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Spinal cord ischemia associated with trauma and surgical procedures including thoraco-abdominal aortic aneurysm repair and thoracic endovascular aortic repair results in devastating clinical deficits in patients. Because spinal cord ischemia is inadequately treated, we studied the effects of [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl) vinyl)-2-methoxy-phenol)] (CNB-001), a novel curcumin-based compound, in a rabbit SCI model. CNB-001 is known to inhibit human 5-lipoxygenase and 15-lipoxygenase and reduce the ischemia-induced inflammatory response. Moreover, CNB-001 can reduce the level of oxidative stress markers and potentiate brain-derived neurotrophic factor and brain-derived neurotrophic factor receptor signaling. The Tarlov scale and quantal analysis technique results revealed that CNB-001 administered as an intravenous dose (bolus) 30 minutes prior to spinal cord ischemia improved the behaviors of female New Zealand White rabbits. The improvements were similar to those produced by the uncompetitive N-methyl-D-aspartate receptor antagonist memantine. At 48 hours after aortic occlusion, there was a 42.7% increase (P < 0.05) in tolerated ischemia duration (n = 14) for rabbits treated with CNB-001 (n = 16), and a 72.3% increase for rabbits treated with the positive control memantine (P < 0.05) (n = 23) compared to vehicle-treated ischemic rabbits (n = 22). CNB-001 is a potential important novel treatment for spinal cord ischemia induced by aortic occlusion. All experiments were approved by the CSMC Institutional Animal Care and Use Committee (IACUC #4311) on November 1, 2012.

Acute ischemic stroke is devastating to patients and their families because of few viable therapeutic options to promote recovery after reperfusion windows close. Recent breakthroughs in biotechnology have resulted in a reproducible patented process for the purification of extracellular vesicles (EVs) from human cardiosphere-derived cells (CDCs). Because CDC-EVs have many features potentially beneficial to treat acute ischemic stroke, CDC-EVs were evaluated in an established small-clot rabbit embolic stroke model, where clinically relevant end points were used to assess recovery in a more translational large animal model. Biodistribution studies with fluorescent DiD-labeled CDC-EVs showed intense uptake in the ischemic region of the brain. In this report, we show that intravenous (IV) CDC-EVs (0.75 mg/kg) administered 1-hour post-embolization significantly attenuate behavioral deficits following an embolic stroke in rabbits. In CDC-EV-treated rabbits, P50 (3.63 ± 1.27 mg, n = 24) was increased by 245% over vehicle control (1.05 ± 0.15 mg, n = 24); by comparison, rt-PA increased P50 by 91% (2.01 ± 0.24 mg, n = 23). Importantly, the therapy was also without adverse effects on intracerebral hemorrhage or survival rate of embolized rabbits. Thus, as a first step toward widespread use, CDC-EVs, given adjunctively to routine reperfusion therapy, merit further investigation as a therapeutic candidate for stroke.

Important questions regarding the conduct of scientific research and data transparency have been raised in various scientific forums over the last 10 years. It is becoming clear, that in spite of published RIGOR guidelines, that improvement in the transparency of scientific research is required to focus on the discovery and drug development process so that a treatment can be provided to stroke patients. We have the unique privilege of conducting research using animal models of a disease so that we can address the development of a new therapy, and we should do this with great care and vigilance. This document identifies valuable resources for researchers to become Good Laboratory Practices compliant and increase and improve data transparency and provides guidelines for accurate data management to continue to propel the translational stroke research field forward while recognizing that there is a shortage of research funds worldwide. While data audits are being considered worldwide by funding agencies and they are used extensively by industry, they are still quite controversial for basic researchers. Due to the special exploratory nature of basic and translational science research, the current challenging funding environment, and independent and individualized laboratory activities, it is debatable if current individualized non-standardized data management and monitoring represents the best approach. Thus, herein, we propose steps to prepare research study data in an acceptable form for archival purposes so that standards for translational research data can be comparable to those that are accepted and adhered to by the clinical community. If all translational research laboratories follow and institute the guidelines while conducting translational research, data from all sources may be more comparable and reliable.

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/cm(2)) 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 (p0.05) increased P50 values by 95% and 56% (p0.05), respectively over control. TLT-tPA increased P50 by 136% over control (p0.05). Embolization reduced cortical ATP content by 39%; decreases that were attenuated by either TLT or tPA treatment (p0.05). 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 (p0.05) 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.

