Rabbit Small Clot Embolic Stroke Research Articles

Abstract TP260: A Novel Neurogenic-Stem Cell Stimulating Curcumin Analog to Treat Acute Ischemic Stroke

While nerve cells rapidly die in stroke, and patients do initiate a neurogenic response to replace these cells, there has been no attempt to develop pleiotropic cytoprotective compounds to further promote neurogenesis in stroke patients, and there are no neurogenic or cytoprotective drugs approved by the FDA. In this study, we focused on the parent compound J-147, a curcumin derivative that is a 5-LOX inhibitor, and has potent neuroprotective and neurogenic activities. J-147 rescues HT22 cells from iodoacetic acid challenge (EC 50 50 nM), and oxytosis (EC 50 115 nM). J-147 is neuroprotective against glutamate excitotoxcity in E18 cortical neuron culture (EC 50 27 nM), and prevents cell death in an embryonic cortical cell trophic factor withdrawal assay (EC 50 25 nM). J-147 can rescue a clone of the hippocampal nerve cell line HT22 expressing TrkB, from serum starvation under conditions where the cells can be protected by BDNF (EC 50 50 nM). In mice, J-147 is neurogenic and can stimulate cell division in germinal regions of the brain (increase BrdU + DCX + labeled cells), enhances endogenous neurotrophic factors and energy metabolism. With this basis, we tested J-147 in 2 translational stroke models. First, using a rodent intraluminal suture model, in combination with a quantal analysis method to determine the effect of increasing ischemia duration (5-90 minutes) on behavioral deficits, IV J-147 treatment (10 mg/kg) administered 5 minutes following the end of occlusion (5-90 minutes), significantly (P=0.045) reduced stroke-induced behavioral deficits and increased the P 50 value to 36.37 ± 1.72 (n=19) minutes from P 50 = 20.1 ± 4.07 minutes (n=19) for the control group. J-147 also reduced infarct volume following IV administration (p<0.05). Second, using the standardized rabbit small clot embolic stroke model, IV J-147 treatment (10mg/kg) administered 1 hour post-embolization increased the P 50 value (behavior) to 1.88 ± 0.10 mg (n=20) compared to P 50 = 1.07 ± 0.07 (n=19) for control(p<0.05). Additional studies showed that J-147 was not mitogenic, or cytotoxic using the CeeTox assay (Raking 90μM) and had a good IV PK profile in rabbits/rats (t 1/2 5.89-8.56 hours). These data support the development of J-147 and neurogenic analogs as a novel class of drug to treat stroke.

Dose-finding study of phototherapy on stroke outcome in a rabbit model of ischemic stroke

GoalWhile transcranial laser therapy (TLT) has been shown to improve clinical outcome in a preclinical model of ischemic stroke, optimal timing and dosing has yet to be tested adequately. The purpose of this study was to assess clinical stroke outcome in the Rabbit Small Clot Embolic Model (RSCEM) with dose escalating TLT. MethodsWe utilized the rabbit small clot embolic stroke model (RSCEM) using dose-escalating regimens. Behavioral analysis was conducted at 24h post-embolization, allowing for the determination of the effective stroke dose (ES50) or clot amount (mg) that produces neurological deficits in 50% of a group of rabbits. Using the RSCEM, a treatment is considered beneficial if it significantly increases the ES50 compared with the control group. FindingsA significant behavioral benefit was seen at triple TLT of 111mW treatment of 2min at 2h post-embolization (6.47±1.06, n=17; p=0.03), compared with the previously used regimen (3.09±0.51, n=15). ConclusionTLT results in significant behavioral improvement when administered 2h post-embolization. Studies are warranted to evaluate this therapy in combination with thrombolysis.

A novel method to promote behavioral improvement and enhance mitochondrial function following an embolic stroke

