Quest for Quality in Translational Stroke Research—A New Dawn for Neuroprotection?

Despite tremendous progress in modern-day stroke therapy, ischemic stroke remains a disease associated with a high socioeconomic burden in industrialized countries. In light of demographic change, these health care costs are expected to increase even further. The current causal therapeutic treatment paradigms focus on successful thrombolysis or thrombectomy, but only a fraction of patients qualify for these recanalization therapies because of therapeutic time window restrictions or contraindications. Hence, adjuvant therapeutic concepts such as neuroprotection are urgently needed. A bench-to-bedside transfer of neuroprotective approaches under stroke conditions, however, has not been established after more than twenty years of research, albeit a great many data have demonstrated several neuroprotective drugs to be effective in preclinical stroke settings. Prominent examples of substances supported by extensive preclinical evidence but which failed clinical trials are tirilazad and disodium 2,4-sulphophenyl-N-tert-butylnitrone (NXY-059). The NXY-059 trial, for instance, was retrospectively shown to have a seriously weak study design, a trial of insufficient quality and a poor statistical analysis, although it initially met the recommendations of the STAIR committee. In light of currently ongoing novel neuroprotective stroke trials, such as ESCAPE-NA, and to avoid the mistakes made in the past, an improvement in study quality in the field of stroke neuroprotection is urgently needed. In the present review, animal models closely reflecting the “typical” stroke patient, occlusion techniques and the appropriate choice of time windows are discussed. In this context, the STAIR recommendations could provide a useful orientation. Taking all of this into account, a new dawn for neuroprotection might be possible.     

Neuroprotection for Ischemic Stroke: Moving Past Shortcomings and Identifying Promising Directions

The translation of neuroprotective agents for ischemic stroke from bench-to-bedside has largely failed to produce improved treatments since the development of tissue plasminogen activator (tPA). One possible reason for lack of translation is the failure to acknowledge the greatest risk factor for stroke, age, and other common comorbidities such as hypertension, obesity, and diabetes that are associated with stroke. In this review, we highlight both mechanisms of studying these factors and results of those that have been addressed. We also discuss the potential role of other lifestyle factors associated with an increased stroke risk such as sleep fragmentation and/or deprivation. Furthermore, many proposed therapeutic agents have targeted molecular mechanisms occurring soon after the onset of ischemia despite data indicating delayed patient presentation following ischemic stroke. Modulating inflammation has been identified as a promising therapeutic avenue consistent with preliminary success of ongoing clinical trials for anti-inflammatory compounds such as minocycline. We review the role of inflammation in stroke and in particular, the role of inflammatory cell recruitment and macrophage phenotype in the inflammatory process. Emerging evidence indicates an increasing role of neuro-immune crosstalk, which has led to increased interest in identification of peripheral biomarkers indicative of neural injury. It is our hope that identification and investigation of factors influencing stroke pathophysiology may lead to improved therapeutics.     

Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy

Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury and is among the leading cause of death among infants. Currently there are no well-established treatments; thus, it is important to understand the pathophysiology of the disease and elucidate complications that are creating a gap between basic science and clinical translation. In the development of neuroprotective strategies and translation of experimental results in HIE, there are many limitations and challenges to master based on an appropriate study design, drug delivery properties, dosage, and use in neonates. We will identify understudied targets after HIE, as well as neuroprotective molecules that bring hope to future treatments such as melatonin, topiramate, xenon, interferon-beta, stem cell transplantation. This review will also discuss some of the most recent trials being conducted in the clinical setting and evaluate what directions are needed in the future.     