Transcranial near-infrared laser therapy originally referred to as transcranial laser therapy (TLT) or near-infrared laser therapy in translational stroke studies remains a potentially important method to promote acute neuroprotection, or cytoprotection and repair following a stroke. However, translational research has shown that the human skull can be an obstacle to the use of TLT to treat stroke. TLT has been studied in the ultimate translational model, the human in three clinical trials, NeuroThera effectiveness and safety trial (NEST)-1, NEST-2, and NEST-3 with ambiguous mixed efficacy-lack of efficacy results that are difficult to explain. Before mounting the NEST trials, there was a large amount of proof of concept efficacy data in animal models, in particular the rabbit embolic stroke model that was central to the development of acute ischemic stroke standard-of-care reperfusion therapy, tissue plasminogen activator. This brief chapter will review the NEST trial design and data, the translational embolic stroke data used to mount the NEST trials, and discuss significant differences in skull thickness and penetration data in rabbit and human in an attempt to explain the disappointing NEST results; and provide a basis for the next phase of TLT studies in humans.

Ischemic stroke is an acute neurodegenerative disease that is extremely devastating to patients, their families and society. Stroke is inadequately treated even with endovascular procedures and reperfusion therapy. Using an extensive translational screening process, we have developed a pleiotropic cytoprotective agent with the potential to positively impact a large population of brain ischemia patients and revolutionize the process used for the development of new drugs to treat complex brain disorders. In this unique translational study article, we document that the novel curcumin-based compound, CNB-001, when administered as a single intravenous dose, has significant efficacy to attenuate clinically relevant behavioral deficits following ischemic events in agyrencephalic rabbits when administered 1 h post-embolization and reduces infarct growth in gyrencephalic non-human primates, when administered 5 min after initiation of middle cerebral artery occlusion. CNB-001 is safe and does not increase morbidity or mortality in either research species. Mechanistically, CNB-001 inhibits human 5- and 15-lipoxygenase in vitro, and can attenuate ischemia-induced inflammatory markers, and oxidative stress markers, while potentially promoting synaptic plasticity mediated by enhanced brain-derived neurotrophic factor (BDNF).

Objective Tissue plasminogen activator (tPA) is administered to acute ischemic stroke victims in a vehicle formulation containing high concentrations of l -arginine (3.5 g/100 mg vial), a well-known nitric oxide synthase (NOS) substrate and precursor to nitric oxide (NO), as well as an enhancer of cerebral blood flow. Methods We studied the effects of tPA vehicle compared to tPA (3.3 mg/kg) formulated in the same vehicle containing l -arginine, normal saline or normal saline containing l -arginine, on behavioral function following small clot embolic strokes in rabbits using clinical rating scores and quantal analysis curves as the primary end point. Treatments were administered intravenously (1 ml/kg; 20% bolus/80% infused over 30 min) starting 1 h following the injection of small-sized blood clots into the brain vasculature and terminal behavior was measured 2 days following embolization. Behavioral rating scores were used to calculate the effective stroke dose ( P 50 in mg) that produces neurological deficits in 50% of the rabbits. Results In this study, tPA significantly ( p = 0.001) improved behavior compared to all other treatments including tPA vehicle, saline and saline- l -arginine, increasing the P 50 by 141% over tPA vehicle. Saline- l -arginine was not significantly different from either saline or tPA vehicle ( p > 0.05). Conclusion This study demonstrates that the l -arginine component of the tPA vehicle does not contribute to the reproducible clinical improvement observed following tPA administration in rabbits. Moreover, the administration of l -arginine was not an effective method to promote behavioral recovery following embolic strokes in the stringent rabbit small clot stroke model, nor did l -arginine exacerbate behavioral deficits or intracerebral hemorrhage in embolized rabbits.

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 (p50 value (behavior) to 1.88 ± 0.10 mg (n=20) compared to P 50 = 1.07 ± 0.07 (n=19) for control(p1/2 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.

The ischemic penumbra is now defined as tissue at risk of becoming fully involved in the evolving neurodegenerative process following an embolic stroke. After an ischemic core is quickly developed following vascular occlusion, there is slow spreading of the ischemic injury from the core to areas immediately surrounding the core until full recruitment is achieved. Forty years ago, seminal electrophysiological studies forming the basis of the penumbral hypothesis were conducted in a large animal model, baboons, an animal that is now rarely used in translational stroke research because it can no longer be justified! Thereafter, the rabbit embolic stroke model led the way for approval of Alteplase® (tissue plasminogen activator, tPA, rt-PA) to treat acute ischemic stroke.