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for stroke; tPA increases cerebral reperfusion, blood flow and improved behavior. Novel transcranial laser therapy (TLT) also enhances cerebral blood flow and activates mitochondrial function. Using the rabbit small clot embolic stroke model (RSCEM), we studied the effects of continuous wave TLT (7.5mW/cm2) alone or in combination with standardized intravenous (IV) tPA (3.3mg/kg) applied 1h post-embolization on 3 endpoints: 1) behavioral function measured 2 days [effective stroke dose (P50 in mg) producing neurological deficits in 50% of embolized rabbits], 2) intracerebral hemorrhage (ICH) rate, and 3) cortical adenosine-5′-triphosphate (ATP) content was measured 6h following embolization. TLT and tPA significantly (p<0.05) increased P50 values by 95% and 56% (p<0.05), respectively over control. TLT-tPA increased P50 by 136% over control (p<0.05). Embolization reduced cortical ATP content by 39%; decreases that were attenuated by either TLT or tPA treatment (p<0.05). TLT-tPA further enhanced cortical ATP levels 22% above that measured in naïve control. TLT and tPA both effectively and safely, without affecting ICH rate, improved behavioral outcome in embolized rabbits; and there was a trend (p>0.05) for the TLT-tPA combination to further increase P50. TLT and tPA both attenuated stroke-induced ATP deficits, and the combination of tPA and TLT produced an additive effect on ATP levels. This study demonstrates that the combination of TLT-tPA enhances ATP production, and suggests that tPA-induced reperfusion in combination with TLT neuroprotection therapy may optimally protect viable cells in the cortex measured using ATP levels as a marker.

Abstract WP282: DS-1040 an Inhibitor of the Activated Thrombin Activatable Fibrinolysis Inhibitor Improves Behavior in Embolized Rabbits

Introduction: DS-1040 is an inhibitor of the activated thrombin-activatable fibrinolysis inhibitor (TAFIa) being developed for the treatment of acute ischemic stroke (AIS). We evaluated the effect of DS-1040 infusiontreatment on rabbit TAFIa activity; plasma concentrations and we also determined if DS-1040 treatment affected behavioral outcome following embolization using the standard rabbit small clot embolic stroke model (RSCEM). Safety was assessed by measuring the effect of DS-1040 on intracerebral hemorrhage (ICH) and mortality. Methods: The inhibitory effect of DS-1040 on TAFIa was evaluated using hippuryl-Arg, a substrate of carboxypeptidase. The study was conducted randomized and blinded per RIGOR guidelines. Male New Zealand white rabbits (2-2.5kg) were embolized by injecting a suspension of small blood clots into the brain via an indwelling carotid catheter. Saline (control), tPA (3.3 mg/kg, i.v., 10% bolus/90% infusion for 0.5 h), high and low doses of DS-1040 (approx. 940 mg/per rabbit and 470 mg/per rabbit, respectively) were given 1 h following embolization as an i.v. bolus followed by a s.c. infusion for 47 h. Behavioral analysis was conducted 48 h following embolization, resulting in the calculation of an effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. ICH was also assessed in all treatment groups. Results: DS-1040 inhibited rabbit TAFIa with an IC50 value of 11.7 ng/mL. Using the dosing regimen described above, the mean plasma concentration of DS-1040 at 48 h was 1838 ng/mL (high dose) and 591 ng/mL (low dose). Rabbits treated with high and low doses of DS-1040 had P50 values of 1.61 ± 0.23 mg (p&lt;0.05) and 0.96 ± 0.32 mg, respectively; only the high dose significantly increased (p&lt;0.05) the group P50 by 206% compared to control group P50 (0.78 ± 0.39 mg). The tPA group had a P50 value of 2.29 ± 0.46 mg (p&lt;0.05). Neither the hemorrhage rate nor mortality rate were significantly affected by DS-1040 treatment. Conclusions: DS-1040 significantly improved behavioral function in small clot embolized rabbits, suggesting that DS-1040 should be further pursued as a treatment for stroke victims.

Abstract TP275: Effect of Transcranial Laser and Thrombolytic Combination Therapy on Clinical Function and ATP Production in Small Clot Embolized Rabbits

Background and Purpose: Transcranial laser therapy (TLT) and tissue plasminogen activator (tPA) improve behavior when administered following clot-induced strokes. tPA increases cerebral reperfusion and blood flow and TLT also enhances cerebral blood flow and activates mitochondrial function. We hypothesize that a combination therapy may be more effective than individual therapies and that efficacy may be mediated by enhancing mitochondrial function. Therefore, the goal of the present study is to evaluate the effect of combined TLT and tPA therapy on behavioral outcome and cellular ATP content using the rabbit small clot embolic stroke model (RSCEM). Methods: The study was conducted randomized and blinded. Male New Zealand white rabbits were embolized by injecting a suspension of small blood clots into brain via a carotid catheter. Using the RSCEM, we studied the effects of continuous wave TLT (7.5 mW/cm2, 2 minutes) alone or in combination with standardized intravenous (IV) tPA (3.3mg/kg) applied 1 hour post-embolization on 3 endpoints: 1) behavioral function measured 2 days [effective stroke dose (P50 in mg) producing neurological deficits in 50% of embolized rabbits], 2) intracerebral hemorrhage (ICH) rate, and 3) cortical adenosine-5'-triphosphate (ATP) content was measured 6 hours following embolization. Results: TLT and tPA significantly (p&lt;0.05) increased P50 values by 95% (p=0.026) and 56% (p=0.026) over control, respectively. TLT-tPA increased P50 by 136% over control (p=0.0038 compared to control). Treatment did not affect ICH incidence. Embolization reduced cortical ATP content by 39%; decreases that were attenuated by both TLT and tPA treatment (p&lt;0.05). TLT-tPA further enhanced cortical ATP levels 22% above that measured in naïve control. Conclusion: TLT and tPA both effectively and safely improve behavioral outcome in embolized rabbits and attenuate stroke-induced ATP deficits; TLT-tPA did not offer additional clinical benefit, but the TLT-tPA combination produced a synergistic effect on ATP levels. The study suggests that tPA-induced reperfusion in combination with TLT neuroprotection therapy may optimally protect viable cells in the cortex measured using ATP levels as a marker.

