids, proteins and DNA. Use of transgenic mutants and selective pharmacological antioxidants has greatly increased understanding of the complex interplay between substrate deprivation and ischemic outcome. Recent evidence that reactive oxygen/nitrogen species play a critical role in initiation of apoptosis, mitochondrial permeability transition and poly(ADP-ribose) polymerase activation provides additional mechanisms for oxidative damage and new targets for post-ischemic therapeutic intervention. Because oxidative stress involves multiple post-ischemic cascades leading to cell death, effective prevention/treatment of ischemic brain injury is likely to require intervention at multiple effect sites.

Simvastatin Increases Endothelial Nitric Oxide Synthase and Ameliorates Cerebral Vasospasm Resulting From Subarachnoid Hemorrhage

Background and Purpose— Endothelial nitric oxide synthase (eNOS) activity is decreased after subarachnoid hemorrhage (SAH). Simvastatin increases eNOS activity. We hypothesized that simvastatin would increase eNOS protein and ameliorate SAH-induced cerebral vasospasm. Methods— Mice were treated with subcutaneous simvastatin or vehicle for 14 days and then subjected to endovascular perforation of the right anterior cerebral artery or sham surgery. Three days later, neurological deficits were scored (5 to 27; 27=normal), and middle cerebral artery diameter and eNOS protein were measured. The study was repeated, but simvastatin treatment was started after SAH or sham surgery. Results— In SAH mice, simvastatin pretreatment increased middle cerebral artery diameter (SAH-simvastatin=74±22 &mgr;m, SAH-vehicle=52±18 &mgr;m, P =0.03; sham-simvastatin=102±8 &mgr;m, sham-vehicle=105±6 &mgr;m). Pretreatment reduced neurological deficits (SAH-simvastatin=25±2, SAH-vehicle=20±2, P =0.005; sham-simvastatin and sham-vehicle=27±0). Simvastatin pretreatment also increased eNOS protein. Simvastatin posttreatment caused a modest increase in middle cerebral artery diameter in SAH mice (SAH-simvastatin=56±12 &mgr;m, SAH-vehicle=45±4 &mgr;m, P =0.03; sham-simvastatin=92±13 &mgr;m, sham-vehicle=99±10 &mgr;m) and reduced neurological deficits (SAH-simvastatin=21±1, SAH-vehicle=19±2, P =0.009). Simvastatin posttreatment did not significantly increase eNOS protein. Conclusions— Simvastatin treatment before or after SAH attenuated cerebral vasospasm and neurological deficits in mice. The mechanism may be attributable in part to eNOS upregulation.

Apolipoprotein E-Deficient Mice Have Increased Susceptibility To Focal Cerebral Ischemia

Recent evidence suggests that apolipoprotein E (ApoE) plays a role in neurologic disease. This experiment compared the neurologic and histologic outcome of ApoE-deficient mutant and wild-type mice subjected to a 60- or 90-minute episode of middle cerebral artery filament occlusion and a recovery interval of 24 hours. With 60 minutes of ischemia, there was no mortality. Apolipoprotein E-deficient mice had larger infarcts (cortex: ApoE deficient = 20 mm3 ± 12, wild-type = 9 ± 7 mm3, P = 0.03; subcortex: ApoE deficient = 22 ± 7 mm3, wild-type = 16 ± 7 mm3, P = 0.07). Hemiparesis was less severe in wild-type animals (P = 0.02). After 90 minutes of ischemia, mortality in ApoE-deficient mice (n = 10) was 40% versus 0% in wild-type mice (n = 10; P = 0.09). Intraparenchymal hemorrhage was found in 3 of the 4 dead mice. No difference in cortical (ApoE deficient = 37 ± 8 mm3; wild-type = 31 ± 18 mm3; P = 0.49) or subcortical (ApoE deficient = 30 ± 11 mm3; wild-type = 32 ± 18 mm3; P = 0.78) infarct volumes was present among survivors. ApoE-deficient mice had a prolonged activated partial thromboplastin time and increased fibrinogen concentration. This data supports the hypothesis that apolipoprotein E plays a role in the pathophysiology of ischemic brain damage.