Type 2 diabetes is growing at epidemic proportions, and pharmacological interventions are being actively sought. This study examined the effect of a novel neuroprotective curcuminoid, CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol], on glucose intolerance and insulin signaling in high-fat diet (HFD)-fed mice. C57BL6 mice (5-6 weeks old) were randomly assigned to receive either a HFD (45% fat) or a low-fat diet (LFD, 10% fat) for 24 weeks, together with CNB-001 (40 mg/kg i.p. per day). Glucose tolerance test revealed that the area under the curve of postchallenge glucose concentration was elevated on HF-feeding, which was attenuated by CNB-001. CNB-001 attenuated body weight gain, serum triglycerides, and IL-6, and augmented insulin signaling [elevated phosphoprotein kinase B (p-Akt), and phosphoinsulin receptor (p-IR)β, lowered endoplasmic reticulum (ER) stress, protein-tyrosine phosphatase 1B (PTP1B)] and glucose uptake in gastrocnemius muscle of HFD-fed mice. Respiratory quotient, measured using a metabolic chamber, was elevated in HFD-fed mice, which was unaltered by CNB-001, although CNB-001 treatment resulted in higher energy expenditure. In cultured myotubes, CNB-001 reversed palmitate-induced impairment of insulin signaling and glucose uptake. Docking studies suggest a potential interaction between CNB-001 and PTP1B. Taken together, CNB-001 alleviates obesity-induced glucose intolerance and represents a potential candidate for further development as an antidiabetic agent.

Tissue plasminogen activator (tPA) is currently used in combination with endovascular procedures to enhance recanalization and cerebral reperfusion and is also currently administered as standard-of-care thrombolytic therapy to patients within 3–4.5 h of an ischemic stroke. Since tPA is not neuroprotective or cytoprotective, adjuvant therapy with a neuroprotective or an optimized cytoprotective compound is required to provide the best care to stroke victims to maximally promote clinical recovery. In this article, we describe the use of a sensitive standardized protease assay with CH3SO2-D-hexahydrotyrosine-Gly-Arg-p-nitroanilide•AcOH, a chromogenic protease substrate that is cleaved to 4-nitroaniline (p-nitroaniline) and measured spectrophotometrically at 405 nm (OD405 nm), and how the assay can be used as an effective screening assay to study drug-tPA interactions. While we focus on two compounds of interest in our drug development pipeline, the assay is broadly applicable to all small molecule neuroprotective or cytoprotective compounds currently being discovered and developed worldwide. In this present study, we found that the specific tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1; 0.25 μM), significantly (p 10 μM for CSMC-19 and CSMC-140, respectively. Using bonafide 4-nitroaniline, we then demonstrated that the reduction of 4-nitroaniline detection was not due to drug-4-nitroaniline quenching of signal detection at OD405 nm. In conclusion, the results suggest that high concentrations of both cytoprotectives reduced 4-nitroaniline production in vitro, but the inhibition only occurs with concentrations 104–1025-fold that of EC50 values in an efficacy assay. Thus, CSMC-19 and CSMC-140 should be further developed and evaluated in embolic stroke models in the absence or presence of a thrombolytic. If necessary, they could be administered once effective tPA thrombolysis has been confirmed to avoid the possibility that the chromone will reduce the efficacy of tPA in patients. Stroke investigator developing new cytoprotective small molecules should consider adding this sensitive assay to their development and screening repertoire to assess possible drug-tPA interactions in vitro as a de-risking step.

Ischemic spinal cord injury (SCI) is one of the most morbid complications of aortic operations and traumatic injury. Numerous animal models have been created to try to understand the pathogenesis of spinal cord injury and to find treatment options. In this chapter, different animal spinal cord injury models are discussed. An overview of potential spinal cord injury therapeutic agents that are studied in the literature is presented as well. Finally, a new minimally invasive rabbit spinal cord ischemia model that uses a transfemoral intra-aortic balloon occlusion and neuromonitoring with motor-evoked potentials (MEPs) is presented. This rabbit model has the potential to aid in novel therapeutic drug development for early ischemic SCI.

Translational neuroprotection research is currently undergoing a rebirth, a much needed revival, in part due to the efficacy of both thrombolytic and endovascular procedures in subpopulations of ischemic stroke patients. Stroke is currently treated with the Food and Drug administration (FDA)-approved thrombolytic, tissue plasminogen activator (rt-PA), and can be treated with endovascular approaches using the MERCI stent retriever or the Solitaire FR stent retriever, with the application of thrombolytic (i.e., rt-PA or urokinase) prior to embolectomy for rt-PA eligible patients. Moreover, from retrospective analysis in rt-PA ineligible stroke patients, embolectomy alone has proven safe and beneficial if completed within 6 h.