Synergistic Effect of AJW200, a von Willebrand Factor Neutralizing Antibody with Low Dose (0.9 mg/mg) Thrombolytic Therapy Following Embolic Stroke in Rabbits.

The von Willebrand factor (vWF) is an acute stroke response protein involved in platelet aggregation, adhesion, inflammation, and thrombus formation, responses that occur following an ischemic stroke. We hypothesize that administration of an anti-vWF antibody (anti-vWF-Ab) may be used as adjunctive therapy with tissue plasminogen activator (tPA) to promote behavioral improvement following an embolic stroke. In this proof-of-concept study, which used a blinded and randomized design, we studied delayed treatment with the anti-vWF-Ab, AJW200 (0.30 mg/kg), alone or in combination with a rabbit low-dose of tPA (0.9 mg/kg) using the rabbit small clot embolic stroke model (RSCEM) with behavioral function as the primary clinically relevant endpoint. To evaluate the quantitative relationship between clot burden in brain and clinical scores, so that an effective stroke dose (P50) could be calculated, logistic sigmoidal quantal analysis curves were constructed. A beneficial treatment significantly increases P50 compared to control. The effect of antibody administration, either alone or with low dose tPA was compared to a "positive control", a standard rabbit optimized dose of tPA (3.3 mg/kg), as a measure of the maximum improvement potential in the RSCEM. The anti-vWF-Ab, AJW200, or control IgG were administered IV 1 hour following embolization, and behavior was measured 48 hours later. AJW200 plus low-dose tPA significantly increased the P50 value by 74% (p<0.05, t=2.612) and 81% (p<0.05, t=2.519) compared to low dose tPA or IgG, respectively, but not the AJW200 group (p>0.05). AJW200 increased the P50 value by 28%, (p>0.05) compared to the control IgG-treated group. Standard dose tPA increased the P50 value by 154% (p<0.05). Statistically, the combination response for AJW200 plus low-dose tPA was not significantly different from standard dose tPA (p=0.26). This study shows that the concomitant administration of the anti-vWF-Ab AJW200 with low dose tPA is synergistic and results in significantly improved behavioral function following embolic stroke. We postulate that neutralization of vWF may suppress or attenuate one or more aspects of the acute phase stroke cascade response including suppression of inflammatory response and reduced leukocyte adhesion.

Incremental treatments with laser therapy augments good behavioral outcome in the rabbit small clot embolic stroke model

Transcranial near-infrared laser therapy (TLT) improves behavioral outcome in animal stroke models when applied as single treatment within the 24h of the stroke onset. It is unknown if the multiple TLT treatments have an added beneficial effect. We aim to determine whether multiple irradiations with TLT would have further improvement in behavioral outcomes in the rabbit small clot embolic stroke model (RSCEM). Using the RSCEM, two and three TLT treatments (7.5-20mW/cm(2)) were compared against single laser treatment alone (7.5-10.8mW/cm(2)). Two sham irradiation groups were added for the control curves. The double treatment group received TLT at 3 and 5h and the triple treatment group at 2, 3, and 4h after embolization. Behavioral analysis was conducted 24h after embolization using a dichotomized behavioral score. The determination of the effective clot amount (milligrams) that produces neurological deficits in 50% of the rabbits (P 50) was used to compare TLT treatments with the sham. The P 50 for double treatment was 5.47 ± 0.90, with n = 39; the corresponding P 50 value for a single treatment was 3.87 ± 0.73, with n = 38; and the corresponding control curve was 3.25 ± 0.4, n = 32. The P 50 for triple treatment was 5.91 ± 0.49, with n = 23; the corresponding P 50 value for a single treatment was 3.09 ± 0.59, with n = 15, and the corresponding control curve was 1.71 ± 0.26, with n = 17. The triple treatment had 91% improvement when compared with the single treatment and 245% improvement when compared with the sham. The present study suggests that the additional TLT treatments provide further behavioral improvement when given during the acute ischemic stroke phase.

