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.

Introduction: Failures to translate pre-clinical results have been discouraging. We have contended that stroke is too heterogeneous with respect to factors influencing outcome to expect small studies to be balanced. It is not only difficult to control for biological and methodological variability but efforts to improve homogeneity, such as minimizing physiological variability, may render results less applicable to humans. Here, we report a predictive outcome model in experimental stroke which incorporates baseline variability and provides statistical thresholds a treatment must exceed to be efficacious in a broad population. Methods: We generated a mathematical model to predict outcome using transient MCA occlusion in 23 unfasted rats. To create baseline variability, we varied occlusion times from 90-120 min, altered baseline glucose with streptozotocin, and assessed neurological outcome 3 days later with a modified Bederson Score (BS; 0-6 functional measure, 7 death). Statistical surfaces in 3 dimensions were generated using Jacobian matrices flanking the model to provide a screening threshold (1 SD) for comparing new therapies against this model. Results: We successfully generated an outcome model from occlusion time, glucose and BS (Fig; R 2 =.49, p=.0003; middle surface is the model surrounded by ±SD surfaces). Outcome was sensitive to change in glucose and time, suggesting small imbalances in these factors between groups may influence outcome, and hence the perceived efficacy of a new therapeutic intervention. At normoglycemia and 90 mins, the lower surface overlapped with no deficit, indicating it would be difficult to reliably demonstrate benefit under those conditions. Conclusions: These results indicate it is feasible to incorporate biological variability to generate more clinically relevant conditions. The method will be tested with other stroke models and modifiers towards a generalized model to screen for therapies worthy of further study.

Introduction: While oxidative stress mediates many of the pathologies in stroke, antioxidant therapies have been unsuccessful. Current antioxidants are limited either by a narrow range of radicals quenched or by low capacity. Some can yield toxic intermediates. We developed a new class of antioxidant, highly modified carbon nanoparticles called PEGylated hydrophilic carbon clusters (PEG-HCCs). These 40 nM particles quench superoxide by a high capacity novel catalytic mechanism (PNAS 2015: 2343-8), are effective against - OH and recouple nitric oxide synthase. Here we report the effect of PEG-HCCs administered during reperfusion after transient MCA suture occlusion in the rat under varying glucose levels. Hyperglycemia occurs in 1/3 of stroke patients and worsens outcome in part by accelerating release of radicals during reperfusion. Methods: Variation in glucose was induced by streptozocin IP 2 days prior to MCAO. At 90 mins, 23 rats were injected IV with PBS or PEG-HCCs (4 mg/kg) and the suture removed. A second dose was given 2 hours later (dosing based on half-life and in-vitro efficacy). At 3 days, infarct size corrected for edema and neurological function (Bederson Score, BS) was obtained. An outcome curve was developed using a range of baseline glucose in controls and the effect of PEG-HCCs compared at rat’s baseline glucose values. Results: We found a strong sigmoidal relationship between infarct size and glucose level in control rats (Fig. R 2 =.93; p=.0001; blue curve: control arm ± 95% confidence intervals). All PEG-HCC treated rats showed infarct size below the confidence interval (red stars; p<.003 vs control). Preliminary results for 120 min occlusion showed improved BS compared to control (3.5±2.4 vs 5.4±1.8; p=.047). Conclusions: This novel, broadly active, high capacity antioxidant was able to improve outcome in a severe test of pre-clinical stroke at clinically relevant times. Studies are ongoing to assess the treatment window and longer term outcomes.

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.

BACKGROUND: While intravenous thrombolysis (IVT) remains the only established therapy for ischemic stroke including beyond 3 hours, there is considerable room for improvement in IVT outcomes. Non-randomized series suggested better outcomes for add-on therapies; however randomized clinical trials (RCTs) were often equivocal. One reason for these discrepancies is that imbalances in important baseline factors between treatment and control arms affect all but the largest trials and can produce misleading results. We developed techniques to identify potentially efficacious therapies by generating a pooled outcome model. Here we tested RCTs attempting to improve upon IVT against this model at each treatment arm’s baseline NIHSS and age. METHODS: We generated outcome models (mortality, mRS 0-1, mRS 0-2) pooled from the thrombolytic arms of all IV tPA RCTs with the novel feature of multi-dimensional statistical intervals. A function was iteratively fitted and individual results of IV, add-on and endovascular arms tested against this model to determine whether outcomes surpassed the + p< .05 surface (see mRS0-2 Fig.). RESULTS: Functions derived from 19 IVT RCT thrombolytic arms representing 2748 subjects were generated (Mortality: r 2 =0.51; mRS0-1: r 2 =0.79; 0-2: r 2 =0.85). No treatment increased mortality. Three hour IVT/ultrasound with (TUCSON) or without (CLOTBUST) microspheres, the neuroprotectant Cerebrolysin and IA tPA alone (SYNTHESIS) had better functional outcomes, as did CTP-guided 6 hr. IV tenecteplase. Notably, 4.5 hour SYNTHESIS-EXPANSION (IA tPA) and IMS III (IV tPA + endovascular) did not. No extended window device showed benefit. CONCLUSION: We found that several treatments show the potential to improve outcome compared to our pooled IVT model, including 3 hour ultrasound, IA tPA and 6 hr. tenecteplase. Consistent with the cardiac experience, there remains little evidence that the 3 hour window can be extended other than by an IV thrombolytic.

