Pervasive Genomic Damage in Experimental Intracerebral Hemorrhage: Therapeutic Potential of a Mechanistic-Based Carbon Nanoparticle.

Therapy for intracerebral hemorrhage (ICH) remains elusive, in part dependent on the severity of the hemorrhage itself as well as multiple deleterious effects of blood and its breakdown products such as hemin and free iron. While oxidative injury and genomic damage have been seen following ICH, the details of this injury and implications remain unclear. Here, we discovered that, while free iron produced mostly reactive oxygen species (ROS)-related single-strand DNA breaks, hemin unexpectedly induced rapid and persistent nuclear and mitochondrial double-strand breaks (DSBs) in neuronal and endothelial cell genomes and in mouse brains following experimental ICH comparable to that seen with γ radiation and DNA-complexing chemotherapies. Potentially as a result of persistent DSBs and the DNA damage response, hemin also resulted in senescence phenotype in cultured neurons and endothelial cells. Subsequent resistance to ferroptosis reported in other senescent cell types was also observed here in neurons. While antioxidant therapy prevented senescence, cells became sensitized to ferroptosis. To address both senescence and resistance to ferroptosis, we synthesized a modified, catalytic, and rapidly internalized carbon nanomaterial, poly(ethylene glycol)-conjugated hydrophilic carbon clusters (PEG-HCC) by covalently bonding the iron chelator, deferoxamine (DEF). This multifunctional nanoparticle, DEF-HCC-PEG, protected cells from both senescence and ferroptosis and restored nuclear and mitochondrial genome integrity in vitro and in vivo. We thus describe a potential molecular mechanism of hemin/iron-induced toxicity in ICH that involves a rapid induction of DSBs, senescence, and the consequent resistance to ferroptosis and provide a mechanistic-based combinatorial therapeutic strategy.

Efficacy of Novel Carbon Nanoparticle Antioxidant Therapy in a Severe Model of Reversible Middle Cerebral Artery Stroke in Acutely Hyperglycemic Rats.

INTRODUCTION: While oxidative stress can be measured during transient cerebral ischemia, antioxidant therapies for ischemic stroke have been clinically unsuccessful. Many antioxidants are limited in their range and/or capacity for quenching radicals and can generate toxic intermediates overwhelming depleted endogenous protection. We developed a new antioxidant class, 40 nm × 2 nm carbon nanoparticles, hydrophilic carbon clusters, conjugated to poly(ethylene glycol) termed PEG-HCCs. These particles are high-capacity superoxide dismutase mimics, are effective against hydroxyl radical, and restore the balance between nitric oxide and superoxide in the vasculature. Here, we report the effects of PEG-HCCs administered during reperfusion after transient middle cerebral artery occlusion (tMCAO) by suture in the rat under hyperglycemic conditions. Hyperglycemia occurs in one-third of stroke patients and worsens clinical outcome. In animal models, this worsening occurs largely by accelerating elaboration of reactive oxygen species (ROS) during reperfusion. METHODS: PEG-HCCs were studied for their protective ability against hydrogen peroxide in b.End3 brain endothelial cell line and E17 primary cortical neuron cultures. In vivo, hyperglycemia was induced by streptozotocin injection 2 days before tMCAO. 58 Male Sprague-Dawley rats were analyzed. They were injected IV with PBS or PEG-HCCs (4 mg/kg 2×) at the time of recanalization after either 90- or 120-min occlusion. Rats were survived for up to 3 days, and infarct volume characteristics and neurological functional outcome (modified Bederson Score) were assessed. RESULTS: PEG-HCCs were protective against hydrogen peroxide in both culture models. In vivo improvement was found after PEG-HCCs with 90-min ischemia with reduction in infarct size (42%), hemisphere swelling (46%), hemorrhage score (53%), and improvement in Bederson score (70%) (p = 0.068-0.001). Early high mortality in the 2-h in the PBS control group precluded detailed analysis, but a trend was found in improvement in all factors, e.g., reduction in infarct volume (48%; p = 0.034) and a 56% improvement in Bederson score (p = 0.055) with PEG-HCCs. CONCLUSION: This nano-antioxidant showed some improvement in several outcome measures in a severe model of tMCAO when administered at a clinically relevant time point. Long-term studies and additional models are required to assess potential for clinical use, especially for patients hyperglycemic at the time of their stroke, as these patients have the worst outcomes.

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters.