Acute ischemic stroke patients now have the benefit of receiving either thrombolytic therapy or endovascular procedure therapy, alone or with an approved thrombolytic to significantly improve clinical outcome measured using the modified Rankin scale. Most recent data indicate that large vessel occlusion stroke victims can achieve a modified Rankin scale score of 0–2, clinically normal, 3–12 months after treatment. Like tissue plasminogen activator (rt-PA), endovascular procedures will be of benefit to a small well-defined patient population, because it appears that a small ischemic core and large penumbral substrate are a prerequisite for neuroprotection and functional recovery. Endovascular procedures and thrombolysis both effectively target reperfusion of the penumbra to promote cell survival, but neither therapy actually targets a specific mechanism directly involved in the ischemic cascade, a complex series of temporally regulated events that result in differential death of cellular populations in the brain. With our constantly evolving understanding of cell death mechanisms, we now have the opportunity to develop neuroprotective or cytoprotective strategies to use as adjuvant therapy with endovascular procedures and rt-PA to further promote function and potentially repair ischemia-damaged cellular pathways. The benefit of testing novel neuroprotective and cytoprotective therapies as an adjuvant in the patient population defined above will take advantage of both large penumbra and reperfusion of the penumbra. Progress to develop new therapies will be inextricably linked to standardization of translational research practices, complete transparency of data, and adherence to STAIR, RIGOR, ARRIVE, and VOW guidelines. Moreover, because the target population will be undergoing reperfusion therapies, the recommendation is for translational research to be conducted in standardized and validated embolic stroke models.

Acute ischemic stroke is inadequately treated in the USA and worldwide due to a lengthy history of neuroprotective drug failures in clinical trials. The majority of victims must endure life-long disabilities that not only affect their livelihood, but also have an enormous societal economic impact. The rapid development of a neuroprotective or cytoprotective compound would allow future stroke victims to receive a treatment to reduce disabilities and further promote recovery of function. This opinion article reviews in detail the enormous costs associated with developing a small molecule to treat stroke, as well as providing a timely overview of the cell-death time-course and relationship to the ischemic cascade. Distinct temporal patterns of cell-death of neurovascular unit components provide opportunities to intervene and optimize new cytoprotective strategies. However, adequate research funding is mandatory to allow stroke researchers to develop and test their novel therapeutic approach to treat stroke victims.

Transcranial near-infrared laser therapy (NILT) or low-level laser(or light) therapy remains a promising noninvasive neuroprotectivemethod to treat acute ischemic stroke (AIS), despite the difficultiesobserved with translation in clinical trials. Three clinical trials-NeuroThera Effectiveness and Safety Trial (NEST)-1, NEST-2, andNEST-3-have evaluated the use of NILT to promote clinical recoveryin patients with AIS; the results have been quite mixed. Can werecover from this failure reminiscent of the SAINT clinical trialfailure?

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 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.

Introduction: After vascular occlusion, there are two major zones of injury: the infarct core that rapidly dies, and the surrounding penumbra, which is hypoxic and at risk for infarction. Restoring blood flow by vascular recanalization (ie. endovascular +/- IV tPA) to prevent the penumbra from infarcting has become the new standard of care for acute ischemic stroke patients presenting with salvageable tissue. However, not all patients benefit from recanalization. Regardless of successful or partial reperfusion, secondary hypoxic events within the rescued penumbra can cause selective neuronal loss (SNL) that may account for suboptimal clinical recovery. OMX is a breakthrough oxygen delivery protein tuned to release oxygen specifically in hypoxic tissue and reduce hypoxia to prevent SNL within the reperfused penumbra. Methods: To mimic the clinical situation whereby occlusion is followed by gradual reperfusion after spontaneous or therapeutic thrombolysis, we used the endothelin-1 induced middle cerebral artery occlusion (MCAO) model. OMX was administered intravenously up to 2h post-MCAO. Short and long-term histological and behavioral outcomes were used to assess hypoxia, apoptosis, gliosis, SNL, infarct volume and sensorimotor functions. Results: Despite reperfusion, hypoxic tissue persists and progressively infarcts in vehicle-treated rats. However, OMX-treated rats showed no infarct expansion over 7d, indicating that OMX prevents infarction of the majority of hypoxic tissue. OMX reduces hypoxia and caspase 3/9 activity in a dose-dependent manner and significantly prevents SNL and gliosis. When administered post-MCAO, OMX significantly improves both sensory and motor functions (∼30-80% improvement, * p Conclusions: OMX is a promising therapeutic candidate that can be administered in combination with reperfusion therapies to stabilize the at-risk hypoxic tissue and ameliorate long-term clinical outcomes in stroke patients.

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 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.