Translational stroke research using a rabbit embolic stroke model: a correlative analysis hypothesis for novel therapy development.

Alteplase (tissue plasminogen activator, tPA) is currently the only FDA-approved treatment that can be given to acute ischemic stroke (AIS) patients if patients present within 3 h of an ischemic stroke. After 14 years of alteplase clinical research, evidence now suggests that the therapeutic treatment window can be expanded 4.5 h, but this is not formally approved by the FDA. Even though there remains a significant risk of intracerebral hemorrhage associated with alteplase administration, there is an increased chance of favorable outcome with tPA treatment. Over the last 30 years, the use of preclinical models has assisted with the search for new effective treatments for stroke, but there has been difficulty with the translation of efficacy from animals to humans. Current research focuses on the development of new and potentially useful thrombolytics, neuroprotective agents, and devices which are also being tested for efficacy in preclinical and clinical trials. One model in particular, the rabbit small clot embolic stroke model (RSCEM) which was developed to test tPA for efficacy, remains the only preclinical model used to gain FDA approval of a therapeutic for stroke. Correlative analyses from existing preclinical translational studies and clinical trials indicate that there is a therapeutic window ratio (ARR) of 2.43-3 between the RSCEM and AIS patients. In conclusion, the RSCEM can be used as an effective translational tool to gauge the clinical potential of new treatments.

Efficacy and safety profile of the carotenoid trans sodium crocetinate administered to rabbits following multiple infarct ischemic strokes: A combination therapy study with tissue plasminogen activator

Trans sodium crocetinate (TSC) is a synthetic small-molecule antioxidant that has the ability to enhance oxygen diffusion to hypoxic tissue. Because TSC is a promising drug candidate to treat acute ischemic stroke (AIS), we tested the hypothesis that TSC may be neuroprotective following cerebral ischemia using a rabbit small clot embolic stroke model (RSCEM) using clinical rating scores as the endpoint. TSC or saline was administered IV following the injection of small blood clots into the brain vasculature. Behavior was measured 24 h following embolization in order to calculate the effective stroke dose (P 50) that produces neurological deficits in 50% of the rabbits. A treatment is considered beneficial if it significantly increases the P 50 compared to control. TSC (0.25 mg/kg) given 5 or 60 min following embolization significantly ( p < 0.05) increased P 50 values by 104% and 181%; but not when given 3 h post-embolization (48% increase, p > 0.05). tPA (3.3 mg/kg) produced a significant increase in P 50 when given 1, but not 3 h following embolization. In combination studies, when TSC was administered 1 h and tPA was given either 1 or 3 h following embolization, the group P 50 values were increased by 291% and 140%, respectively. In addition, TSC plus tPA administered 3 h following embolization significantly ( p < 0.05) increased the group P 50 value by 90%. There were no significant effects ( p > 0.05) of either TSC alone or TSC administered in combination with tPA on intracerebral hemorrhage incidence. This study suggests that TSC may be used for the treatment of AIS either alone or when administered before or concomitant with tPA to improve clinical rating scores with a therapeutic window for TSC therapy up to 3 h in rabbits. Moreover, it appears that TSC can be administered with tPA, since the combination did not result in any significant change in intracerebral hemorrhage incidence.

Transcranial near infrared laser treatment (NILT) increases cortical adenosine-5′-triphosphate (ATP) content following embolic strokes in rabbits

