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This review surveys the efforts taken to achieve clinically efficacious protection of the ischemic brain and underscores the necessity of expanding our purview to include the essential role of cerebral perfusion and the collateral circulation. We consider the development of quantitative strategies to measure cerebral perfusion at the regional and local levels and the application of these methods to elucidate flow-related thresholds of ischemic viability and to characterize the ischemic penumbra. We stress that the modern concept of neuroprotection must consider perfusion, the necessary substrate upon which ischemic brain survival depends. We survey the major mechanistic approaches to neuroprotection and review clinical neuroprotection trials, focusing on those phase 3 multicenter clinical trials for acute ischemic stroke that have been completed or terminated. We review the evolution of thrombolytic therapies; consider the lessons learned from the initial, negative multicenter trials of endovascular therapy; and emphasize the highly successful positive trials that have finally established a clinical role for endovascular clot removal. As these studies point to the brain's collateral circulation as key to successful reperfusion, we next review the anatomy and pathophysiology of collateral perfusion as it relates to ischemic infarction, as well as the molecular and genetic influences on collateral development. We discuss the current MR and CT-based diagnostic methods for assessing the collateral circulation and the prognostic significance of collaterals in ischemic stroke, and we consider past and possible future therapeutic directions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background and Purpose: The ALIAS (Albumin in Acute Ischemic Stroke) part 1 and 2 trials evaluated whether 25% human serum albumin improves clinical outcomes after acute ischemic stroke above and beyond standard of care using similar protocols. The part 1 trial ended prematurely because of safety concerns, and the part 2 trial terminated early because of futility of finding a statistically significant effect of albumin over saline (control) administration. We combine the subject-level data of the part 1 and 2 trials to reevaluate the efficacy and safety outcomes with the larger sample size., Methods: The combined data analyses closely follow those conducted in the part 2 trial. The primary outcome is the composite of the modified Rankin Scale and the National Institutes of Health Stroke Scale defined as a composite of modified Rankin Scale score 0 to 1 and National Institutes of Health Stroke Scale score 0 to 1 at 90 days from randomization. The unadjusted analyses use a simple Chi-square test, and those adjusting for baseline covariates use a generalized linear model with log link (to obtain relative risks)., Results: The participant characteristics at baseline were generally similar between the treatment groups and between the trials; however, thrombolysis use was greater in part 2 (84% versus 75%), and the upper age limit imposed in part 2 resulted in a younger sample (mean age in part 1 was 69 versus 64 in part 2). In the combined sample, the proportions of good outcome in the 2 treatment groups were identical (41%). Similar results were observed in all secondary efficacy outcomes. Pulmonary edema was a consistent safety concern, with a 6-fold increase in the albumin arm (13%) compared with saline (2%; relative risk =7.76, 95% confidence interval 3.87-15.57)., Conclusions: Treatment with intravenous albumin 25% at 2 g/kg was not associated with improved outcome at 90 days and was associated with increased rates of intracerebral hemorrhage and pulmonary edema., Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00235495., (© 2016 American Heart Association, Inc.)

Background: Albumin treatment of ischemic stroke was associated with cardiopulmonary adverse events in previous studies and a low incidence of intracranial hemorrhage. We sought to describe the neurological and cardiopulmonary adverse events in the ALIAS Part 2 Multicenter Trial., Methods: Ischemic stroke patients, aged 18-83 and a baseline NIHSS ≥ 6, were randomized to treatment with ALB or saline control within 5 hours of stroke onset. Neurological adverse events included symptomatic intracranial hemorrhage, hemicraniectomy, neurological deterioration and neurological death. Cardiopulmonary adverse events included pulmonary edema/congestive heart failure, acute coronary syndromes, atrial fibrillation, pneumonia and pulmonary thromboembolism., Results: Among 830 patients, neurological and cardiopulmonary adverse events were not differentially associated with poor outcome between ALB and saline control subjects. The rate of symptomatic intracranial hemorrhage in the first 24h was low overall (2.9%, 24/830) but more common in the ALB treated subjects (RR = 2.4, CI95 1.01-5.8). The rate of pulmonary edema/CHF in the first 48h was 7.9% (59/830) and was more common among ALB treated subjects (RR = 10.7, CI95 4.3-26.6); this complication was expected and was satisfactorily managed with mandated diuretic administration and intravenous fluid guidelines. Troponin elevations in the first 48h were common, occurring without ECG change or cardiac symptoms in 52 subjects (12.5%)., Conclusions: ALB therapy was associated with an increase in symptomatic ICH and pulmonary edema/congestive heart failure but this did not affect final outcomes. Troponin elevation occurs routinely in the first 48 hours after acute ischemic stroke., Trial Registration: ClincalTrials.gov NCT00235495.