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Hyperglycemia worsens outcome after rt-PA primarily in the large-vessel occlusive stroke subtype.

Hyperglycemia at the time of ischemic stroke has been associated with poorer outcomes. Preclinical literature suggests that hyperglycemia is an independent prognostic factor and the vasculature is more vulnerable to reperfusion injury. We applied a method to match subjects on important baseline factors to test whether, independent of stroke severity, stroke subtype influences the effect of hyperglycemia on outcome after recombinant tissue plasminogen activator (rt-PA). We reanalyzed the NINDS rt-PA dataset with respect to matching variables baseline NIHSS, age, and investigator-determined stroke subtypes small-vessel occlusive stroke (SVS), large-vessel occlusive stroke (LVS), and cardioembolic stroke (CES), above and below a glucose threshold of 150 mg/dl. Ninety-day outcomes were compared. Post hoc baseline matching was excellent in most cases. Hyperglycemia was associated with worsened functional outcome mostly in the LVS subtype with increased mortality in the placebo arm (15.3% mortality normoglycemia vs. 30.6% hyperglycemia; p = .046), worse functional outcome in the rt-PA arm (modified Rankin Score (mRS) 0-1; 46.3 vs. 22.0%; p = .034), and no improvement in functional outcome with rt-PA compared to placebo (mRS 0-1; 25% in both groups). Among hyperglycemic subjects, CES subjects showed significant improvement following rt-PA (p = .027). After matching for baseline severity, the influence of hyperglycemia on outcome was primarily in the LVS subtype, especially after rt-PA. This finding is consistent with a deleterious effect of hyperglycemia on ischemia/reperfusion of symptomatic large arteries. If confirmed, the particular vulnerability of the LVS subtype is important in understanding the role of stroke subtype in the mechanism of worsening and potential treatment of hyperglycemic stroke patients.

Design of poly(ethylene glycol)-functionalized hydrophilic carbon clusters for targeted therapy of cerebrovascular dysfunction in mild traumatic brain injury.

Traumatic brain injury (TBI) involves the elaboration of oxidative stress that causes cerebrovascular dysfunction, including impairment of autoregulation of cerebral blood flow. Currently, there is no clinically effective antioxidant treatment for these pathologies. Most currently available antioxidants act through mechanisms in which the antioxidant either transfers the radical or requires regeneration, both of which are impaired in the toxic post-TBI environment. We previously reported that single-walled carbon nanotubes (SWCNTs) and ultrashort SWCNTs possess antioxidant activity, and their characteristics suggest that radical annihilation is the major mechanism. We have now developed a biologically compatible class of carbon-based nanovectors, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs) that can be further functionalized with antibodies, and hence show promise as targeted drug delivery platforms. Here we report that PEG-HCCs possess innate antioxidant activity and can be rapidly targeted via an antibody to the P-selectin antigen in a model of injured cultured brain endothelial cells. One immediate application of this therapy is to vascular dysfunction that accompanies TBI and worsens outcome in the face of systemic hypotension. These in vitro results support the need for further investigation in animal models.

Antioxidant carbon particles improve cerebrovascular dysfunction following traumatic brain injury.

Injury to the neurovasculature is a feature of brain injury and must be addressed to maximize opportunity for improvement. Cerebrovascular dysfunction, manifested by reduction in cerebral blood flow (CBF), is a key factor that worsens outcome after traumatic brain injury (TBI), most notably under conditions of hypotension. We report here that a new class of antioxidants, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), which are nontoxic carbon particles, rapidly restore CBF in a mild TBI/hypotension/resuscitation rat model when administered during resuscitation--a clinically relevant time point. Along with restoration of CBF, there is a concomitant normalization of superoxide and nitric oxide levels. Given the role of poor CBF in determining outcome, this finding is of major importance for improving patient health under clinically relevant conditions during resuscitative care, and it has direct implications for the current TBI/hypotension war-fighter victims in the Afghanistan and Middle East theaters. The results also have relevancy in other related acute circumstances such as stroke and organ transplantation.

Hyperglycemia accentuates persistent "functional uncoupling" of cerebral microvascular nitric oxide and superoxide following focal ischemia/reperfusion in rats.