Transcranial near infrared laser therapy (NILT) improves behavioral outcome following embolic strokes in embolized rabbits and clinical rating scores in acute ischemic stroke (AIS) patients; however, the cellular mechanism(s) involved in NILT neuroprotection have not been elucidated. It has been proposed that mitochondrial energy production may underlie a response to NILT, but this has not been demonstrated using an in vivo embolic stroke model. Thus, we evaluated the effect of NILT on cortical ATP content using the rabbit small clot embolic stroke model (RSCEM), the model originally used to demonstrate NILT efficacy and initiate the NEST-1 clinical trial. Five minutes following embolization, rabbits were exposed to 2 min of NILT using an 808 nm laser source, which was driven to output either continuous wave (CW), or pulsed wave modes (PW). Three hours after embolization, the cerebral cortex was excised and processed for the measurement of ATP content using a standard luciferin-luciferase assay. NILT-treated rabbits were directly compared to sham-treated embolized rabbits and naïve control rabbits. Embolization decreased cortical ATP content in ischemic cortex by 45% compared to naive rabbits, a decrease that was attenuated by CW NILT which resulted in a 41% increase in cortical ATP content compared to the sham embolized group ( p > 0.05). The absolute increase in ATP content was 22.5% compared to naive rabbits. Following PW NILT, which delivered 5 (PW1) and 35 (PW2) times more energy than CW, we measured a 157% (PW1 p = 0.0032) and 221% (PW2 p = 0.0001) increase in cortical ATP content, respectively, compared to the sham embolized group. That represented a 41% and 77% increase in ATP content compared to naive control rabbits. This is the first demonstration that embolization can decrease ATP content in rabbit cortex and that NILT significantly increases cortical ATP content in embolized rabbits, an effect that is correlated with cortical fluence and the mode of NILT delivery. The data provide new insight into the molecular mechanisms associated with clinical improvement following NILT.

Effect of internal carotid artery reperfusion in combination with Tenecteplase on clinical scores and hemorrhage in a rabbit embolic stroke model

In the present study, we used a modification of the rabbit small clot embolic stroke model (RSCEM), a multiple infarct ischemia model to achieve reperfusion (REP) through the internal carotid artery (ICA) following small clot embolization. We determined if increasing regional cortical blood flow (RCBF) following an embolic stroke is beneficial to neurological outcome. We compared this to cerebral reperfusion induced by the administration of the thrombolytic Tenecteplase (TNK, 1.5 mg/kg, IV bolus) in the presence or absence of REP. In this study, we also measured the incidence of ICH following REP and thrombolytic treatment. Following embolization, RCBF was reduced to 48–55% of baseline. When REP was induced by removal of a CCA ligature, RCBF initially increased to 185% of baseline. REP (P 50 = 1.18 ± 0.43 mg) had no effect on embolization-induced behavior measured 24 h following embolization compared to control (P 50 = 1.01 ± 0.48 mg). However, TNK treatment (2-hours post-embolization) in the absence or presence of REP (initiated 2 h following embolization) significantly ( p < 0.05) increased the group P 50 to 2.92 ± 0.55 mg and 2.42 ± 0.40 mg, respectively. In addition, ICH was increased in the REP (42%, p < 0.05) and REP-TNK (35%, p > 0.05) group compared to either the control group (5.5%) or TNK group (10%). This study show that reperfusion of ICA can increase RCBF following embolization, but this is not associated with improved neurological outcome measured using quantal analysis. However, TNK administration significantly increased behavioral outcome when given 2 h following embolization; an increase that is not affected by combining TNK with REP.

Cosmic Implications of NXY-059

The SAINT studies on NXY-0591,2 have reinvigorated debate over the momentous issues of neuroprotection and the development of novel stroke therapeutics. Some may argue there is little new ground that NXY-059 has unearthed and, in fact, the clinical trials merely confirm that as a strategy, neuroprotection has not been shown to be effective. Editorials in high-profile journals have sent out the clarion call to terminate research on neuroprotection given decades of work, which has uniformly led to failure after failure in clinical studies.3 These sentiments are premature and ignore studies showing that hypothermia improves outcome in patients after cardiac arrest, a major cause of global cerebral ischemia.4,5 Proof for the neuroprotection concept has therefore already been established. Despite decades of neutral clinical trials, there still remains optimism among the stroke academic community. A majority of university-affiliated stroke neurologists in the United States in a recent survey still believe neuroprotection is a viable therapeutic strategy, although admittedly, that survey was conducted between the SAINT I and SAINT II publications.6 However, the question whether neuroprotection will ever work in stroke has been overshadowed by the larger issue of whether animal studies, which serve as the foundation for the development of stroke therapeutics, are even relevant to our patients with acute stroke. In the discussion section of the SAINT II article, the authors write, “it is possible that the animal models of acute focal infarction are not relevant to the patient population; they certainly are insufficient to guarantee a positive clinical-trial result.” Have all prior neuroprotective agents failed in clinical studies because rodent stroke is not relevant to human stroke? This is a key question. Substantial reductions in infarct size in rodents with stroke treated with various types of purported neuroprotective agents would seem to support this view. There …

The lipophilic multifunctional antioxidant edaravone (radicut) improves behavior following embolic strokes in rabbits: A combination therapy study with tissue plasminogen activator