Introduction: We studied the pharmacokinetics and pharmacology of intravenously administered CNB-001, a pleiotropic anti-inflammatory molecule with neurotrophic properties in two rabbit neuro-ischemia models. Hypothesis: In vitro, we have found that CNB-001 can significantly reduce cell death mediated by glutamate, iodoacetic acid, ATP depletion, oxidative stress (glutathione depletion) and oxygen-glucose depletion. We tested the hypothesis that CNB-001 would attenuate ischemia-induced behavioral deficits in vivo. Methods: Abiding by RIGOR guidelines, the study was randomized and the neuro-examiners were naive (blinded) to treatment. CNB-001 (10mg/kg) was administered IV 45 minutes prior to balloon of occlusion of the infrarenal aorta, which reliably produces spinal cord ischemia (SCI) depending upon the occlusion time. Using TARLOV scores and quantal analysis, we quantified the ischemia duration (P50 in minutes) producing SCI in 50% of study rabbits. Second, using a rabbit embolic stroke model (RSCEM), CNB-001(1-25mg/kg) was applied IV 1 hour post-ischemia and behavioral function measured. We calculated the effective stroke clot dose (P50 in mg) producing neurological deficits in 50% of embolized rabbits. Control groups and positive-control drugs were also studied in parallel in both rabbit ischemia models. All behavioral endpoints were measured 2days after ischemia. Results: Pharmacokinetic analysis of CNB-001 in normal male rabbits indicated that the t1/2 was 3.37 ± 0.52 hours and clearance rate was 44.2 ml/min/kg in rabbits. In the SCI model, CNB-001 (10mg/kg) significantly increased P50 values to 19.38 ± 2.03 min (n=16) from 13.58 ± 0.90min (n=14; p Conclusions: CNB-001 can be provided to patients prior to cardiaovascular surgery to reduce the probability of SCI. Moreover, CNB-001 can reduce behavioral deficits associated with brain ischemia when given after an embolic stroke.

Hemorrhagic transformation (HT) is an important complication of ischemic stroke and is responsible for most of the mortality associated with acute reperfusion therapy. Although many important publications address the preclinical models of ischemic stroke, there are no current recommendations on the conduct of research aimed at understanding the mechanisms and consequences of HT. The purpose of this review is to present the various models used in HT research, the clinical correlates, and the experimental variables known to influence the quantitation of HT in preclinical investigation. Lastly, recommendations for the conduct of preclinical research in HT are provided.

Stroke continues to be a serious and significant health problem in the USA and worldwide. This article will emphasize the need for good laboratory practices, transparent scientific reporting, and the use of translational research models representative of the disease state to develop effective treatments. This will allow for the testing and development of new innovative strategies so that efficacious therapies can be developed to treat ischemic and hemorrhagic stroke. This article recommends guidelines for effective translational research, most importantly, the need for study blinding, study group randomization, power analysis, accurate statistical analysis, and a conflict of interest statement. Additional guidelines to ensure reproducibility of results and confirmation of efficacy in multiple species are discussed.

Acute ischemic stroke (AIS) is a major cause of morbidity and mortality in the aging population worldwide. Alteplase, a recombinant tissue plasminogen activator, is the only Food and Drug Administration-approved thrombolytic agent for the treatment of AIS. Only 2–5% of patients with stroke receive thrombolytic treatment, mainly due to delay in reaching the hospital. Edaravone is a free radical scavenger marketed in Japan to treat patients with AIS, who present within 24 h of the onset of symptoms. When used in combination with alteplase, edaravone may have three useful effects: enhancement of early recanalization, inhibition of alteplase-induced hemorrhagic transformation and extension of the therapeutic time window for alteplase. This is the first review of the literature evaluating the clinical efficacy of edaravone, aiming to clarify whether edaravone should be further evaluated for clinical use worldwide. This review covers both clinical and experimental studies conducted between 1994 and 2012. Edaravone is a potentially useful neurovascular protective agent, used in combination with thrombolytic agents to treat >15 million patients devastated by stroke worldwide annually. Additional clinical studies are necessary to verify the efficacy of edaravone when used in combination with a thrombolytic agent.

Stroke is the 4th leading cause of death and disability in adults in the USA. However, in the majority of patients, the detrimental effects of an ischemic insult go untreated because of the lack of efficacious neuroprotective compounds. Using naturally occurring compounds as a building block to create efficacious neuroprotective compounds that cross the blood brain barrier may eventually benefit the stroke patients. However, historically, the development of novel compounds has been fraught with problems including lack of significant pleiotropic activity, inability to effectively cross the blood brain barrier and poor bioavailability. This article details, in a stepwise manner, the synthesis and in vitro screening steps used to produce new flavonoids that have increased neuroprotective activity compared to the parent compound fisetin. Moreover, as a preliminary de-risking step, select compounds have been screened in a transfected Madin Darby canine kidney cell assay as a measure of blood brain barrier penetration. Initial de-risking steps have allowed us to identify a series of BBB-penetrating neuroprotective candidates for further development.

One of the most promising methods to treat neurodegeneration is noninvasive transcranial near-infrared laser therapy (NILT), which appears to promote acute neuroprotection by stimulating mitochondrial function, thereby increasing cellular energy production. NILT may also promote chronic neuronal function restoration via trophic factor-mediated plasticity changes or possibly neurogenesis. Clearly, NILT is a treatment that confers neuroprotection or neurorestoration using pleiotropic mechanisms. The most advanced application of NILT is for acute ischemic stroke based upon extensive preclinical and clinical studies. In laboratory settings, NILT is also being developed to treat traumatic brain injury, Alzheimer's disease and Parkinson's disease. There is some intriguing data in the literature that suggests that NILT may be a method to promote clinical improvement in neurodegenerative diseases where there is a common mechanistic component, mitochondrial dysfunction and energy impairment. This article will analyze and review data supporting the continued development of NILT to treat neurodegenerative diseases.