Background: In animal models of ischaemic stroke, 25% albumin reduced brain infarction and improved neurobehavioural outcome. In a pilot clinical trial, albumin doses as high as 2 g/kg were safely tolerated. We aimed to assess whether albumin given within 5 h of the onset of acute ischaemic stroke increased the proportion of patients with a favourable outcome., Methods: We did a randomised, double-blind, parallel-group, phase 3, placebo-controlled trial between Feb 27, 2009, and Sept 10, 2012, at 69 sites in the USA, 13 sites in Canada, two sites in Finland, and five sites in Israel. Patients aged 18-83 years with ischaemic (ie, non-haemorrhagic) stroke with a baseline National Institutes of Health stroke scale (NIHSS) score of 6 or more who could be treated within 5 h of onset were randomly assigned (1:1), via a central web-based randomisation process with a biased coin minimisation approach, to receive 25% albumin (2 g [8 mL] per kg; maximum dose 750 mL) or the equivalent volume of isotonic saline. All study personnel and participants were masked to the identity of the study drug. The primary endpoint was favourable outcome, defined as either a modified Rankin scale score of 0 or 1, or an NIHSS score of 0 or 1, or both, at 90 days. Analysis was by intention to treat. Thrombolytic therapies were permitted. This trial is registered with ClinicalTrials.gov, number NCT00235495., Findings: 422 participants were randomly assigned to receive albumin and 419 to receive saline. On Sept 12, 2012, the trial was stopped early for futility (n=841). The primary outcome did not differ between patients in the albumin group and those in the saline group (186 [44%] vs 185 [44%]; risk ratio 0·96, 95% CI 0·84-1·10, adjusted for baseline NIHSS score and thrombolysis stratum). Mild-to-moderate pulmonary oedema was more common in patients given albumin than in those given saline (54 [13%] of 412 vs 5 [1%] of 412 patients); symptomatic intracranial haemorrhage within 24 h was also more common in patients in the albumin group than in the placebo group (17 [4%] of 415 vs 7 [2%] of 414 patients). Although the rate of favourable outcome in patients given albumin remained consistent at 44-45% over the course of the trial, the cumulative rate of favourable outcome in patients given saline rose steadily from 31% to 44%., Interpretation: Our findings show no clinical benefit of 25% albumin in patients with ischaemic stroke; however, they should not discourage further efforts to identify effective strategies to protect the ischaemic brain, especially because of preclinical literature showing convincing proof-of-principle for the possibility of this outcome., Funding: National Institute of Neurological Disorders and Stroke, US National Institutes of Health; and Baxter Healthcare Corporation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Abundant preclinical studies have identified multiple mechanisms of ischemic brain injury and have provided proof of principle that strategies designed to counter these mechanisms can protect the ischemic brain. This review article emphasizes the translation of these strategies from the laboratory to clinical trials. It is a disappointing fact that many agents have been brought to clinical trial despite only modest or inconsistent preclinical evidence of neuroprotective efficacy. Preclinical investigations require rigorous attention to a variety of variables that may influence outcome. The widely touted STAIR criteria represent constructive guidelines for preclinical testing but, as experience has shown, do not increase the likelihood of translational success. Of the approximately 160 clinical trials of neuroprotection for ischemic stroke conducted as of late 2007, only approximately 40 represent larger-phase completed trials, and fully one half of the latter utilized a window to treatment of >6 hours, despite strong preclinical evidence that this delay exceeds the likely therapeutic window of efficacy in acute stroke. Other shortcomings of these trials include the use of agents lacking robust, consistent preclinical efficacy; inability to achieve adequate dosing in humans; and suboptimal clinical and statistical design features. Taken together, these factors identify areas of needed improvement for future trials.