Edaravone is a lipophilic drug with multiple mechanisms of action. Because edaravone is a promising drug candidate for the treatment of stroke, we tested the hypothesis that edaravone would be neuroprotective following cerebral ischemia using a rabbit embolic stroke model with a well-defined behavioral endpoint. Using the rabbit small clot embolic stroke model (RSCEM), a drug or drug combination is considered beneficial if it significantly increases the amount of microclots (mg) measured in brain that produce neurologic dysfunction in 50% of a group of animals (P 50) compared to the control group. Edaravone (100 mg/kg, SC), increased the P 50 value to 1.80 ± 0.24 mg ( p < 0.05) when administered 5 min following embolization and increased P 50 values by 195% and 161% (compared to control) when administered 1 and 3 h following embolization, respectively, but was inactive when applied 6 h following embolization, compared to the cumulative control group (P 50 = 0.93 ± 0.16 mg). To simulate the design of current clinical trials, edaravone was also given following a standard tPA regimen, which by itself increased the P 50 value to 2.72 ± 0.28 mg. When tPA was infused 1 h following embolization and edaravone was given 3 h following embolization, the P 50 was 2.68 ± 0.56 mg. This study indicates that edaravone may have substantial therapeutic benefit for the treatment of AIS since it had a therapeutic widow of at least 3 h in rabbits. Edaravone can also be administered with a thrombolytic to improve behavior.

Therapeutic window for nonerythropoietic carbamylated-erythropoietin to improve motor function following multiple infarct ischemic strokes in New Zealand white rabbits

Carbamylated erythropoietin (CEPO) is a novel neuroprotective agent that does not bind to the classical erythropoietin receptor or affect hematocrit. Since CEPO has not been systematically studied in a fully blinded and randomized manner in an embolic stroke model, we determined if CEPO would be useful to attenuate clinical deficits associated with multiple infarct ischemia using the rabbit small clot embolic stroke model (RSCEM). Rabbits were embolized and treated with vehicle or CEPO within 6 h of embolization and behavioral analysis was conducted 48 h after embolization . Using quantal analysis, we determined the quantity of blood clot (mg) in brain that produce neurologic dysfunction in 50% of the rabbits ( P 50), with intervention considered beneficial if it increased the P 50 compared to controls. CEPO administered between 5 min and 3 h after embolization significantly ( p < 0.05) improved behavioral function and increased the P 50 value by 55–216%. However, CEPO administration did not improve behavior when administered 6 h following embolization. In conclusion, in the RSCEM, CEPO had a therapeutic window of at least 3 h, where it effectively improved clinical rating scores and motor function. Our results suggest that CEPO may be useful to treat acute ischemic stroke and supports the study of CEPO in stroke patients.

A Critique of SAINT II

Marc Fisher MD Kennedy Lees MD Section Editors: The neutral results of the SAINT II study1 dashed the hopes for a second ischemic stroke drug in the near future. Although this is certainly disappointing for stroke neurologists, a careful analysis of the NXY-059 development program reveals several deficiencies, which if properly addressed, might revitalize and strengthen the field of neuroprotection for acute ischemic stroke. This critique points out several potential reasons for the failure of SAINT II to replicate the positive findings of SAINT 1,2 discusses the preclinical problems with NXY-059, and provides a perspective on the development of future neuroprotective agents for acute ischemic stroke. The SAINT II trial1 investigated the efficacy of the free-radical–trapping agent, NXY-059, for the treatment of acute ischemic stroke and failed to confirm the positive results of SAINT I.2 In the latter study with 1699 patients, NXY-059 showed for the first time in a large phase III trial that a drug other than tissue plasminogen activator (t-PA) might lead to significant improvement on the modified Rankin Scale (mRS) using a novel and controversial outcome analysis (Rankin shift analysis). The larger SAINT II trial with 3306 patients not only failed to replicate improvement on the same primary end point but all secondary end points (neurological deficits on the National Institutes of Health Stroke Scale (NIHSS) at 90 days and activities of daily living on the Barthel Index at 90 days) were identical between treatment groups. Besides the obvious possibility that the drug itself was inactive, which may be considered due to the known instability of free-radical–trapping agents, several additional problems were evident. Both trials were designed, in part, as combination studies, testing the addition of NXY-059 to standard therapy compared with t-PA alone in the 3-hour time window. SAINT II gave …

Baicalein, an antioxidant 12/15-lipoxygenase inhibitor improves clinical rating scores following multiple infarct embolic strokes