In the present study, we used a comprehensive panel of in vitro assays to evaluate the efficacy and safety of stilbazulenyl nitrone (STAZN) as a lead compound to treat acute ischemic stroke. First, we measured neuroprotection in vitro using two different HT22 hippocampal nerve cell assays. Secondly, to de-risk drug development, we used CeeTox analysis with the H4IIE rat hepatoma cell line to determine the acute toxicity profile of STAZN. Third, STAZN was tested in microsomes from four species for measures of metabolic stability. Last, we determined the Ames test genotoxicity profile of STAZN using Salmonella typhimurium TA989 and TA100. In vitro, STAZN was neuroprotective against toxicity induced by iodoacetic acid, and oxytosis-induced glutathione depletion was initiated by glutamate, with an EC(50) value of 1-5 μM. Secondly, using CeeTox analysis, the estimated C(Tox) value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) for STAZN was calculated to be 260 μM. Third, the half-life of STAZN in humans, dogs, and rats was 60-78 min. Last, the genotoxicity profile showed that STAZN did not induce bacterial colony growth under any conditions tested, indicating the lack of mutagenicity with this compound. STAZN appears to be a multi-target neuroprotective compound that has an excellent safety profile in both the CeeTox and Ames mutagenicity assays. STAZN may have significant potential as a novel neuroprotective agent to treat stroke and should be pursued in clinically relevant embolic stroke models.

The development of new treatments for stroke remains a formidable challenge as emphasized in the article by Frank Sharp and colleagues [1]. However, with the recent advances in genomic and proteomic profiling [2–4], in vitro screening methodologies [5–7], predictive animal models [8–12], and a greater understanding of the complexity of processes and mechanisms involved in cellular degeneration and behavioral deficits following a stroke [13,14], we now have the basic tools in our repertoire to develop and systematically test new candidates in a preclinical translational setting. Moreover, with more sophisticated adaptive clinical trial design [15–18], therapy can be targeted to specific stroke patient populations to ensure some extent of efficacy, rather than complete failure. If the best that we can expect to achieve is to treat some patients, then we should accept that fact!

J. Neurochem. (2011) 116, 122–131. Abstract Acute ischemic stroke is a major risk for morbidity and mortality in our aging population. Currently only one drug, the thrombolytic tissue plasminogen activator, is approved by the US Food and Drug Administration to treat stroke. Therefore, there is a need to develop new drugs that promote neuronal survival following stroke. We have synthesized a novel neuroprotective molecule called CNB-001 (a pyrazole derivative of curcumin) that has neurotrophic activity, enhances memory, and blocks cell death in multiple toxicity assays related to ischemic stroke. In this study, we tested the efficacy of CNB-001 in a rigorous rabbit ischemic stroke model and determined the molecular basis of its in vivo activity. CNB-001 has substantial beneficial properties in an in vitro ischemia assay and improves the behavioral outcome of rabbit ischemic stroke even when administered 1 h after the insult, a therapeutic window in this model comparable to tissue plasminogen activator. In addition, we elucidated the protein kinase pathways involved in neuroprotection. CNB-001 maintains the calcium-calmodulin-dependent kinase signaling pathways associated with neurotrophic growth factors that are critical for the maintenance of neuronal function. On the basis of its in vivo efficacy and novel mode of action, we conclude that CNB-001 has a great potential for the treatment of ischemic stroke as well as other CNS pathologies.

Transcranial near-infrared laser therapy (NILT) has been investigated as a novel neuroprotective treatment for acute ischemic stroke (AIS), for approximately 10 years. Two clinical trials, NeuroThera Effectiveness and Safety Trial (NEST)-1 and NEST-2, have evaluated the use of NILT to promote clinical recovery in patients with AIS. This review covers preclinical, translational, and clinical studies documented during the period 1997-2010. The primary aim of this article is to detail the development profile of NILT to treat AIS. Secondly, insight into possible mechanisms involved in light therapy will be presented. Lastly, possible new directions that should be considered to improve the efficacy profile of NILT in AIS patients will be discussed. The use of NILT was advanced to clinical trials based upon extensive translational research using multiple species. NILT, which may promote functional and behavioral recovery via a mitochondrial mechanism and by enhancing cerebral blood flow, may eventually be established as an Food and Drug Administration (FDA)-approved treatment for stroke. The NEST-3 trial, which is the pivotal trial for FDA approval, should incorporate hypotheses derived from translational studies to ensure efficacy in patients. Future NILT studies should consider administration of a thrombolytic to enhance cerebral reperfusion alongside NILT neuroprotection.