Neuroprotection for ischemic stroke refers to strategies, applied singly or in combination, that antagonize the injurious biochemical and molecular events that eventuate in irreversible ischemic injury. There has been a recent explosion of interest in this field, with over 1000 experimental papers and over 400 clinical articles appearing within the past 6 years. These studies, in turn, are the outgrowth of three decades of investigative work to define the multiple mechanisms and mediators of ischemic brain injury, which constitute potential targets of neuroprotection. Rigorously conducted experimental studies in animal models of brain ischemia provide incontrovertible proof-of-principle that high-grade protection of the ischemic brain is an achievable goal. Nonetheless, many agents have been brought to clinical trial without a sufficiently compelling evidence-based pre-clinical foundation. At this writing, around 160 clinical trials of neuroprotection for ischemic stroke have been initiated. Of the approximately 120 completed trials, two-thirds were smaller early-phase safety-feasibility studies. The remaining one-third were typically larger (>200 subjects) phase II or III trials, but, disappointingly, only fewer than one-half of these administered neuroprotective therapy within the 4-6h therapeutic window within which efficacious neuroprotection is considered to be achievable. This fact alone helps to account for the abundance of "failed" trials. This review presents a close survey of the most extensively evaluated neuroprotective agents and classes and considers both the strengths and weakness of the pre-clinical evidence as well as the results and shortcomings of the clinical trials themselves. Among the agent-classes considered are calcium channel blockers; glutamate antagonists; GABA agonists; antioxidants/radical scavengers; phospholipid precursor; nitric oxide signal-transduction down-regulator; leukocyte inhibitors; hemodilution; and a miscellany of other agents. Among promising ongoing efforts, therapeutic hypothermia, high-dose human albumin therapy, and hyperacute magnesium therapy are considered in detail. The potential of combination therapies is highlighted. Issues of clinical-trial funding, the need for improved translational strategies and clinical-trial design, and "thinking outside the box" are emphasized.

Background and Purpose: Results of our recent pilot clinical trial suggest that the efficacy of thrombolytic therapy in acute ischemic stroke may be enhanced by the coadministration of high-dose albumin. Here, we explored the microvascular hemodynamic effects of this combined therapy in a laboratory model of cortical arteriolar thrombosis., Methods: We studied the cortical microcirculation of physiologically monitored rats in vivo by two-photon laser-scanning microscopy after plasma-labeling with fluorescein-dextran. We induced focal thrombosis in 30- to 50-microm cortical arterioles by laser irradiation and measured arteriolar flow velocity by repeated line-scanning. At 30 minutes post-thrombosis, we treated animals with the thrombolytic agent, reteplase, which was coadministered with either human albumin, 2 g/kg, or with saline control., Results: Baseline arteriolar flow velocity averaged 3.8+/-0.7 mm/s, was immediately reduced by thrombosis to 22% to 25% of control values, and remained unchanged before treatment. Subthrombolytic doses of reteplase combined with saline led to a median increase in flow velocity to 37% of control distal to the thrombus (P=nonsignificant versus pretreatment). By contrast, reteplase combined with albumin therapy resulted in a prompt, highly significant increase of median flow velocity to 58% of control levels (P=0.013 versus reteplase+saline), which remained significantly higher than the reteplase+saline group at multiple time-points over the subsequent hour., Conclusions: The beneficial effect of subthrombolytic doses of reteplase on microvascular hemodynamics distal to a cortical arteriolar thrombosis is markedly enhanced by the coadministration of high-dose albumin therapy; these results have important clinical implications for the management of patients with acute ischemic stroke.