The present study assessed whether baicalein (5,6,7-trihydroxyflavone), a polyphenolic antioxidant 12/15-lipoxygenase inhibitor would attenuate oxidative cell death in vitro using a mouse hippocampal HT22 cell assay. Moreover, we determined if baicalein would be useful to attenuate behavioral deficits associated with multiple infarct ischemic events in vivo using a rabbit small clot embolic stroke model (RSCEM). Using HT22 cell in vitro, baicalein was shown to significantly promote cell survival with an estimated dose for 50% cell survival of 2 μM following incubation in the presence of iodoacetic acid (20 μM), an irreversible inhibitor of the glycolytic pathway that results in the free radical production, lipid peroxidation and cell death. Since baicalein was neuroprotective and attenuated iodoacetic acid (IAA) toxicity in vitro, we studied its effects in vivo in an embolic stroke model using behavioral measures as the endpoint. Quantal analysis for each treatment in the embolism model identifies the quantity of microclots (mg) that produce neurologic dysfunction in 50% of a group of animals (P 50), with intervention considered neuroprotective if it increases the P 50 compared with controls. Baicalein (100 mg/kg, s.c.) injected 5 and 60 min post-embolization significantly ( P<0.05) improved behavioral function. The calculated P 50 values were 2.85±0.64 mg ( n=21) and 2.15±0.12 mg ( n=14), respectively compared with 1.37±0.20 mg ( n=23) for the control group. In conclusion, we have shown that baicalein effectively attenuated cell death in vitro using HT22 cells and also significantly reduced behavioral deficits in rabbits when given up to 1 h following an embolic stroke. The results suggest that baicalein, or derivatives of baicalein with multiple pharmacological activities may be useful to develop as novel treatments for acute ischemic stroke.

Transcranial near-infrared light therapy improves motor function following embolic strokes in rabbits: An extended therapeutic window study using continuous and pulse frequency delivery modes

Photon or near-infrared light therapy (NILT) may be an effective neuroprotective method to reduce behavioral dysfunction following an acute ischemic stroke. We evaluated the effects of continuous wave (CW) or pulse wave (P) NILT administered transcranially either 6 or 12 h following embolization, on behavioral outcome. For the studies, we used the rabbit small clot embolic stroke model (RSCEM) using three different treatment regimens: 1) CW power density of 7.5 mW/cm 2; 2) P1 using a frequency of 300 μs pulse at 1 kHz or 3) P2 using a frequency of 2 ms pulse at 100 Hz. Behavioral analysis was conducted 48 h after embolization, allowing for the determination of the effective stroke dose (P 50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a treatment is considered beneficial if it significantly increases the P 50 compared with the control group. Quantal dose–response analysis showed that the control group P 50 value was 1.01±0.25 mg ( n=31). NILT initiated 6 h following embolization resulted in the following P 50 values: (CW) 2.06±0.59 mg ( n=29, P=0.099); (P1) 1.89±0.29 mg ( n=25, P=0.0248) and (P2) 1.92±0.15 mg ( n=33, P=0.0024). NILT started 12 h following embolization resulted in the following P 50 values: (CW) 2.89±1.76 mg ( n=29, P=0.279); (P1) 2.40±0.99 mg ( n=24, P=0.134). At the 6-h post-embolization treatment time, there was a statistically significant increase in P 50 values compared with control for both pulse P1 and P2 modes, but not the CW mode. At the 12-h post-embolization treatment time, neither the CW nor the P1 regimens resulted in statistically significant effect, although there was a trend for an improvement. The results show that P mode NILT can result in significant clinical improvement when administered 6 h following embolic strokes in rabbits and should be considered for clinical development.

The phenylpropanoid micronutrient chlorogenic acid improves clinical rating scores in rabbits following multiple infarct ischemic strokes: Synergism with tissue plasminogen activator