The Development of Novel Drug Treatments for Stroke Patients: A Review

Acute ischemic stroke is a critical condition that can result in disability and death. The consequences of this medical condition depend on various factors, including the size of the stroke, affected brain region, treatment onset, and the type of treatment. The primary objective of stroke treatment is to restart ischemic penumbra tissue perfusion and reduce infarct volume by sustaining blood flow. Recent research on the condition’s pathological pathways and processes has significantly improved treatment options beyond restoring perfusion. Many studies have concentrated on limiting injury severity via the manipulation of molecular mechanisms of ischemia, particularly in animal research. This article reviews completed and ongoing research on the development of acute ischemic stroke drugs. This study focuses on three main categories of antithrombotic drugs, thrombolytic drugs, and neuroprotective agents. The paper outlines findings from animal and clinical trials and explores the working mechanisms of these drugs.     

Promising Strategies for the Development of Advanced In Vitro Models with High Predictive Power in Ischaemic Stroke Research

Although stroke is one of the world’s leading causes of death and disability, and more than a thousand candidate neuroprotective drugs have been proposed based on extensive in vitro and animal-based research, an effective neuroprotective/restorative therapy for ischaemic stroke patients is still missing. In particular, the high attrition rate of neuroprotective compounds in clinical studies should make us question the ability of in vitro models currently used for ischaemic stroke research to recapitulate human ischaemic responses with sufficient fidelity. The ischaemic stroke field would greatly benefit from the implementation of more complex in vitro models with improved physiological relevance, next to traditional in vitro and in vivo models in preclinical studies, to more accurately predict clinical outcomes. In this review, we discuss current in vitro models used in ischaemic stroke research and describe the main factors determining the predictive value of in vitro models for modelling human ischaemic stroke. In light of this, human-based 3D models consisting of multiple cell types, either with or without the use of microfluidics technology, may better recapitulate human ischaemic responses and possess the potential to bridge the translational gap between animal-based in vitro and in vivo models, and human patients in clinical trials.     

Blood–Brain Barrier Transporters: Opportunities for Therapeutic Development in Ischemic Stroke

Globally, stroke is a leading cause of death and long-term disability. Over the past decades, several efforts have attempted to discover new drugs or repurpose existing therapeutics to promote post-stroke neurological recovery. Preclinical stroke studies have reported successes in identifying novel neuroprotective agents; however, none of these compounds have advanced beyond a phase III clinical trial. One reason for these failures is the lack of consideration of blood–brain barrier (BBB) transport mechanisms that can enable these drugs to achieve efficacious concentrations in ischemic brain tissue. Despite the knowledge that drugs with neuroprotective properties (i.e., statins, memantine, metformin) are substrates for endogenous BBB transporters, preclinical stroke research has not extensively studied the role of transporters in central nervous system (CNS) drug delivery. Here, we review current knowledge on specific BBB uptake transporters (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents); organic cation transporters (OCTs in humans; Octs in rodents) that can be targeted for improved neuroprotective drug delivery. Additionally, we provide state-of-the-art perspectives on how transporter pharmacology can be integrated into preclinical stroke research. Specifically, we discuss the utility of in vivo stroke models to transporter studies and considerations (i.e., species selection, co-morbid conditions) that will optimize the translational success of stroke pharmacotherapeutic experiments.     

Neuroprotective Strategies for Traumatic Brain Injury: Improving Clinical Translation

Traumatic brain injury (TBI) induces secondary biochemical changes that contribute to delayed neuroinflammation, neuronal cell death, and neurological dysfunction. Attenuating such secondary injury has provided the conceptual basis for neuroprotective treatments. Despite strong experimental data, more than 30 clinical trials of neuroprotection in TBI patients have failed. In part, these failures likely reflect methodological differences between the clinical and animal studies, as well as inadequate pre-clinical evaluation and/or trial design problems. However, recent changes in experimental approach and advances in clinical trial methodology have raised the potential for successful clinical translation. Here we critically analyze the current limitations and translational opportunities for developing successful neuroprotective therapies for TBI.     

Molecular Hydrogen Neuroprotection in Post-Ischemic Neurodegeneration in the Form of Alzheimer’s Disease Proteinopathy: Underlying Mechanisms and Potential for Clinical Implementation—Fantasy or Reality?