Data has emerged, largely from non-thromboembolic animal models of stroke, that suggests that statins, which have efficacy in preventing strokes when given pre-ischemically, may have a positive effect on stroke even when given post-ischemically, possibly through pleitropic cerebrovascular effects. The goal of this study was to characterize the effects of IV tPA in a clinically relevant model of stroke utilizing a vascular occlusion with a freshly formed clot, and evaluate the effects of post-ischemic administration of simvastatin on stroke outcome in this model. Neurological deficit, clot burden, and lesion volume were assessed after treatment with tPA in one experiment, and after treatment with simvastatin in another. In the tPA experiment, treatment with 10 mg/kg of tPA IV (with 20% given as an initial bolus, and 80% given as an infusion over the remaining 30 min), starting within an hour after stroke, resulted in significant reductions, compared with control animals, in neurological deficit (mean ± SD neuroscores of 21.5 ± 21.1 and 30 ± 29.3, respectively, p = 0.005), clot burden (p = 0.010) and lesion volume (p = 0.049) at 24 h. In the simvastatin experiment on the other hand, treatment with a 20 mg/kg of simvastatin as a single intraperitoneal dose within an hour after stroke resulted in no salutary effects on neurological deficit, clot burden or lesion volume compared with controls at 24 h. These results suggest that more research needs to be done to fully ascertain the therapeutic potential and optimal dosing paradigm of a post-ischemic treatment with a statin.

The rabbit large clot embolic stroke model has been used for over 23 years to study methods to manipulate hemorrhage and to test drugs and devices for safety, because the rabbit model is particularly sensitive to embolism-induced hemorrhage. This study refined the original embolization procedure using an automated, pump-assisted injection method to introduce large blood clots or macroscopic emboli into the middle cerebral artery (MCA) via an indwelling carotid artery catheter. The study shows that rapid injection of blood clots (3ml/30 seconds) produced a model where there is a high hemorrhage incidence rate (79%) and a high stroke success rate (63%), compared to a low stroke success rate (19%) with no hemorrhages when clots were injected at a slow rate (3ml/90 seconds). The rapid injection method, which produces a high hemorrhage rate is particularly useful to study neuroprotective agents to attenuate embolism-induced hemorrhage. In addition, we show that manual injection of blood clots, which produces a lower baseline hemorrhage rate (41%) with a similar stroke success rate (65%), may allow investigators to study pharmacological agents to either up or down-regulate hemorrhage incidence. Lastly, we show that in the rabbit embolic stroke model, hemorrhages are adjacent to areas of 2,3,5-triphenyltetrazolium (TTC)-negative tissue, normally associated with infarcted or ischemic tissue. Thus, there is clear separation of ischemia and hemorrhage in the model, suggesting that therapeutics that are neuroprotective may also be useful to limit the evolution of ischemic damage associated with a hemorrhage, if not attenuate hemorrhage itself.

In the present study, we used a comprehensive cellular toxicity (CeeTox) analysis panel to determine the toxicity profile for CNB-001 [4-((1E)-2-(5-(4-hydroxy-3-methoxystyryl-)-1-phenyl-1H-pyrazoyl-3-yl)vinyl)-2-methoxy-phenol)], which is a hybrid molecule created by combining cyclohexyl bisphenol A, a molecule with neurotrophic activity and curcumin, a spice with neuroprotective activity. CNB-001 is a lead development compound since we have recently shown that CNB-001 has significant preclinical efficacy both in vitro and in vivo. In this study, we compared the CeeTox profile of CNB-001 with two neuroprotective molecules that have been clinically tested for efficacy: the hydrophilic free radical spin trap agent NXY-059 and the hydrophobic free radical scavenger edaravone (Radicut). CeeTox analyses using a rat hepatoma cell line (H4IIE) resulted in estimated C Tox value (i.e., sustained concentration expected to produce toxicity in a rat 14-day repeat dose study) of 42 μM for CNB-001 compared with >300 μM for both NXY-059 and Radicut. The CeeTox panel suggests that CNB-001 produces some adverse effects on cellular adenosine triphosphate content, membrane toxicity, glutathione content, and cell mass (or number), but only with high concentrations of the drug. After a 24-h exposure, the drug concentration that produced a half-maximal response (TC50) on the measures noted above ranges from 55 to 193 μM. Moreover, all CNB-001-induced changes in the markers were coincident with loss of cell number, prior to acute cell death as measured by membrane integrity, suggesting a cytostatic effect of CNB-001. NXY-059 and Radicut did not have acute toxic effects on H4IIE cells. We also found that CNB-001 resulted in an inhibition of ethoxyresorufin-o-deethylase activity, indicating that the drug may affect cytochrome P4501A activity and that CNB-001 was metabolically unstable using a rat microsome assay system. For CNB-001, an estimated in vitro efficacy/toxicity ratio is 183–643-fold, suggesting that there is a significant therapeutic safety window for CNB-001 and that it should be further developed as a novel neuroprotective agent to treat stroke.

