Experimental Stroke Research Research Articles

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre’s behavioral evaluations before project’s interventional phase.

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Abstract P746: Sex Differences in Cognitive and Psychological Outcomes of Stroke: Impact of Diabetes

Diabetes increases risk and severity of post-stroke cognitive impairment (PSCI), a major cause of disability worldwide. While it is known that females suffer more from PSCI, psychological outcomes and underlying reasons are poorly understood. From a preclinical perspective, potential explanations include 1) use of otherwise healthy animals in experimental stroke research without integration of common comorbid diseases like diabetes into the study design, and 2) optimization of most behavioral tests for sensorimotor and cognitive functions using only male animal models. Our hypothesis is that post-stroke outcomes are sex and comorbid disease-dependent. To test this, we validated the Novel Object Recognition (NOR), Y-maze, and Passive Avoidance (PAT) behavioral paradigms in Ctrl and Diabetic (DM) male (M) and female (F) rats pre- and post-stroke (S) via 60 min. middle cerebral artery occlusion (MCAO). We tested the PAT paradigm with a multi-trial method where the animals were habituated to the dark/light chambers without foot shock and then trained in 3 trials where they received foot shock upon entering the dark. We then tested retention following MCAO for their memory of foot shock 2 weeks prior. Multitrial results suggested that there was no difference between groups in learning to associate the dark chamber with the shock, so we revised the multitrial method into a single-trial method for ongoing retention tests to compare the impact of stroke on shock memory recall. PAT revealed (Table 1) disease- and sex-dependent responses to aversive stimulus. NOR revealed that M-DM-S and F-DM-S rats have decreased exploration time, suggesting that they are unmotivated or depressed. Y-maze indicated that males displayed spatial memory recovery, while females remained impaired. In summary, we have observed numerous sex- and disease-dependent post-stroke outcomes with standard behavioral paradigms, causing us to carefully consider how we evaluate preclinical outcomes.

Detection of cytokine-induced sickness behavior after ischemic stroke by an optimized behavioral assessment battery

Stroke causes severe and long-lasting symptoms in patients. Besides focal deficits such as speech impairment and limb weakness, stroke also results in neuropsychiatric symptoms, including fatigue, anxiety, and depression, which are debilitating and often impair post-stroke rehabilitation. However, in experimental stroke research, the study of neuropsychiatric symptoms and their therapeutic targeting has so far been largely neglected, which can be mainly attributed to the lack of appropriate tools to investigate such deficits in mice.Here, we report that neuropsychiatric symptoms can be differentiated from focal deficits and specifically modulated independent of treating the primary lesion. In order to achieve this, we developed a novel behavior analysis tool by assessing test performance of various tests, combining outcome parameters to cover functional domains of focal and neuropsychiatric symptoms, and finally weighted results into a time point-specific score. This weighted score enabled us to clearly differentiate focal deficits and neuropsychiatric symptoms and detect these until the chronic phase after stroke. Using this analysis tool, we detected that neutralizing systemic cytokines (TNF-α, IL-1β and IL-6) specifically ameliorated neuropsychiatric symptoms but did not affect focal deficits or lesion volume. Hence, most conventional studies analyzing only focal deficits and lesion volume as primary outcome measures would have missed these significant and translationally relevant therapeutic effects. We anticipate that these findings will encourage more detailed analyses of neuropsychiatric symptoms particularly for anti-inflammatory therapies in stroke and that the presented weighted composite score will facilitate this development.

Refined Ischemic Penumbra Imaging with Tissue pH and Diffusion Kurtosis Magnetic Resonance Imaging.