Background: Proven options to treat ischemic stroke patients ineligible for IV t-PA are limited. Our focus is on treatments to spare the penumbral microcirculation. Evidence indicates that Glycoprotein (GP) IIb/IIIa inhibitors when given with heparin, as used in coronary intervention, increases macro- and distal micro-vascular patency in the heart and animal stroke models. We hypothesized that the cardiac dose of IV abciximab (ABX) used in the halted AbESTT trial was too high leading to hemorrhage, while exclusion of heparin reduced efficacy. We previously showed that a 30% lower ABX dose achieved target GP IIb/IIIa platelet inhibition of 92% in stroke patients and hypothesized a lower dose when given with short term heparin would be safe and effective. Methods: After consent, patients with non-lacunar stroke ineligible for t-PA were offered low dose IV ABX (bolus:0.2 mg/kg & infusion:0.05 ug/kg/hr) along with IV heparin (36 hrs) for ant. circulation strokes within 6 hrs and post. within 12 hrs of onset. In place of a control arm, we adapted our pPAIRS matching strategy to obtain best matches from non-lacunar placebo patients in the NINDS dataset at the 2 hr post-randomization NIHSS. Age, glucose and gender were other factors that were matched. Results: Forty five patients have been treated. t-PA ineligibility was mostly due to time of onset, recent surgery or anticoagulation. Mean time to treatment was 5.2 h (Ant:4.1h; Post:9.3h). Matching with NINDS placebo subjects was excellent: median NIHSS: ABX: 18 vs NINDS: 17; age: ABX: 66.6 ± 13 vs NINDS: 66.3 ± 11; and glucose: ABX:148 ± 58 vs NINDS:144 ± 52; all p>0.05. There was 1 symptomatic hemorrhage in ABX patients (2.2%) vs none in NINDS; p=1.0. Ninety day mRS 0-1 and 0-2 in the ABX arm (42.2% and 55.6%) were higher than in NINDS matched placebo subjects (15.6% and 31.1%); all p<0.05. Mortality was 24% after ABX and 40% in NINDS; p>.05. Conclusion: Low dose ABX appeared safe and potentially effective when compared with NINDS placebo subjects assessed 2 hrs after their initial presentation so as to provide a comparable baseline. Since low dose ABX achieves sufficient platelet inhibition and the combination with short term heparin appeared well tolerated, we suggest these findings deserve confirmation in a prospective RCT.

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.

This study was conducted to identify plasma membrane Ca2+-transporting ATPases present in rat kidney. Characterization of the cDNAs of the plasma membrane Ca2+-ATPases revealed a family of proteins with regions of highly conserved amino acid sequence. To examine the extent of the diversity of rat renal plasma membrane Ca2+-ATPases, we used the polymerase chain reaction to detect additional gene products in rat kidney mRNA that shared these conserved regions. Sequences corresponding to three previously known rat plasma membrane Ca2+-ATPases were obtained. In addition, we found sequence corresponding to a new putative plasma membrane Ca2+-ATPase. Our results demonstrate that the rat kidney contains at least four different plasma membrane Ca2+-ATPases and the complexity of this multigene family is greater than previously thought.

The clearance of creatinine has been shown to overestimate GFR in patients with renal disease and in kidney transplant recipients. Alternate means for measuring GFR involve the use of constant infusion and, frequently, of radioisotopes. We report herein the measurement of GFR with the use of a radiocontrast agent (diatrizoate meglumine) injected subcutaneously with a small amount of epinephrine (0.05 ml of 1:1000). The concentrations of this marker of glomerular filtration in blood and in urine were measured with high-performance liquid chromatography. The results demonstrate that with this method of administration stable blood levels are observed for at least three hours. Furthermore, creatinine clearance overestimated GFR by 50% and by 68% in patients with renal disease and in renal transplant recipients, respectively. This method offers several advantages: 1) the use of commonly available and inexpensive materials; 2) obviates the need for the use of constant infusions; and 3) avoids the use of radioisotopes.

Previous studies have shown that captopril (CP) inhibits ADH-stimulated osmotic water permeability (Pf) in the toad bladder by potentiating endogenous bradykinin (BK). The present studies examine the effect of CP on ADH-stimulated Pf in isolated, perfused rabbit cortical collecting tubules (CCT). CP (10(-4) M) reversibly inhibited Pf, stimulated by maximal concentrations of ADH (10 microU/ml). Pretreatment of CCT's with 5 microM indomethacin, however, abolished the effect of CP. Inhibition of BK production by the kallikrein inhibitors, aprotinin and benzamidine, failed to enhance Pf stimulated by submaximal concentrations of ADH (2.5 microU/ml). Since ADH exerts its effects by activation of adenylyl cyclase (AC), further experiments were performed to identify the site at which CP inhibits this cascade. CP significantly inhibited forskolin (10(-4) M) stimulated Pf; however, it had no effect on cyclic AMP (10(-5) M) stimulated Pf, suggesting that the site of action is on the catalytic subunit or one of the GTP regulatory proteins of AC. To further localize the site of CP's action, CCT's were pre-incubated with pertussis toxin (0.5 microgram/ml) to inactivate the inhibitory, guanosine triphosphate (GTP) regulatory protein, Gi. In these tubules, CP failed to inhibit the action of ADH. We conclude that CP stimulates prostaglandin production which in turn activates Gi and inhibits AC activity. We further suggest that CP stimulates PG's directly, not via BK.