Currently, there is a lot of public interest in naturally occurring substances with medicinal properties that are minimally toxic, readily available and have an impact on health. Over the past decade, molecular hydrogen has gained the attention of both preclinical and clinical researchers. The death of pyramidal neurons in especially the CA1 area of the hippocampus, increased permeability of the blood-brain barrier, neuroinflammation, amyloid accumulation, tau protein dysfunction, brain atrophy, cognitive deficits and dementia are considered an integral part of the phenomena occurring during brain neurodegeneration after ischemia. This review focuses on assessing the current state of knowledge about the neuroprotective effects of molecular hydrogen following ischemic brain injury. Recent studies in animal models of focal or global cerebral ischemia and cerebral ischemia in humans suggest that hydrogen has pleiotropic neuroprotective properties. One potential mechanism explaining some of the general health benefits of using hydrogen is that it may prevent aging-related changes in cellular proteins such as amyloid and tau protein. We also present evidence that, following ischemia, hydrogen improves cognitive and neurological deficits and prevents or delays the onset of neurodegenerative changes in the brain. The available evidence suggests that molecular hydrogen has neuroprotective properties and may be a new therapeutic agent in the treatment of neurodegenerative diseases such as neurodegeneration following cerebral ischemia with progressive dementia. We also present the experimental and clinical evidence for the efficacy and safety of hydrogen use after cerebral ischemia. The therapeutic benefits of gas therapy open up new promising directions in breaking the translational barrier in the treatment of ischemic stroke.     

Glibenclamide for the Treatment of Ischemic and Hemorrhagic Stroke

Ischemic and hemorrhagic strokes are associated with severe functional disability and high mortality. Except for recombinant tissue plasminogen activator, therapies targeting the underlying pathophysiology of central nervous system (CNS) ischemia and hemorrhage are strikingly lacking. Sur1-regulated channels play essential roles in necrotic cell death and cerebral edema following ischemic insults, and in neuroinflammation after hemorrhagic injuries. Inhibiting endothelial, neuronal, astrocytic and oligodendroglial sulfonylurea receptor 1–transient receptor potential melastatin 4 (Sur1–Trpm4) channels and, in some cases, microglial K_ATP (Sur1–Kir6.2) channels, with glibenclamide is protective in a variety of contexts. Robust preclinical studies have shown that glibenclamide and other sulfonylurea agents reduce infarct volumes, edema and hemorrhagic conversion, and improve outcomes in rodent models of ischemic stroke. Retrospective studies suggest that diabetic patients on sulfonylurea drugs at stroke presentation fare better if they continue on drug. Additional laboratory investigations have implicated Sur1 in the pathophysiology of hemorrhagic CNS insults. In clinically relevant models of subarachnoid hemorrhage, glibenclamide reduces adverse neuroinflammatory and behavioral outcomes. Here, we provide an overview of the preclinical studies of glibenclamide therapy for CNS ischemia and hemorrhage, discuss the available data from clinical investigations, and conclude with promising preclinical results that suggest glibenclamide may be an effective therapeutic option for ischemic and hemorrhagic stroke.     

The Role of Matrix Metalloproteinase Polymorphisms in Ischemic Stroke

Stroke remains the fifth leading cause of mortality in the United States with an annual rate of over 128,000 deaths per year. Differences in incidence, pathogenesis, and clinical outcome have long been noted when comparing ischemic stroke among different ethnicities. The observation that racial disparities exist in clinical outcomes after stroke has resulted in genetic studies focusing on specific polymorphisms. Some studies have focused on matrix metalloproteinases (MMPs). MMPs are a ubiquitous group of proteins with extensive roles that include extracellular matrix remodeling and blood-brain barrier disruption. MMPs play an important role in ischemic stroke pathophysiology and clinical outcome. This review will evaluate the evidence for associations between polymorphisms in MMP-1, 2, 3, 9, and 12 with ischemic stroke incidence, pathophysiology, and clinical outcome. The role of polymorphisms in MMP genes may influence the presentation of ischemic stroke and be influenced by racial and ethnic background. However, contradictory evidence for the role of MMP polymorphisms does exist in the literature, and further studies will be necessary to consolidate our understanding of these multi-faceted proteins.     