Stilbazulenyl nitrone (STAZN) is a potent antioxidant that, in a rat model of transient focal cerebral ischemia, confers significant enduring functional and morphological neuroprotection. This study investigated the influence of dose and time of administration on the neuroprotective effects of STAZN in the intraluminal suture model of middle cerebral artery occlusion (MCAo). Dose response: At 2 and 4 h after the onset of MCAo, animals received intravenously either STAZN (low dose=0.07 mg/kg, n=8; medium dose=0.7 mg/kg, n=9; high dose=3.5 mg/kg, n=9), an equivalent volume of vehicle (30% Solutol HS15 and 70% isotonic saline, 0.37 ml/kg, n=5) or saline (0.37 ml/kg, n=5). Only the medium dose improved scores (p<0.05) on a standardized neurobehavioral test at 1, 2 and 3 days after MCAo. Only the medium dose reduced the total infarction (51%, p=0.014) compared to controls. These results indicate that STAZN exhibits maximal neuroprotection at the 0.7 mg/kg dose. Therapeutic window: STAZN (0.6 mg/kg) dissolved in dimethylsulfoxide was given intra-peritoneally at 2 and 4 h (n=11), 3 and 5 h (n=10), 4 and 6 h (n=10) or 5 and 7 h (n=7) after the onset of MCAo. Additional doses were given at 24 and 48 h. Vehicle (dimethylsulfoxide, 2.0 ml/kg, n=6) was administered at 3, 5, 24 and 48 h. STAZN treatment initiated at 2 or 3 h after the onset of MCAo improved neurological scores (p<0.001) and reduced total infarction (42.2%, p<0.05) compared to controls.

The SAINT II Trial, a large randomized multicenter clinical trial of the putative neuroprotectant, NXY-059, failed to demonstrate a treatment benefit in acute ischemic stroke. The further development of this agent was suspended. The implications of this outcome are considered from several perspectives, including: (1) the marginally positive antecedent trial, SAINT I, and the critical commentary stimulated by it, which called attention to its interpretively challenging primary outcome measure--a shift in the full-scale modified Rankin scale score--and to other statistical shortcomings; (2) the cogency of the STAIR recommendations, to which the development of NXY-059 closely adhered; and (3) the inherent physiochemical shortcomings of NXY-059 as a neuroprotective agent--its polar, nonlipophilic nature, poor blood-brain barrier penetrability, nonphysiological oxidation potential, and low potency. Caution is urged, however, regarding the unwarranted adoption of a nihilistic view toward neuroprotection on the part of the stroke community in view of the abundant preclinical evidence demonstrating proof-of-principle of the feasibility of neuroprotection, as well as the multiplicity of biochemical and molecular neuroprotective targets. The author offers the personal example of a translational journey in which a promising neuroprotectant agent targeting multiple injury mechanisms, high-dose albumin therapy, has proceeded successfully from preclinical studies that established efficacy through a pilot clinical trial that demonstrated safety and offered strong suggestions of clinical efficacy, leading to a large multicenter clinical trial currently in progress.

Background and Purpose: High-dose human albumin (ALB) is robustly neuroprotective in rodent stroke models. A phase I dose-escalation study was conducted to assess the safety of ALB therapy in ischemic stroke. We analyzed the data for preliminary evidence of treatment efficacy., Methods: Eighty-two subjects with acute ischemic stroke (NIH Stroke Scale [NIHSS] of 6 or above) received 25% ALB beginning within 16 hours of stroke onset. Six successive ALB dose tiers were assessed (range, 0.34 to 2.05 g/kg). Forty-two patients also received standard-of-care intravenous tissue plasminogen activator (tPA). Efficacy outcomes were determined at 3 months. We compared the highest three, putatively therapeutic ALB dose tiers (1.37 to 2.05 g/kg) with the lowest three, presumed subtherapeutic doses (0.34 to 1.03 g/kg) and with historical cohort data derived from the NINDS rt-PA Stroke Study., Results: After adjusting for the tPA effect, the probability of good outcome (defined as modified Rankin Scale 0 to 1 or NIH Stroke Scale 0 to 1 at 3 months) at the highest three ALB doses was 81% greater than in the lower dose-tiers (relative risk [RR], 1.81; 95% confidence interval [CI], 1.11 to 2.94) and was 95% greater than in the comparable NINDS rt-PA Stroke Study cohort (RR, 1.95; 95% CI, 1.47 to 2.57). The tPA-treated subjects who received higher-dose ALB were three times more likely to achieve a good outcome than subjects receiving lower-dose ALB, suggesting a positive synergistic effect between ALB and tPA., Conclusions: Our data suggest that high-dose ALB therapy may be neuroprotective after ischemic stroke. These results have led to a multicenter, randomized, placebo-controlled efficacy trial of ALB in acute ischemic stroke-the ALIAS Phase III Trial.