Apolipoprotein E Isoform-Specific Differences in Outcome from Focal Ischemia in Transgenic Mice:

Apolipoprotein E (apoE), a 34-kD glycosylated lipid-binding protein, is expressed as three common isoforms in humans (E2, E3, or E4). Clinical evidence suggests that the apoE genotype (APOE) may be a risk factor for poor outcome after acute central nervous system injury. This was examined further in transgenic mice constructed with the human APOE3 or APOE4 gene under the control of human promoter and tissue expression elements. Presence of human apoE3 and apoE4 proteins in brains of human APOE homozygous transgenic mice was confirmed by Western blotting. APOE3 (n = 12) and APOE4 (n = 10) mice underwent 60 minutes of middle cerebral artery occlusion. After 24-hour recovery, infarct size was measured. Infarct volumes (mean ± standard deviation) were smaller in the APOE3 group (cortex: APOE3 = 18 ± 4 mm3; APOE4 = 30 ± 11 mm3, P = 0.04; subcortex: APOE3 = 12 ± 4 mm3; APOE4 = 18 ± 4 mm3, P = 0.003). Hemiparesis was less severe in APOE3 mice (P = 0.02). These data indicate that human isoform-specific effects of apoE are relevant to acute pathomechanisms of focal ischemic brain damage when examined in the mouse. APOE transgenic mice may provide an appropriate model to examine the mechanistic basis for the differential effects of human apoE isoforms in acute central nervous system injury.

Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular/subependymal zone (SVZ/SEZ) generate Doublecortin (DCX)+ neuroblasts that migrate and integrate into olfactory bulb circuitry1,2. Continuous production of neuroblasts is controlled by SVZ microenvironmental niche3,4. It is generally believed that enhancing neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear if there are conditions that favor astrogenesis over neurogenesis in the SVZ niche, and if astrocytes produced there exhibit different properties from others in the brain. We have uncovered that SVZ-generated astrocytes express high levels of Thrombospondin-4 (Thbs4)5,6, a secreted homopentameric glycoprotein, in contrast to cortical astrocytes which are Thbs4low. We found that localized photothrombotic/ischemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-CreERtm4 lineage-tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not DCX+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation, modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production7. Consequently, Thbs4KO/KO animals showed severe defects in cortical injury-induced SVZ astrogenesis, instead producing cells expressing DCX from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular hemorrhage into the brain parenchyma of Thbs4KO/KO animals. Taken together, these findings have significant implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members play important roles8,9.Postnatal/adult neural stem cells (NSCs) within the rodent subventricular zone (SVZ; also called subependymal zone) generate doublecortin (Dcx)+ neuroblasts that migrate and integrate into olfactory bulb circuitry. Continuous production of neuroblasts is controlled by the SVZ microenvironmental niche. It is generally thought that enhancing the neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear whether there are conditions that favour astrogenesis over neurogenesis in the SVZ niche, and whether astrocytes produced there have different properties compared with astrocytes produced elsewhere in the brain. Here we show in mice that SVZ-generated astrocytes express high levels of thrombospondin 4 (Thbs4), a secreted homopentameric glycoprotein, in contrast to cortical astrocytes, which express low levels of Thbs4. We found that localized photothrombotic/ischaemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-creERtm4 lineage tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not Dcx+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production. Consequently, Thbs4 homozygous knockout mice (Thbs4KO/KO) showed severe defects in cortical-injury-induced SVZ astrogenesis, instead producing cells expressing Dcx migrating from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular haemorrhage into the brain parenchyma of Thbs4KO/KO mice. Taken together, these findings have important implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members have important roles.

Mice overexpressing extracellular superoxide dismutase have increased resistance to focal cerebral ischemia.

Transgenic mice, which had been transfected with the human extracellular superoxide dismutase gene, causing an approximate five-fold increase in brain parenchymal extracellular superoxide dismutase activity, were used to investigate the role of extracellular superoxide dismutase in ischemic brain injury. Transgenic (n = 21) and wild-type (n = 19) mice underwent 90 min of intraluminal middle cerebral artery occlusion and 24 h of reperfusion. Severity of resultant hemiparesis and cerebral infarct size were measured. Wild-type mice had larger infarcts (cortex: wild type =37+/-14 mm3, transgenic = 27+/-13 mm3, P=0.03; subcortex: wild type = 33+/-14 mm3, transgenic = 23+/-10 mm3, P = 0.02). Neurological scores, however, were similar (P = 0.29). Other mice underwent autoradiographic determination of intra-ischemic cerebral blood flow. The volume of tissue at risk of infarction (defined as volume of tissue where blood flow was <25 ml/100g/min) was similar between groups (cortex: wild type = 51+/-15 mm3, transgenic = 47+/-9 mm3, P=0.65; subcortex: wild type = 39+/-16 mm3, transgenic= 37+/-17 mm3, P=0.81). These results indicate that antioxidant scavenging of free radicals by extracellular superoxide dismutase plays an important role in the histological response to a focal ischemic brain insult.

Isoflurane provides long-term protection against focal cerebral ischemia in the rat.