Role of Regular Physical Activity in Neuroprotection against Acute Ischemia

One of the major obstacles that prevents an effective therapeutic intervention against ischemic stroke is the lack of neuroprotective agents able to reduce neuronal damage; this results in frequent evolution towards a long-term disability with limited alternatives available to aid in recovery. Nevertheless, various treatment options have shown clinical efficacy. Neurotrophins such as brain-derived neurotrophic factor (BDNF), widely produced throughout the brain, but also in distant tissues such as the muscle, have demonstrated regenerative properties with the potential to restore damaged neural tissue. Neurotrophins play a significant role in both protection and recovery of function following neurological diseases such as ischemic stroke or traumatic brain injury. Unfortunately, the efficacy of exogenous administration of these neurotrophins is limited by rapid degradation with subsequent poor half-life and a lack of blood–brain-barrier permeability. Regular exercise seems to be a therapeutic approach able to induce the activation of several pathways related to the neurotrophins release. Exercise, furthermore, reduces the infarct volume in the ischemic brain and ameliorates motor function in animal models increasing astrocyte proliferation, inducing angiogenesis and reducing neuronal apoptosis and oxidative stress. One of the most critical issues is to identify the relationship between neurotrophins and myokines, newly discovered skeletal muscle-derived factors released during and after exercise able to exert several biological functions. Various myokines (e.g., Insulin-Like Growth Factor 1, Irisin) have recently shown their ability to protects against neuronal injury in cerebral ischemia models, suggesting that these substances may influence the degree of neuronal damage in part via inhibiting inflammatory signaling pathways. The aim of this narrative review is to examine the main experimental data available to date on the neuroprotective and anti-ischemic role of regular exercise, analyzing also the possible role played by neurotrophins and myokines.     

Identification of Novel Circulating miRNAs in Patients with Acute Ischemic Stroke

Ischemic strokes are associated with significant morbidity and mortality, but currently there are no reliable prognostic or diagnostic blood biomarkers. MicroRNAs (miRNAs) regulate various molecular pathways and may be used as biomarkers. Using RNA-Seq, we conducted comprehensive circulating miRNA profiling in patients with ischemic stroke compared with healthy controls. Samples were collected within 24 h of clinical diagnosis. Stringent analysis criteria of discovery (46 cases and 95 controls) and validation (47 cases and 96 controls) cohorts led to the identification of 10 differentially regulated miRNAs, including 5 novel miRNAs, with potential diagnostic significance. Hsa-miR-451a was the most significantly upregulated miRNA (FC; 4.8, FDR; 3.78 × 10⁻⁸⁵), while downregulated miRNAs included hsa-miR-574-5p and hsa-miR-142-3p, among others. Importantly, we computed a multivariate classifier based on the identified miRNA panel to differentiate between ischemic stroke patients and healthy controls, which showed remarkably high sensitivity (0.94) and specificity (0.99). The area under the ROC curve was 0.97 and it is superior to other current available biomarkers. Moreover, in samples collected one month following stroke, we found sustained upregulation of hsa-miR-451a and downregulation of another 5 miRNAs. Lastly, we report 3 miRNAs that were significantly associated with poor clinical outcomes of stroke, as defined by the modified Rankin scores. The clinical translation of the identified miRNA panel may be explored further.     

The Potential of Adaptive Design in Animal Studies

Clinical trials are the backbone of medical research, and are often the last step in the development of new therapies for use in patients. Prior to human testing, however, preclinical studies using animal subjects are usually performed in order to provide initial data on the safety and effectiveness of prospective treatments. These studies can be costly and time consuming, and may also raise concerns about the ethical treatment of animals when potentially harmful procedures are involved. Adaptive design is a process by which the methods used in a study may be altered while it is being conducted in response to preliminary data or other new information. Adaptive design has been shown to be useful in reducing the time and costs associated with clinical trials, and may provide similar benefits in preclinical animal studies. The purpose of this review is to summarize various aspects of adaptive design and evaluate its potential for use in preclinical research.     