Imaging has played a vital role in our mechanistic understanding of acute ischemia and the management of acute stroke patients. The most recent DAWN and DEFUSE-3 trials showed that endovascular therapy could be extended to a selected group of late-presenting stroke patients with the aid of imaging. Although perfusion and diffusion MRI have been commonly used in stroke imaging, the approximation of their mismatch as the penumbra is oversimplified, particularly in the era of endovascular therapy. Briefly, the hypoperfusion lesion includes the benign oligemia that does not proceed to infarction. Also, with prompt and effective reperfusion therapy, a portion of the diffusion lesion is potentially reversible. Therefore, advanced imaging that provides improved ischemic tissue characterization may enable new experimental stroke therapeutics and eventually further individualize stroke treatment upon translation to the clinical setting. Specifically, pH imaging captures tissue of altered metabolic state that demarcates the hypoperfused lesion into ischemic penumbra and benign oligemia, which remains promising to define the ischemic penumbra's outer boundary. On the other hand, diffusion kurtosis imaging (DKI) differentiates the most severely damaged and irreversibly injured diffusion lesion from the portion of diffusion lesion that is potentially reversible, refining the inner boundary of the penumbra. Altogether, the development of advanced imaging has the potential to not only transform the experimental stroke research but also aid clinical translation and patient management.

A Semiquantitative Non-invasive Measurement of PcomA Patency in C57BL/6 Mice Explains Variance in Ischemic Brain Damage in Filament MCAo.

Numerous studies on experimental ischemic stroke use the filament middle cerebral artery occlusion (fMCAo) model in C57BL/6 mice, but lesion sizes in this strain are highly variable. A known contributor is variation in the posterior communicating artery (PcomA) patency. We therefore aimed to provide a semiquantitative non-invasive in vivo method to routinely assess PcomA patency. We included 43 male C57BL/6 mice from four independent studies using a transient 45 min fMCAo model. Edema-corrected lesion sizes were measured by magnetic resonance (MR) imaging 24 h after reperfusion. Time-of-flight MR angiography was performed 7 days before and 24 h after fMCAo. Scores of PcomA size measured 24 h after, but not scores measured 7 days before fMCAo were negatively correlated with lesion size. Variability in PcomA patency explained 30% of the variance in our cohort (p < 0.0001, coefficient of determination r2 = 0.3). In a simulation using parameters typical for experimental stroke research, the power to detect a true effect of d = 1 between two groups increased by 15% when an according covariate was included in the statistical model. We have demonstrated that in vivo measurement of PcomA size is feasible and can lead to increased accuracy in assessing the effect of treatments.

The role of peptidase neurolysin in neuroprotection and neural repair after stroke.

Current experimental stroke research has evolved to focus on detailed understanding of the brain’s self-protective and restorative mechanisms, and harness this knowledge for development of new therapies. In this context, the role of peptidases and neuropeptides is of growing interest. In this focused review, peptidase neurolysin (Nln) and its extracellular peptide substrates are briefly discussed in relation to pathophysiology of ischemic stroke. Upregulation of Nln following stroke is viewed as a compensatory cerebroprotective mechanism in the acute phase of stroke, because the main neuropeptides inactivated by Nln are neuro/cerebrotoxic (bradykinin, substance P, neurotensin, angiotensin II, hemopressin), whereas the peptides generated by Nln are neuro/cerebroprotective (angiotensin-(1–7), Leu-/Met-enkephalins). This notion is confirmed by experimental studies documenting aggravation of stroke outcomes in mice after inhibition of Nln following stroke, and dramatic improvement of stroke outcomes in mice overexpressing Nln in the brain. The role of Nln in the (sub)chronic phase of stroke is less clear and it is likely, that this peptidase does not have a major role in neural repair mechanisms. This is because, the substrates of Nln are less uniform in modulating neurorestorative mechanisms in one direction, some appearing to have neural repair enhancing/stimulating potential, whereas others doing the opposite. Future studies focusing on the role of Nln in pathophysiology of stroke should determine its potential as a cerebroprotective target for stroke therapy, because its unique ability to modulate multiple neuropeptide systems critically involved in brain injury mechanisms is likely advantageous over modulation of one pathogenic pathway for stroke pharmacotherapy.