Background and Purpose: In preclinical stroke models, high-dose human albumin confers robust neuroprotection. We investigated the safety and tolerability of this therapy in patients with acute ischemic stroke., Methods: The ALIAS (Albumin in Acute Stroke) Pilot Clinical Trial used a multiple-tier, open-label, dose-escalation design. Subjects with acute ischemic stroke (NIH Stroke Scale [NIHSS] of 6 or above) received a 2-hour infusion of 25% human albumin (ALB) beginning within 16 hours of stroke onset. Six successive ALB dose tiers were assessed ranging from 0.34 to 2.05 g/kg. Neurologic and cardiac function was sequentially monitored. At 3 months, the NIHSS, modified Rankin Scale, and Barthel Index were measured., Results: Eighty-two subjects (mean age, 65 years) received ALB at 7.8+/-3.4 hours after stroke onset (mean+/-standard deviation). Forty-two patients also received standard-of-care intravenous tissue plasminogen activator (tPA). Vital signs were unaltered by ALB treatment. Dose-related increases in plasma albumin and mild hemodilution were maximal at 4 to 12 hours. Age-related plasma brain natriuretic peptide levels increased at 24 hours after ALB but did not predict cardiac adverse events. The sole ALB-related adverse event was mild or moderate pulmonary edema in 13.4% of subjects, which was readily managed with diuretics. In the tPA-treated subgroup, symptomatic intracranial hemorrhage occurred in only one of 42 subjects., Conclusions: Twenty-five percent human albumin in doses ranging up to 2.05 g/kg was tolerated by patients with acute ischemic stroke without major dose-limiting complications. tPA therapy did not affect the safety profile of ALB. The companion article presents neurologic outcome data and efficacy analysis in these subjects.

The effects of oligemia (moderate ischemia) on the brain need to be explored because of the potential role of subtle microvascular changes in vascular cognitive impairment and dementia. Chronic bilateral common carotid artery occlusion (BCCAO) in adult rats has been used to study effects of oligemia (hypoperfusion) using neuropathological and neurochemical analysis as well as behavioral tests. In this study, BCCAO was induced for 1 week, or 2, 4, and 6 months. Sensitive immunohistochemistry with marker proteins was used to study reactions of astrocytes (GFAP, nestin), and lectin binding to study microglial cells during BCCAO. Overt neuronal loss was visualized with NeuN antibodies. Astrocytes reacted to changes in the optic tract at all time points, and strong glial reactions also occurred in the target areas of retinal fibers, indicating damage to the retina and optic nerve. Astrocytes indicated a change in the corpus callosum from early to late time points. Diffuse increases in GFAP labeling occurred in parts of the neocortex after 1 week of BCCAO, in the absence of focal changes of neuronal marker proteins. No significant differences emerged in the cortex at longer time points. Nestin labeling was elevated in the optic tract. Reactions of microglia cells were seen in the cortex after 1 week. Measurements of the basilar artery indicated a considerable hypertrophy, indicative of macrovascular compensation in the chronic occlusion model. These results indicate that chronic BCCAO and, by inference, oligemia have a transient effect on the neocortex and a long-lasting effect on white matter structures.

Protein kinase C (PKC) isozymes have been known to mediate a variety of complex and diverse cellular functions. deltaPKC has been implicated in mediating apoptosis. Using two models of cerebral ischemia, cardiac arrest in rats and oxygen glucose deprivation (OGD) in organotypic hippocampal slices, we tested whether an ischemic insult promoted deltaPKC cleavage during the reperfusion and whether the upstream pathway involved release of cytochrome c and caspase 3 cleavage. We showed that cardiac arrest/OGD significantly enhanced deltaPKC translocation and increased its cleavage at 3 h of reperfusion. Since deltaPKC is one of the substrates for caspase 3, we next determined caspase 3 activation after cardiac arrest and OGD. The maximum decrease in levels of procaspase 3 was observed at 3 h of reperfusion after cardiac arrest and OGD. We also determined cytochrome c release, since it is upstream of caspase 3 activation. Cytochrome c in cytosol increased at 1 h of reperfusion after cardiac arrest/OGD. Inhibition of either deltaPKC/caspase 3 during OGD and early reperfusion resulted in neuroprotection in CA1 region of hippocampus. Our results support the deleterious role of deltaPKC in reperfusion injury. We propose that early cytochrome c release and caspase 3 activation promote deltaPKC translocation/cleavage.