Statins have pleiotropic neuroprotective effects in the central nervous system. In this study, we assessed the pharmacological effects of simvastatin on measures of behavior in New Zealand white rabbits embolized using a suspension of small-sized blood clots. For these studies, simvastatin was administered up to 3 hours following embolization, and behavior was measured 48 hours following embolization to calculate the dose of emboli (P(50) in mg) that produces neurological deficits in 50% of the rabbits. A treatment is considered neuroprotective if it significantly increases the P(50) compared to control. Simvastatin treatment (20mg/kg, bolus subcutaneous injection) significantly improved clinical function and increased the P(50) by 143% when administered 1 hour following embolization but was ineffective at 3 hours. In combination studies with the thrombolytic, tissue plasminogen activator (tPA) using a standard intravenous dose of 3.3mg/kg (20% bolus, 80% infused), we found that simvastatin could be safely administered with tPA to improve clinical scores; however, the maximum behavioral improvement with the combination treatment was similar to either monotherapy alone, both of which significantly improved behavior (p0.05). It has been proposed that Simvastatin neuroprotection may be related to a variety of signaling pathways including Rho-kinase (ROCK). To determine if a ROCK mechanism is involved in simvastatin-induced neuroprotection following embolic strokes, we used pharmacological intervention with the ROCK inhibitor, fasudil. When fasudil was administered 30 minutes before simvastatin (given at 1 hour), there was an additional significant (p=0.0217) synergistic increase in behavioral function. However, fasudil as a monotherapy did not affect behavioral function in embolized rabbits. The study suggests that there may be an interaction between simvastatin treatment and the ROCK signaling pathway that should be further explored. Our results suggest that simvastatin treatment may have clinical benefit when used alone or in the presence of tPA, but the therapeutic window using a single-dose regimen is narrow.

Edaravone (Radicut) is a free radical scavenger marketed in Japan by Mitsubishi Tanabe Pharma Corp. to treat acute ischemic stroke (AIS) patients presenting within 24 h of the attack. Injectable edaravone ampoules (30 mg b.i.d., i.v., 14 days) were first approved on 23 May 2001. On 19 January 2010, as a new innovation, the Radicut BAG (Intravenous BAG) was approved by the Japanese Ministry of Health and Welfare. Efficacy of edaravone ranges from large significant clinical improvements to only modest improvements in clinical function measured using standard stroke scales when administered 6-72 h following an ischemic stroke. With almost 17 years of edaravone clinical experience, a few adverse events--including acute renal failure--have been noted.This is the only article to date to critically review available clinical efficacy and toxicology data published in the literature to ascertain whether edaravone should be further pursued as a candidate for development worldwide.This review covers clinical studies carried out over the period 1993-2008.Edaravone may be a useful neuroprotective agent to treat the15 million victims worldwide who are devastated by stroke annually. Additional clinical studies are necessary to verify the efficacy of edaravone.

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 (p0.05) increased P(50) values by 104% and 181%; but not when given 3 h post-embolization (48% increase, p0.05). 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 (p0.05) increased the group P(50) value by 90%. There were no significant effects (p0.05) 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 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 naive 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.

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, s.c.), increased the P(50) value to 1.80+/-0.24 mg (p0.05) 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.

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

Development of drugs and devices for the treatment of stroke is not exempt from current translational research standards, which include Stroke Treatment Academic Industry Roundtable (STAIR) criteria and RIGOR guidelines. Near-infrared laser therapy (NILT) was developed to treat stroke in an era when STAIR criteria were not adhered to, thus NILT was not optimized in multiple species, nor was it optimized for efficacy across barriers in translational animal models before proceeding to expensive and extensive clinical trials. Moreover, the majority of rodent studies did not adhere to RIGOR guidelines. This ultimately led to failure in the NeuroThera Effectiveness and Safety Trial-3. Because NILT remains a promising therapeutic approach to treat stroke, we designed a systematic study to determine laser light penetration profiles across the skull of four different species with increasing skull thickness: mouse, rat, rabbit, and human.Our study demonstrates that NILT differentially penetrates the skulls. There is especially extensive attenuation of light energy penetration across the human calvaria, compared with animal skulls, which suggests that the power density setting used in stroke clinical trials may not have optimally stimulated neuroprotection and repair pathways. The results of our study suggest that NILT cannot be sufficiently optimized in "small" animals and directly translated to humans because of significant variances of skull thickness and penetration characteristics across species. NILT neuroprotection should be further studied using a research design that endeavors to incorporate human skull characteristics (thickness) into the development plan to increase the probability of success in stroke victims.