Peptidase neurolysin is an endogenous cerebroprotective mechanism in acute neurodegenerative disorders

Stroke and traumatic brain injury (TBI) are significant clinical problems characterized by high rate of mortality and long-lasting disabilities, and an unmet need for new treatments. Current experimental stroke and TBI research are evolving to focus more on understanding the brain’s self-protective mechanisms to meet the critical need of developing new therapies for these disorders. In this hypothesis-based manuscript, I provide several lines of evidence that peptidase neurolysin (Nln) is one of the brain’s potent, self-protective mechanisms promoting preservation and recovery of the brain after acute injury. Based on published experimental observations and ongoing studies in our laboratory, I posit that Nln is a compensatory and cerebroprotective mechanism in the post-stroke/TBI brain that functions to process a diverse group of extracellular neuropeptides and by that to reduce excitotoxicity, oxidative stress, edema formation, blood brain barrier hyper-permeability, and neuroinflammation. If this hypothesis is correct, Nln could potentially serve as a single therapeutic target to modulate the function of multiple targets, the involved neuropeptide systems, critically involved in various mechanisms of brain injury and cerebroprotection/restoration. Such multi-pathway target would be highly desired for pharmacotherapy of stroke and TBI, because targeting one pathophysiological pathway has proven to be ineffective for such complex disorders.

Abstract WMP75: A Modified, Recombinant Tissue Plasminogen Activator-Responding Photothrombotic Stroke Model

Introduction: Photodynamical dye-based stroke models (e.g. Rose Bengal RB/photothrombosis) have several attractive features, including simple surgical procedures, consistent infarct size, and low mortality rate. Yet, because RB/photothrombosis mainly induces platelet-rich but fibrin-poor clots, it responds to recombinant tissue plasminogen activator (rtPA)-based lytic therapy poorly making it less suitable for studying reperfusion injury or improving lytic therapy. Hypothesis: We hypothesize the addition of thrombin to Rose Bengal dye in photothrombosis will increase the fibrin component in clots, making it more susceptible to rtPA-thrombolysis. Methods: Based on the standard RB/photothrombotic stroke procedures (intravenous injection of 50 mg/kg RB, we mixed a subthreshold level of bovine thrombin with RB to create a thrombin-Rose Bengal (TRB)/photothrombotic stroke model. We compared the clot composition, effects of rtPA treatment on cerebral blood flow reperfusion, and the infarct size following rtTA treatment initiated at 30, 60, or 120 min after photothrombosis. Results: We report four sets of results. First, the addition of thrombin did not increase mortality rate or infarct size, when compared to standard RB/photothrombosis. Second, immunostaining showed a scant amount of fibrin at the border of RB/photothrombosis-induced clots. In contrast, fibrin was mixed with erythrocytes throughout TRB/photothrombosis-induced clots. Third, laser speckle contrast imaging indicated that intravenous infusion of rtPA (10 mg/kg) at 30 min after TRB/photothrombosis improved blood flow in the MCA-supplied area, but the same treatment had little effect in RB/photothrombosis. Finally, the therapeutic window of rtPA-treatment is at least 2 h in TRB/photothrombosis (n&gt;10 in each group). Conclusions: Our results confirm the notion by Dr. Brant Watson et al. that rtPA lytic treatment confers little benefits in RB/photothrombosis, likely due to its fibrin-poor clots. Yet, the addition of subthreshold thrombin (TRB/photothrombosis) elevates the fibrin component in clot, making it far more responsive to rtPA-thrombolysis. The modified, rtPA-responding thrombotic stroke model may have broad applications in experimental stroke research.

Multiscale x-ray phase-contrast tomography in a mouse model of transient focal cerebral ischemia

Cerebral ischemia is associated with a lack of oxygen and high-energy phosphates within the brain tissue, leading to irreversible cell injury. Visualizing these cellular injuries has long been a focus of experimental stroke research with application of immunohistochemistry as one of the standard approaches. It is, however, a destructive imaging technique with non-isotropic resolution, as only the two-dimensional tissue structure of a thin brain section is visualized using optical microscopy and specific stainings. Herein, we extend the structural analysis of mouse brain tissue after cerebral ischemia to the third dimension via microfocus computed tomography (µ-CT). Contrast of the weakly absorbing unstained brain tissue is enhanced by phase contrast. We show that recordings at two different magnifications and fields of view can be combined as a single approach for visualization of the associated structural alterations at isotropic resolution, from the level of the whole organ down to single cells.