The present study assessed whether chlorogenic acid (CGA), a phenylpropanoid molecule that has multiple mechanisms of action would be useful to attenuate behavioral deficits associated with embolic strokes using the rabbit small clot embolic stroke model (RSCEM). Quantal analysis for each treatment determines the quantity of microclots (mg) that produce neurologic dysfunction in 50% of a group of animals ( P 50), with intervention considered beneficial if it increases the P 50 compared to controls. CGA (50 mg/kg) injected 5 min post-embolization significantly increased behavioral function and the P 50 to 3.61 ± 0.52 mg ( n = 19) compared to 1.58 ± 0.15 mg ( n = 26) in controls. In addition, CGA also increased the P 50 to 2.57 ± 0.28 mg ( n = 18) when administered 1 h post-embolization, but was ineffective when given 3 h following embolization ( P 50 = 1.22 ± 0.24 mg, n = 18). For combination studies with the thrombolytic tissue plasminogen activator (tPA), we used tPA at a standard dose of 3.3 mg/kg, which significantly increased the P 50 to 2.89 ± 0.29 mg ( n = 17) when administered 1 h after embolization, but not 3 h after embolization ( P 50 = 1.54 ± 0.27 mg, n = 18). However, when tPA (3.3 mg/kg) was combined with CGA (50 mg/kg) and administered 3 h following embolization, there was a significant increase in behavioral function as evidenced by an increase in the P 50 value to 3.40 ± 0.76 mg ( n = 23). In conclusion, as a mono-therapy CGA effectively reduced behavioral deficits when given up to 1 h following embolic strokes in rabbits. Moreover, there was a synergistic effect of the combination of tPA with CGA when administered 3 h following embolization. The results show that the therapeutic window for a standard effective dose of tPA could be increased by administration of CGA, suggesting that it may be most useful as a co-therapy with a standard thrombolytic treatment regimen.

3α-OL-5-β-pregnan-20-one hemisuccinate, a steroidal low-affinity NMDA receptor antagonist improves clinical rating scores in a rabbit multiple infarct ischemia model: Synergism with tissue plasminogen activator

We previously showed that the neuroactive steroid 3α-ol-5-β-pregnan-20-one hemisuccinate (ABHS), a low-affinity NMDA receptor antagonist, improves behavior in rabbits following spinal cord ischemia. The present study assessed whether this also occurs following cerebral ischemia. We used the rabbit small clot embolic stroke model (RSCEM), a model of multiple infarct ischemia, which has a well-defined behavioral endpoint. Quantal analysis for each treatment determines microclots (mg) that produce neurologic dysfunction in 50% of a group of animals ( P 50), with treatment considered beneficial if it increases the P 50 compared to controls. ABHS (25 mg/kg) injected 5 min post-embolization significantly ( P = 0.046) increased the P 50 to 2.60 ± 0.69 mg compared to 1.15 ± 0.19 mg in controls but was ineffective at longer intervals. For combination studies with tissue plasminogen activator (tPA), a cumulative control curve ( P 50 = 1.18 ± 0.25 mg) was used for statistical comparisons. Low-dose tPA (0.9 mg/kg) and ABHS, given 60 min post-embolization, improved behavior synergistically ( P 50 = 2.44 ± 0.44 mg), compared to tPA ( P 50 = 1.25 ± 0.25 mg) or ABHS ( P 50 = 1.05 ± 0.24 mg) alone. A standard tPA dose (3.3 mg/kg) significantly increased the P 50 at 60 (2.69 ± 0.19 mg) but not 180 min (1.28 ± 0.31 mg) post-embolization. However, when combined with ABHS, an effect was evident even 180 min post-embolization ( P 50 = 2.31 ± 0.48 mg). Although the therapeutic window for ABHS is limited, it significantly enhanced neuroprotection by low-dose tPA and increased the therapeutic window for tPA, suggesting that it may be most useful as a co-therapy with thrombolytics for stroke.

Transcranial infrared laser therapy improves clinical rating scores after embolic strokes in rabbits.

Because photon energy delivered using a low-energy infrared laser may be useful to treat stroke, we determined whether transcranial laser therapy would improve behavioral deficits in a rabbit small clot embolic stroke model (RSCEM). In this study, the behavioral and physiological effects of laser treatment were measured. The RSCEM was used to assess whether low-energy laser treatment (7.5 or 25 mW/cm2) altered clinical rating scores (behavior) when given to rabbits beginning 1 to 24 hours postembolization. Behavioral analysis was conducted from 24 hours to 21 days after embolization, allowing for the determination of the effective stroke dose (P50) or clot amount (mg) that produces neurological deficits in 50% of the rabbits. Using the RSCEM, a treatment is considered beneficial if it significantly increases the P50 compared with the control group. In the present study, the P50 value for controls were 0.97+/-0.19 mg to 1.10+/-0.17 mg; this was increased by 100% to 195% (P50=2.02+/-0.46 to 2.98+/-0.65 mg) if laser treatment was initiated up to 6 hours, but not 24 hours, postembolization (P50=1.23+/-0.15 mg). Laser treatment also produced a durable effect that was measurable 21 days after embolization. Laser treatment (25 mW/cm2) did not affect the physiological variables that were measured. This study shows that laser treatment improved behavioral performance if initiated within 6 hours of an embolic stroke and the effect of laser treatment is durable. Therefore, transcranial laser treatment may be useful to treat human stroke patients and should be further developed.