Stilbazulenyl nitrone (STAZN) is a potent lipophilic second-generation azulenyl nitrone antioxidant, which is highly neuroprotective in rodent models of cerebral ischemia and trauma. This study was conducted to establish whether the neuroprotection induced by STAZN persists with chronic survival and to characterize STAZN's pharmacokinetics. Physiologically regulated rats received a 2-h middle cerebral artery occlusion by intraluminal suture and were treated with either STAZN [four 0.6 mg/kg doses i.p. administered at 2 (i.e., onset of recirculation), 4, 24, and 48 h; n = 16] or dimethyl sulfoxide vehicle (n = 11). They received sequential neurobehavioral examinations followed by quantitative neuropathology at 30 days. STAZN improved neurological deficits compared with vehicle controls, beginning within <2 h of the first dose and persisting throughout a 30-day survival. Large cystic necrotic infarcts were common in vehicle-treated rats but infrequent in STAZN-treated rats, and noninfarcted forebrain tissue was increased on average by 15%. In normal rats administered 5 mg/kg STAZN i.v. in Solutol HS 15/ethanol/saline vehicle, STAZN blood levels exhibited a biexponential decline, with an initial half-life of 28 min and a subsequent slow decay with half-life of approximately 7 h. STAZN tissue levels at 2 to 3 h were, on average, 2.5% of blood levels in forebrain, 56% in myocardium, and 41% in kidney. STAZN was concentrated in liver with initial concentrations averaging 5.2-fold above blood levels and a subsequent linear decline of 40% between 24 and 72 h. These results establish that STAZN confers enduring ischemic neuroprotection, has a long circulating half-life, and penetrates well into brain and other organs-characteristics favoring its potential therapeutic utility.

In recent experimental studies, a selective antagonist of endothelin ET(A) receptors, SB 234551, improved neurological and histological outcome in both head trauma and transient focal cerebral ischemia. The present study was conducted to ascertain the degree to which hemodynamic alterations are responsible for this therapeutic effect in a model of permanent middle cerebral artery occlusion (MCAo) in rats. Anesthetized Sprague-Dawley rats were subjected to permanent MCAo by insertion of an intraluminal nylon suture coated with poly-L-lysine. The agent (SB 234551, 30 microg/kg/min = 1.8 mg/kg/h) or vehicle (PBS; 0.6 ml/h) was administered by i.v. infusion beginning 15 min after onset of MCAo and lasting for 23.75 h. Autoradiographic measurement of local cerebral blood flow (lCBF) was performed at 24 h. Physiological data were similar among groups. SB 234551 augmented perfusion by 1.7- to 1.8-fold in both the ischemic hemisphere and in the contralateral (non-ischemic) hemisphere when compared to vehicle-treated ischemic animals. In the ischemic hemisphere, the brain regions significantly benefited were those lying outside the zone of most dense ischemia (i.e., paramedian cortex and thalamus), while in the non-ischemic hemisphere all regions measured showed significant lCBF augmentation. This study demonstrates that SB 234551 therapy results in significant improvement of local cerebral perfusion in the ischemic as well as in the non-ischemic hemispheres after permanent MCAo.

Background and Purpose: Darbepoetin alfa is a novel erythropoiesis-stimulating protein developed for treating anemia. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental cerebral ischemia. In this study, we determined whether darbepoetin alfa would protect in a rat model of transient focal cerebral ischemia., Methods: Rats received 2-hour middle cerebral artery suture-occlusion. The drug (darbepoetin alfa, 10 microg/kg) or vehicle was administered intraperitoneally 2 hours after onset of middle cerebral artery occlusion. Animals were allowed to survive for 3 or 14 days. Behavioral tests were performed sequentially. Infarct volumes and brain swelling were determined., Results: Darbepoetin alfa-treated rats showed improved neuroscores relative to vehicle-treated animals beginning within 1 hour of treatment and persisting throughout the 14-day survival period. Darbepoetin alfa significantly reduced corrected total (cortical + subcortical) infarct volume (56.3+/-20.6 and 110.8+/-6.8 mm3, respectively) and total infarct areas at multiple levels compared with vehicle in the 14-day survival group. Brain swelling was not affected by treatment., Conclusions: Darbepoetin alfa confers behavioral and histological neuroprotection after focal ischemia in rats.