Inter- and intra-rater reliability of computer-assisted planimetry in experimental stroke research

BackgroundComputer-assisted planimetry is widely used in experimental stroke research to assess the size of the ischemic lesion or hemispheric volume. New methodOnly insufficient data exist on the training required to achieve sufficient reliability in planimetry. Therefore, planimetry was performed over 15 months by two blinded raters who were initially inexperienced in the method. For inter-rater reliability, the hemispheric and lesional volume of 227 male Wistar Unilever rats subjected to middle cerebral artery occlusion were determined in diffusion- and T2-weighted sequences. For the intra-rater agreement, one investigator assessed the hemispheric and lesional volume in 87 T2-weighted sequences twice within a six-week interval. The correlation was calculated using Krippendorff’s alpha and Bland-Altman plots illustrated the agreement. ResultsInter-rater agreement increased during the first seven weeks and remained at high values (Krippendorff’s alpha > 0.88). For intra-rater agreement, Krippendorff’s alpha was 0.84 for hemispheric and 0.85 for lesional volume. The Bland-Altman plot indicated solid agreement between raters in the absence of systematic errors. Comparison with existing methodsSimplified geometrical models or automated methods for planimetry can be used to determine lesional volume, but both approaches are inappropriate to assess hemispheric volume. ConclusionComputer-assisted planimetry can be an appropriate method to determine hemispheric or ischemic lesion volume in rodents but requires a sufficiently long learning period of approximately two months. Even an experienced investigator can generate data with serious variation. Inter- and intra-rater-dependent bias should be considered during the design and performance of respective studies.

State-of-the-Art Techniques to Causally Link Neural Plasticity to Functional Recovery in Experimental Stroke Research.

Current experimental stroke research faces the same challenge as neuroscience: to transform correlative findings in causative ones. Research of recent years has shown the tremendous potential of the central nervous system to react to noxious stimuli such as a stroke: Increased plastic changes leading to reorganization in form of neuronal rewiring, neurogenesis, and synaptogenesis, accompanied by transcriptional and translational turnover in the affected cells, have been described both clinically and in experimental stroke research. However, only minor attempts have been made to connect distinct plastic remodeling processes as causative features for specific behavioral phenotypes. Here, we review current state-of the art techniques for the examination of cortical reorganization and for the manipulation of neuronal circuits as well as techniques which combine anatomical changes with molecular profiling. We provide the principles of the techniques together with studies in experimental stroke research which have already applied the described methodology. The tools discussed are useful to close the loop from our understanding of stroke pathology to the behavioral outcome and may allow discovering new targets for therapeutic approaches. The here presented methods open up new possibilities to assess the efficiency of rehabilitative strategies by understanding their external influence for intrinsic repair mechanisms on a neurobiological basis.

Contractile Responses in Spontaneously Hypertensive Rats after Transient Middle Cerebral Artery Occlusion

Stroke is one of the leading causes of mortality and morbidity worldwide, and few therapeutic treatments have shown beneficial effect clinically. One reason for this could be the lack of risk factors incorporated into the preclinical stroke research. We have previously demonstrated phenotypic receptor changes to be one of the injurious mechanisms occurring after stroke but mostly in healthy rats. The aim of this study was to investigate if hypertension has an effect on vasoconstrictive receptor responses to endothelin 1, sarafotoxin 6c and angiotensin II after stroke by inducing transient middle cerebral artery occlusion in spontaneously hypertensive rats and Wistar-Kyoto rats using the wire-myograph. We demonstrated an increased contractile response to endothelin 1 and extracellular potassium as well as an increased carbachol-induced dilator response in the middle cerebral arteries from hypertensive rats after stroke. This study demonstrates the importance of including risk factors in experimental stroke research.

Characterization of long-term gait deficits in mouse dMCAO, using the CatWalk system