Neuroglobin, a Novel Target for Endogenous Neuroprotection against Stroke and Neurodegenerative Disorders

Brain neurons and tissues respond to sublethal injury by activating endogenous protective pathways. Recently, following the failure of a large number of clinical trials for protective strategies against stroke that aim to inhibit a specific ischemia response pathway, endogenous neuroprotection has emerged as a more promising and hopeful strategy for development of therapeutics against stroke and neurodegenerative disorders. Neuroglobin (Ngb) is an oxygen-binding globin protein that is highly and specifically expressed in brain neurons. Accumulating evidence have clearly demonstrated that Ngb is an endogenous neuroprotective molecule against hypoxic/ischemic and oxidative stress-related insults in cultured neurons and animals, as well as neurodegenerative disorders such as Alzheimer's disease, thus any pharmacological strategy that can up-regulate endogenous Ngb expression may lead to novel therapeutics against these brain disorders. In this review, we summarize recent studies about the biological function, regulation of gene expression, and neuroprotective mechanisms of Ngb. Furthermore, strategies for identification of chemical compounds that can up-regulate endogenous Ngb expression for neuroprotection against stroke and neurodegenerative disorders are discussed.     

Application of Perinatal Derivatives on Oncological Preclinical Models: A Review of Animal Studies

The increasing cancer incidence has certified oncological management as one of the most critical challenges for the coming decades. New anticancer strategies are still needed, despite the significant advances brought to the forefront in the last decades. The most recent, promising therapeutic approaches have benefitted from the application of human perinatal derivatives (PnD), biological mediators with proven benefits in several fields beyond oncology. To elucidate preclinical results and clinic outcomes achieved in the oncological field, we present a narrative review of the studies resorting to animal models to assess specific outcomes of PnD products. Recent preclinical evidence points to promising anticancer effects offered by PnD mediators isolated from the placenta, amniotic membrane, amniotic fluid, and umbilical cord. Described effects include tumorigenesis prevention, uncontrolled growth or regrowth inhibition, tumor homing ability, and adequate cell-based delivery capacity. Furthermore, PnD treatments have been described as supportive of chemotherapy and radiological therapies, particularly when resistance has been reported. However, opposite effects of PnD products have also been observed, offering support and trophic effect to malignant cells. Such paradoxical and dichotomous roles need to be intensively investigated. Current hypotheses identify as explanatory some critical factors, such as the type of the PnD biological products used or the manufacturing procedure to prepare the tissue/cellular treatment, the experimental design (including human-relevant animal models), and intrinsic pathophysiological characteristics. The effective and safe translation of PnD treatments to clinical practice relies on the collaborative efforts of all researchers working with human-relevant oncological preclinical models. However, it requires proper guidelines and consensus compiled by experts and health workers who accurately describe the methodology of tissue collection, PnD isolation, manufacturing, preservation, and delivery to the final user.     

Using Noninvasive Electrophysiology to Determine Time Windows of Neuroprotection in Optic Neuropathies

The goal of neuroprotection in optic neuropathies is to prevent loss of retinal ganglion cells (RGCs) and spare their function. The ideal time window for initiating neuroprotective treatments should be the preclinical period at which RGCs start losing their functional integrity before dying. Noninvasive electrophysiological tests such as the Pattern Electroretinogram (PERG) can assess the ability of RGCs to generate electrical signals under a protracted degenerative process in both clinical conditions and experimental models, which may have both diagnostic and prognostic values and provide the rationale for early treatment. The PERG can be used to longitudinally monitor the acute and chronic effects of neuroprotective treatments. User-friendly versions of the PERG technology are now commercially available for both clinical and experimental use.     