Immunochemical staining techniques are commonly used to assess neuronal, astrocytic and microglial alterations in experimental neuroscience research, and in particular, are applied to tissues from animals subjected to ischemic stroke. Immunoreactivity of brain sections can be measured from digitized immunohistology slides so that quantitative assessment can be carried out by computer-assisted analysis. Conventional methods of analyzing immunohistology are based on image classification techniques applied to a specific anatomic location at high magnification. Such micro-scale localized image analysis limits one for further correlative studies with other imaging modalities on whole brain sections, which are of particular interest in experimental stroke research. This report presents a semi-automated image analysis method that performs convolution-based image classification on micro-scale images, extracts numerical data representing positive immunoreactivity from the processed micro-scale images and creates a corresponding quantitative macro-scale image. The present method utilizes several image-processing techniques to cope with variances in intensity distribution, as well as artifacts caused by light scattering or heterogeneity of antigen expression, which are commonly encountered in immunohistology. Micro-scale images are composed by a tiling function in a mosaic manner. Image classification is accomplished by the K-means clustering method at the relatively low-magnification micro-scale level in order to increase computation efficiency. The quantitative macro-scale image is suitable for correlative analysis with other imaging modalities. This method was applied to different immunostaining antibodies, such as endothelial barrier antigen (EBA), lectin, and glial fibrillary acidic protein (GFAP), on histology slides from animals subjected to middle cerebral artery occlusion by the intraluminal suture method. Reliability tests show that the results obtained from immunostained images at high magnification and relatively low magnification are virtually the same.

Background and Purpose: High-dose human albumin therapy is strongly neuroprotective in models of brain ischemia and trauma and is currently being studied in a pilot-phase clinical stroke trial. Among its actions in ischemia, albumin induces the systemic mobilization of n-3 polyunsaturated fatty acids and may help to replenish polyunsaturated fatty acids lost from neural membranes., Methods: We complexed 25% human albumin to docosahexaenoic acid (DHA; 22:6n-3) and compared its neuroprotective efficacy with that of native albumin in rats with 2-hour focal ischemia produced by intraluminal suture-occlusion of the middle cerebral artery., Results: In animals treated with DHA-albumin, 0.63 g/kg, the improvement in neurobehavioral scores at 72 hours significantly exceeded that of other treatment groups, and the extent of histological protection (86% reduction in cortical infarction) was highly significant and tended to surpass the degree of cortical protection produced by native albumin at 1.25 g/kg (65%). DHA-albumin 0.63 g/kg, but not native albumin, also significantly reduced subcortical infarction and markedly diminished brain swelling. Lipidomic analysis of DHA-albumin-treated postischemic brains revealed a large accumulation of the neuroprotective DHA metabolite, 10,17S-docosatriene, in the ipsilateral hemisphere., Conclusions: The high-grade neuroprotection afforded by the DHA-albumin complex at relatively low albumin doses is clinically advantageous in that it might reduce the likelihood of acute intravascular volume overload and congestive heart failure sometimes induced when patients with compromised cardiovascular function are treated with high-dose albumin.

The direction of the chemical reaction of ATP synthetase is reversible. The present study was designed to determine whether mitochondria produce or consume ATP during ischemia. For this purpose, changes in mitochondrial membrane potential were measured in vivo at the site of a direct current (DC) electrode using a potentiometric dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and a rat model of focal ischemia. Two microL of dye (control group) or dye with oligomycin, an ATP synthetase inhibitor (oligomycin group), was injected into the parietotemporal cortex through the DC electrode. With the initiation of ischemia, a decrease in mitochondrial potential was observed within 20 seconds in the oligomycin group (earlier than the onset of DC deflection, P = 0.02). In contrast, in the control group, mitochondrial potential was maintained at 91 +/- 5% of the preischemia level for 118 +/- 38 seconds before showing full depolarization simultaneously with DC deflection. During the period of ischemia, the mitochondrial potential was higher in the control group (66 +/- 9%) than in the oligomycin group (46 +/- 8%, P = 0.0002), whereas DC potential was lower in the control group (-18 +/- 3) than in the oligomycin group (-15 +/- 2 mV, P = 0.04). These observations suggest that mitochondria consume ATP during ischemia by reversing ATP synthetase activity, which compromises cellular membrane potential by consuming ATP.