Evaluation of functional outcome is widely used across species to assess the recovery process following various pathological conditions, including spinal cord injury, musculo-skeletal injury, mithochondrial disease, neuropathic cancer, Huntington's disease, chronic pain, cortical lesion, and olivocerebellar degeneration among others. The Stroke Therapy Academic Industry Roundtable (STAIR) recommends multiple endpoints for behavioral studies in pre-clinical stroke research, to demonstrate their clinical relevance. One of the more challenging tasks in experimental stroke research is measuring long-term functional outcome in mice. It is, however, becoming more important, since transgenic mice are increasingly used for modeling human neurological disorders. Using CatWalk, we characterized long-lasting gait/locomotion deficits following mouse distal middle cerebral artery occlusion (dMCAO). The post-dMCAO assessment was performed at 7, 14, 21, and 28days after experimental ischemia. When compared to sham-operated mice, dMCAO animals displayed a statistically significant decrease in Spatial parameters (such as Paw Area), while the Temporal parameters (Stand, Initial and Terminal Dual Stances) were significantly increased for three weeks after surgery. Kinetic parameters were significantly decreased in dMCAO animals at 7days after dMCAO. The Interlimb coordination group of parameters displayed the strongest deficits at 21days. While CatWalk variables were altered in all paws, the degree of change was greatest for the parameters measured from the Right Front Paw (contralateral to the lesion). All parameters measured in dMCAO and Sham-operated groups reached similar levels at four weeks after the experimental insult, which reflects a spontaneous post-ischemic recovery. Based on our investigation, we conclude that CatWalk represents a relevant and sensitive analysis, which allows long-term characterization of animal functional recovery in the dMCAO model of experimental ischemia.

Anesthesia in Experimental Stroke Research.

Anesthetics have enabled major advances in development of experimental models of human stroke. Yet, their profound pharmacologic effects on neural function can confound the interpretation of experimental stroke research. Anesthetics have species-, drug-, and dose-specific effects on cerebral blood flow and metabolism, neurovascular coupling, autoregulation, ischemic depolarizations, excitotoxicity, inflammation, neural networks, and numerous molecular pathways relevant for stroke outcome. Both preconditioning and postconditioning properties have been described. Anesthetics also modulate systemic arterial blood pressure, lung ventilation, and thermoregulation, all of which may interact with the ischemic insult as well as the therapeutic interventions. These confounds present a dilemma. Here, we provide an overview of the anesthetic mechanisms of action and molecular and physiologic effects on factors relevant to stroke outcomes that can guide the choice and optimization of the anesthetic regimen in experimental stroke.

Stroke research at the crossroads - where are we heading?

Stroke causes 5.7 million deaths annually. This ranks stroke as the second most common cause of death and, additionally, it is a major cause of disability. Because of an ageing population, stroke incidence and costs will greatly increase in the future. This makes stroke an ongoing social and economic burden, in contrast to the only very limited therapeutic options. In the last decade vast sums were spent on translational research focused on neuroprotective strategies in the acute phase of ischaemic stroke. A plethora of candidate agents were tested in experimental models and preclinical studies, but none was proven effective in clinical trials. This gave rise to discussions about the possible reasons for this failure, ending up mainly with criticism of methodological aspects of the preclinical and clinical studies, or of the relevance of animal studies in drug development. Indeed, the question could rather be whether neuroprotection is the right target for successful stroke treatment. In this context, a paradigm change can currently be observed: the focus of experimental and translational stroke research is shifting from early neuroprotection to delayed mechanisms such as stroke-associated comorbidities, regeneration and plasticity. In this review we highlight a few recently emerging fields in translational stroke research. One such topic is the crosstalk between immunity and the injured brain as key pathomechanism in stroke. On one hand, innate and adaptive immune cells play an important role in the fate of injured brain tissue after stroke; on the other, peripheral immune alterations are critically involved in post-stroke comorbidities. Another emerging research area is the analysis of mechanisms involved in regeneration and neuronal plasticity after stroke. Here, we discuss the current understanding of basic mechanisms involved after brain injury, clinical imaging approaches and therapeutic strategies to promote regeneration in stroke patients.