Background:Long-term neuroprotection by isoflurane has been questioned. The authors examined factors in experimental models potentially critical to definition of enduring isoflurane neuroprotection. Methods:Rats were prepared for temporary middle cerebral artery occlusion (MCAO). Pericranial normothermia was maintained. Neurologic deficits (range, 0–48; 0 = no deficit) and cerebral infarct volumes were measured. In experiment 1, rats underwent 50 or 80 min MCAO while awake or anesthetized with 1.8% isoflurane. Blood pressure was controlled with phenylephrine. Outcome was evaluated 2 weeks later. In experiment 2, rats underwent 50 min MCAO while awake or anesthetized with isoflurane, with outcome evaluated 8 weeks later. In experiment 3, rats underwent 50 min MCAO while awake or anesthetized with isoflurane and 2 weeks recovery. Effects of phenylephrine and the mitochondrial adenosine triphosphate–sensitive K+ channel antagonist 5-hydroxydecanoate were studied. In experiment 4, isoflurane-anesthetized rats underwent 50 min MCAO with permanent or temporary common carotid artery occlusion, with outcome evaluated 2 weeks later. Results:In experiment 1, isoflurane reduced neurologic deficit (median ± interquartile range; awake vs. isoflurane: 11 ± 12 vs. 8 ± 6 for 80 min and 13 ± 4 vs. 3 ± 9 for 50 min; P = 0.0006) and infarct size (160 ± 97 vs. 84 ± 62 mm3 for 80 min and 169 ± 78 vs. 68 ± 61 mm3 for 50 min; P < 0.0001). In experiment 2, isoflurane protection persisted at 8 weeks after ischemia. In experiment 3, there was no effect of phenylephrine or 5-hydroxydecanoate. In experiment 4, permanent common carotid ligation increased infarct size threefold versus temporary occlusion. Conclusions:Isoflurane repeatedly improved long-term neurologic and histologic outcome from focal ischemia independent of ischemia duration, perfusion pressure, or pretreatment with 5-hydroxydecanoate.

Possible Role for Vascular Cell Proliferation in Cerebral Vasospasm After Subarachnoid Hemorrhage

Background and Purpose— During vasospasm after subarachnoid hemorrhage (SAH), cerebral blood vessels show structural changes consistent with the actions of vascular mitogens. We measured platelet-derived vascular growth factors (PDGFs) in the cerebrospinal fluid (CSF) of patients after SAH and tested the effect of these factors on cerebral arteries in vivo and in vitro. Methods— CSF was sampled from 14 patients after SAH, 6 patients not suffering SAH, and 8 normal controls. ELISA was performed for PDGF-AB, transforming growth factor-&bgr;1, and vascular endothelial growth factor. A mouse model was used to compare cerebral vascular cell proliferation and PDGF staining in SAH compared with sham-operated controls. Normal human pial arteries were incubated for 7 days in vitro, 2 groups with human blood clot and 1 with and 1 without PDGF antibodies. Results— PDGF-AB concentrations in CSF from SAH patients were significantly higher than those from non-SAH patients and normal controls, both during the first week after SAH and for all time points measured. Smooth muscle and fibroblast proliferation was observed after SAH in the mouse model, and this cellular replication was observed in conjunction with PDGF protein at the sites of thrombus. In human pial arteries, localized thrombus stimulated vessel wall proliferation, and proliferation was blocked by neutralizing antibodies directed against PDGFs. Conclusions— Vascular mitogens are increased in the CSF of patients after SAH. Proliferation of cells in the vascular wall is associated with perivascular thrombus. Cellular proliferation and subsequent vessel wall thickening may contribute to the syndrome of delayed cerebral vasospasm.

Transient Global Cerebral Ischemia Induces a Massive Increase in Protein Sumoylation

A new group of proteins, small ubiquitin-like modifier (SUMO) proteins, has recently been identified and protein sumoylation has been shown to play a major role in various signal transduction pathways. Here, we report that transient global cerebral ischemia induces a marked increase in protein sumoylation. Mice were subjected to 10 mins severe forebrain ischemia followed by 3 or 6 h of reperfusion. Transient cerebral ischemia induced a massive increase in protein sumoylation by SUMO2/3 both in the hippocampus and cerebral cortex. SUMO2/3 conjugation was associated with a decrease in levels of free SUMO2/3. After ischemia, protein levels of the SUMO-conjugating enzyme Ubc9 were transiently decreased in the cortex but not in the hippocampus. We also exposed HT22 cells to arsenite, a respiratory poison that impairs cytoplasmic function and induces oxidative stress. Arsenite exposure induced a marked rise in protein sumoylation, implying that impairment of cytoplasmic function and oxidative stress may be involved in the massive post-ischemic activation of SUMO conjugation described here. Sumoylation of transcription factors has been shown to block their activation, with some exceptions such as the heat-shock factor and the hypoxia-responsive factor, where sumoylation blocks their degradation, and the nuclear factor-κB (NF-κB) essential modulator where sumoylation leads to an activation of NF-κB. Because protein sumoylation is known to be involved in the regulation of various biologic processes, the massive post-ischemic increase in protein sumoylation may play a critical role in defining the final outcome of neurons exposed to transient ischemia.

Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia

Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t(1/2) was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O(2)/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.