The 19 kD interacting protein 3, Nip3/BNIP3, is a pro-apoptotic member of the Bcl-2 family induced during hypoxia via the hypoxia-inducible factor (HIF) 1. BNIP3 has been linked to both apoptotic and necrotic cell death involving mitochondrial permeability transition. Since apoptotic and necrotic mechanisms may occur in brain ischemia, immunohistochemical changes of BNIP3 were studied at 1, 2, 3 and 7 days after transient global brain ischemia (12.5 min) in ventilated normothermic rats. In control brains, BNIP3-like immunoreactivity was moderately strong in neuronal processes or cytoplasm and absent in the nucleus. In the ischemia-vulnerable CA1 neurons, BNIP3-positive granules were seen in the nucleus at 1 and 2 days, and these neurons were damaged at 3 and 7 days. The resistant CA3 neurons showed nuclear BNIP3 labeling by 1 day and then returned to the normal state. BNIP3-positive granules did not overlap with the nucleolus. Constitutively expressed BNIP3 may participate in apoptotic and necrotic processes after brain ischemia. Nuclear location of BNIP3 after brain ischemia indicates a novel role for the regulation of cell survival in neurons or a general disturbance of the nuclear envelope.

Focal brain ischemia is followed by changes in gene expression as reflected by altered mRNA levels. DNA microarray analysis can be used to survey thousands of genes for differential expression triggered by ischemic metabolic stress. In this study, Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) using an intravascular poly-L-lysine-coated filament, and brains were removed after 3 h of recirculation for mRNA isolation. A differential measurement of mRNAs from post-ischemic and sham control animals was performed using the Mouse UniGene 1 microarray. Established values for differential expression were used (> or =1.7 or < or =-1.7 fold), and hits (n=2-3 arrays) divided into known 'ischemia-hypoxia response' genes and 'newly connected' annotated genes. n=28 ischemia-hypoxia response genes were up-regulated and n=6 were down-regulated. Regulated genes comprised immediate early genes, heat shock proteins, anti-oxidative enzymes, trophic factors, and genes involved in RNA metabolism, inflammation and cell signaling. Based on the ability of the microarray to replicate known changes in gene expression, n=35 newly connected genes were found up-regulated and n=41 down-regulated. DNA microarray analysis allows one to develop novel working hypotheses for responses to brain ischemia based on the regulation of annotated genes.

Previous studies have demonstrated that sublethal ischemic insults protect from subsequent ischemia in the intact brain. There are two windows for the induction of tolerance by ischemic preconditioning (IPC). One occurs within 1 h following IPC, and the other one develops from 1 to 3 days after IPC. The goal of this study was to determine whether IPC neuroprotection may be mediated by expression of known neuroprotective genes and to characterize the temporal and spatial expression patterns of these genes. IPC was produced by bilateral carotid artery occlusions and hypotension (50 mmHg) for 2 min. After various survival times, the expression of MAP-2, brain-derived neurotrophic factor (BDNF), c-jun, c-fos, nerve growth factor (NGF) and HSP70 was assessed by in situ hybridization of coronal brain sections with 35S labeled probes. BDNF, NGF, and c-jun were significantly upregulated in the hippocampus. c-fos was detected in the hippocampus, cortex and striatum. HSP70 mRNA was induced in the cortex, hippocampus, striatum, and thalamus. MAP-2 showed no change in expression, confirming previous studies that no cell death occurs following IPC. The increase in expression of these stress-related, neurotrophic and immediate early genes in response to a mild preconditioning insult may help mediate the protection of vulnerable neurons to subsequent lethal ischemic insults., (Copyright 2002 Elsevier Science B.V.)