Hyperglycemia worsens outcome in stroke patients and in a variety of stroke models, most prominently following prolonged ischemia and reperfusion. Vascular dysfunction has been associated with this worsened outcome, manifested by reduced reperfusion cerebral blood flow (CBF), as well as increased hemorrhagic transformation, edema, and mortality. The phenomenon of "uncoupling" of endothelial nitric oxide synthase (eNOS) has been associated with hyperglycemia in the periphery and results in a dysfunctional eNOS-generating superoxide radical ([Formula: see text]) in lieu of nitric oxide (NO). A net result of NOS uncoupling is reduction in blood flow and generation of a pro-thrombotic, inflammatory vascular phenotype that could explain many of the features of hyperglycemic stroke. Because the sources of increased [Formula: see text] and mechanisms of reduced NO are difficult to identify under in vivo pathological conditions, we have termed the presence of perivascular excess of [Formula: see text] expression relative to NO as "functional uncoupling." We hypothesized that hyperglycemia would induce a state of functional uncoupling in the cerebral microvasculature that would be exacerbated by transient focal ischemia. We examined the relative expression of systemically injected radical sensitive dyes in cerebral microvessel profiles in the basal and 24 h post-stroke brain in a rat model of middle cerebral artery occlusion and recanalization (MCAO/R) in controls and after hyperglycemia. We focused on the peri-infarct region because of its importance in penumbra extension and edema. Differential expression of fluorescent dyes sensitive to [Formula: see text] and NO in microvessel profiles were assessed in the peri-infarct region. Hyperglycemia was induced by streptozotocin 48 h prior to MCAO/R. We found that hyperglycemia resulted in an increase in [Formula: see text] relative to NO, a pattern consistent with functional uncoupling. This ratio was accentuated 24 h after MCA/O in both groups. Hyperglycemic rats showed a synergistic increase in the [Formula: see text]/NO ratio as well as reduced acute reperfusion CBF, increased infarct size, and enhanced expression of nitrotyrosine. While effects of hyperglycemia on oxidative radicals is well known, we showed for the first time that hyperglycemia synergistically worsened functional uncoupling in the peri-infarct microvasculature and that it persisted for the 24-h duration of the experiment. Persistent generation of microvascular [Formula: see text] and reduction in NO expression suggest potential late therapeutic targets to restore microvessel function and improve vascular outcomes in hyperglycemic stroke.

A Pilot Trial of Low-Dose Intravenous Abciximab and Unfractionated Heparin for Acute Ischemic Stroke: Translating GP IIb/IIIa Receptor Inhibition to Clinical Practice.

Thrombolysis remains a mainstay in the treatment of ischemic stroke. While not usually considered in the spectrum of clot lysis, experimental data show that inhibition of the platelet glycoprotein (GP) IIb/IIIa receptor can reduce as well as reverse thrombus formation and improve microvascular flow in stroke models. However, a recent clinical trial of GP IIb/IIIa inhibition in stroke did not demonstrate clinical benefit and was associated with increased hemorrhage. Based on an understanding of the relationship between GP IIb/IIIa receptor inhibition, efficacy and hemorrhage, we hypothesized that a lower dose of abciximab would achieve a favorable range of platelet inhibition and potentially good clinical outcomes. Forty-four patients with suspected large vessel occlusion, who were not eligible for rt-PA were offered treatment with approximately 30% lower total dose of intravenous abciximab if within 6 h for anterior circulation or 24 h for posterior circulation stroke (later modified to 12 h). Concomitant anticoagulation, usually with unfractionated heparin was employed. The extent of platelet inhibition was measured in 21 patients. Hemorrhage rate and 90-day functional outcomes and mortality were obtained. A matching algorithm involving finding the nearest neighbor from individual subjects in the control arm of the NINDS rt-PA database was used to compare outcomes at similar baseline characteristics and gender. Mean platelet inhibition was 92.1 ± 6.7% vs inhibition reported with percutaneous coronary intervention (PCI) of 96 ± 10; p = 0.08. Successful matching to NINDS controls was accomplished: after outlier elimination, median and mean NIHSS of the abciximab subjects compared to NINDS controls was 16.5 vs 15.5 (p = 0.92) and 16.3 vs 16.0 (p = 0.86). Mean age was 67.2 vs 67.1 (p = 0.97). Mean glucose was 141 vs 142 (p = 0.92). There was one symptomatic hemorrhage; minor hemorrhages occurred in 9%. The percent of patients who achieved an mRS 0-2 or died in the treated vs matched NINDS control patients was 63 vs 38 (p = .02) and 23 vs 23 (p = 1.0). Our pilot results indicated that a lower dose of abciximab results in platelet inhibition similar to that achieved in the coronary vascular bed during PCI. Comparison to matched historical controls suggests that this lower dose in combination therapy may be safe and effective therapy for thrombotic stroke and a randomized trial is warranted.

Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia.

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.

A decoy oligonucleotide inhibiting nuclear factor-kappaB binding to the IgGkappaB consensus site reduces cerebral injury and apoptosis in neonatal hypoxic-ischemic encephalopathy.

We examined the effect of treatment with intraventricular injection of a decoy oligonucleotide that binds and inhibits nuclear factor-kappaB on cytokine expression, ICAM-1 expression, neutrophil recruitment, apoptosis, and tissue injury in a model of neonatal hypoxic-ischemic cerebral injury with varying degrees of hypoxia. We found a reduction of interleukin-1beta, tumor necrosis factor-alpha, soluble ICAM-1, neutrophil counts, and activity after 2 hr of hypoxia, but not with 90 min of hypoxia. By contrast, a significant reduction of apoptosis was seen in animals treated after 90 min of hypoxia but not in those treated after 2 hr of hypoxia. Overall evidence of an inflammatory response was sparse, with low levels of ICAM-1 expression and neutrophil recruitment even in the more severe hypoxic ischemic injury. It is likely that the decoy oligonucleotide affects cerebral injury and apoptosis not through suppression of downstream elements of the inflammatory response but through other mechanisms, one of which is the reduction of transcription and synthesis of cytokines, which are known to affect other responses to cellular injury.

Potential role for white matter lysosome expansion in HIV-associated dementia.

Expansion of the lysosomal apparatus occurs in subcortical white matter in brains from persons with AIDS. This study examined whether HIV-associated subcortical dementia (HAD) is significantly related to this lysosomal anomaly. Brain cortex and adjacent white matter from the middle frontal gyrus were obtained from the National NeuroAIDS Tissue Consortium. Lysosomal hydrolase activity was assayed in 57 subjects who underwent neuropsychological testing within 6 months prior to autopsy. Decedents were evaluated from 4 geographical sites in the United States: Galveston/Houston, Texas (n = 36), Los Angeles, California (n = 5), New York, New York (n = 5), and San Diego, California (n = 11). Increased beta-glucuronidase activity, a representative lysosomal glycosidase, was correlated with the amount of neurocognitive impairment. Significant correlation was present in 5 of 7 functional testing domains, including some that draw upon frontal lobe output (r = 0.419; P < 0.002). The biochemical anomaly was negligible in cerebral cortex and cerebrospinal fluid and was not correlated with brain dysfunction in those compartments. Glycosidase activation was associated significantly with increased HIV RNA concentration in brain tissue (r = 0.469; P < 0.021) and possibly with HIV RNA in cerebrospinal fluid (r = 0.266; P < 0.067). HIV RNA in blood plasma was not correlated. These results support the suggestion that abnormal metabolism in white matter glial cells contributes to cognitive slowing in persons with HAD. Because membrane turnover is routed through the endosome-lysosome apparatus, these data are in agreement with brain spectroscopic data that have suggested that there is an increase in membrane turnover in white matter glia.

Extracellular superoxide concentration increases following cerebral hypoxia but does not affect cerebral blood flow.

Abnormalities of cerebral blood flow during and following hypoxia and ischemia contribute to the progression of tissue injury. Oxidative stress during and following hypoxia is known to markedly increase superoxide anion concentration. There is conflicting evidence that the concentration of superoxide anion regulates cerebral blood flow through its effect on vascular tone, although difficulties in measurement of superoxide anion complicate these studies. In order to test the hypothesis that changes in cerebral blood flow during and following hypoxia are due to changes in extracellular superoxide anion levels, we examined tissue oxygen levels by fiberoptic oximetry and superoxide anion levels using a previously validated cytochrome c coated electrode on the cortical surface and correlated these measurements to cerebral blood flow measured by laser Doppler in rats subjected to 20 min of hypoxia followed by hyperoxic reoxygenation recovery. The results showed a burst of superoxide anion with the onset of reoxygenation that temporally correlated with a transient peak in tissue oxygen tension lasting 10 min. and was eliminated by pretreatment with Cu-Zn superoxide dismutase conjugated to polyethylene glycol. Cerebral blood flow did not differ during hypoxia or recovery in the polyethylene glycol conjugated superoxide dismutase and control treatment groups. This study demonstrated no effect of increased superoxide anion concentration on cerebral blood flow during hyperoxic recovery following hypoxia.