Spontaneous and Engineered Large Animal Models of Neurofibromatosis Type 1

Animal models are crucial to understanding human disease biology and developing new therapies. By far the most common animal used to investigate prevailing questions about human disease is the mouse. Mouse models are powerful tools for research as their small size, limited lifespan, and defined genetic background allow researchers to easily manipulate their genome and maintain large numbers of animals in general laboratory spaces. However, it is precisely these attributes that make them so different from humans and explains, in part, why these models do not accurately predict drug responses in human patients. This is particularly true of the neurofibromatoses (NFs), a group of genetic diseases that predispose individuals to tumors of the nervous system, the most common of which is Neurofibromatosis type 1 (NF1). Despite years of research, there are still many unanswered questions and few effective treatments for NF1. Genetically engineered mice have drastically improved our understanding of many aspects of NF1, but they do not exemplify the overall complexity of the disease and some findings do not translate well to humans due to differences in body size and physiology. Moreover, NF1 mouse models are heavily reliant on the Cre-Lox system, which does not accurately reflect the molecular mechanism of spontaneous loss of heterozygosity that accompanies human tumor development. Spontaneous and genetically engineered large animal models may provide a valuable supplement to rodent studies for NF1. Naturally occurring comparative models of disease are an attractive prospect because they occur on heterogeneous genetic backgrounds and are due to spontaneous rather than engineered mutations. The use of animals with naturally occurring disease has been effective for studying osteosarcoma, lymphoma, and diabetes. Spontaneous NF-like symptoms including neurofibromas and malignant peripheral nerve sheath tumors (MPNST) have been documented in several large animal species and share biological and clinical similarities with human NF1. These animals could provide additional insight into the complex biology of NF1 and potentially provide a platform for pre-clinical trials. Additionally, genetically engineered porcine models of NF1 have recently been developed and display a variety of clinical features similar to those seen in NF1 patients. Their large size and relatively long lifespan allow for longitudinal imaging studies and evaluation of innovative surgical techniques using human equipment. Greater genetic, anatomic, and physiologic similarities to humans enable the engineering of precise disease alleles found in human patients and make them ideal for preclinical pharmacokinetic and pharmacodynamic studies of small molecule, cellular, and gene therapies prior to clinical trials in patients. Comparative genomic studies between humans and animals with naturally occurring disease, as well as preclinical studies in large animal disease models, may help identify new targets for therapeutic intervention and expedite the translation of new therapies. In this review, we discuss new genetically engineered large animal models of NF1 and cases of spontaneous NF-like manifestations in large animals, with a special emphasis on how these comparative models could act as a crucial translational intermediary between specialized murine models and NF1 patients.     

Preventing Brain Injury in the Preterm Infant—Current Controversies and Potential Therapies

Preterm birth is associated with a high risk of morbidity and mortality including brain damage and cerebral palsy. The development of brain injury in the preterm infant may be influenced by many factors including perinatal asphyxia, infection/inflammation, chronic hypoxia and exposure to treatments such as mechanical ventilation and corticosteroids. There are currently very limited treatment options available. In clinical trials, magnesium sulfate has been associated with a small, significant reduction in the risk of cerebral palsy and gross motor dysfunction in early childhood but no effect on the combined outcome of death or disability, and longer-term follow up to date has not shown improved neurological outcomes in school-age children. Recombinant erythropoietin has shown neuroprotective potential in preclinical studies but two large randomized trials, in extremely preterm infants, of treatment started within 24 or 48 h of birth showed no effect on the risk of severe neurodevelopmental impairment or death at 2 years of age. Preclinical studies have highlighted a number of promising neuroprotective treatments, such as therapeutic hypothermia, melatonin, human amnion epithelial cells, umbilical cord blood and vitamin D supplementation, which may be useful at reducing brain damage in preterm infants. Moreover, refinements of clinical care of preterm infants have the potential to influence later neurological outcomes, including the administration of antenatal and postnatal corticosteroids and more accurate identification and targeted treatment of seizures.