Regulatory T Cells in Post-stroke Immune Homeostasis

The secondary neuroinflammatory response has come into focus of experimental stroke research. Immunological mechanisms after acute stroke are being investigated in the hope to identify novel and druggable pathways that contribute to secondary infarct growth after stroke. Among a variety of neuroimmunological events after acute brain ischemia, including microglial activation, brain leukocyte invasion, and secretion of pro-inflammatory factors, lymphocytes have been identified as the key leukocyte subpopulation driving the neuroinflammatory response and contributing to stroke outcome. Several studies have shown that pro-inflammatory lymphocyte subpopulations worsen stroke outcome and that inhibiting their invasion to the injured brain is neuroprotective. In contrast to the effector functions of pro-inflammatory lymphocytes, regulatory T cells (Treg) are critically involved in maintaining immune homeostasis and have been characterized as disease-limiting protective cells in several inflammatory conditions, particularly in primary inflammatory diseases of the central nervous system (CNS). However, due to the complex function of regulatory cells in immune homeostasis and disease, divergent findings have been described for the role of Treg in stroke models. Emerging evidence suggests that this discrepancy arises from potentially differing functions of Treg depending on the predominant site of action within the neurovascular unit and the surrounding inflammatory milieu. This article will provide a comprehensive review of current findings on Treg in brain ischemia models and discuss potential reasons for the observed discrepancies.

Impact of Comorbidities on Acute Injury and Recovery in Preclinical Stroke Research: Focus on Hypertension and Diabetes.

Human ischemic stroke is very complex, and no single preclinical model can comprise all the variables known to contribute to stroke injury and recovery. Hypertension, diabetes, and hyperlipidemia are leading comorbidities in stroke patients. The use of predominantly young adult and healthy animals in experimental stroke research has created a barrier for translation of findings to patients. As such, more and more disease models are being incorporated into the research design. This review highlights the major strengths and weaknesses of the most commonly used animal models of these conditions in preclinical stroke research. The goal is to provide guidance in choosing, reporting, and executing appropriate disease models that will be subjected to different models of stroke injury.

Developing New Stroke Treatments Using Preclinical Randomized Controlled Trials

Figure 2. Results are pooled from multiple centers performing 2 different stroke models. Anti-CD49d did not show a benefit in the fMCAO model while it did show a benefit in the combined data of the cMCAO model. Preclinical stroke research has suffered from a lack of translatability into human clinical trials. With the exception of tissue plasminogen activator, none of the countless therapies that have shown promise in animal models have demonstrated a beneficial effect in actual patients. As a result of multiple high-profile failures, pharmaceutical companies have eliminated investment in this area of research, forcing a critical evaluation of all stages of the drug discovery pathway. In 1999, the Stroke Therapy Academic Industry Roundtable (STAIR) was established to identify problems in stroke research methodology and make recommendations to guide researchers and funders in the evaluation of experimental quality. A large body of literature has since been produced on improving and standardizing animal testing for stroke therapies. In 2006, O’Collins et al. reviewed the history of experimental stroke research, identifying 1026 experimental stroke therapies published between 1955 and 2003. They reviewed the methodology and results of 912 therapies that were tested in animal models, 17 in only human trials and 97 in both animal and human trials. Each study was given a grade ranging from 0 to 10 on the basis of how many of the STAIR criteria were fulfilled, called the “scope of testing.” They observed that poor scope of testing correlated with a higher reported percentage of neuroprotection (Figure 1). They also noted that whether or not a drug advanced to clinical trials did not correlate with efficacy or scope of testing. With the STAIR preclinical testing recommendations and the poor historical record summarized by critical papers like O’Collins et al., funding for clinical trials has required a higher standard of preclinical evidence. Preclinical studies, however, still lacked the homogeneity and regulation to minimize bias and maximize accuracy. Several international research consortia including MultiPART (Multicentre Preclinical Animal Research Team), funded by the European Union for Stroke Research, and CAESAR (Consortium for PreclinicAl assESsment of cARdioprotective therapies), funded by the National Institutes of Health, have called for preclinical randomized controlled trials (pRCTs) and revealed inadequate data reporting, statistical flaws, and missing crossvalidation of positive results. In August, Llovera et al. published the first pRCT in any disease process. Six centers with demonstrated proficiency in 2 murine models of middle cerebral artery occlusion (MCAO) tested natalizumab, an anti-CD49d antibody and possible neuroprotective agent (Figure 2). The drug had passed all of the old hurdles of an optimal drug candidate to advance to human trials including minimal side-effect profile,multiple years already on themarket for a different disease process, and demonstrated efficacy in an animal model. The preclinical studies, however, were performed at individual institutions and produced conflicting resultswith 4 positive studies and