Gary K. Steinberg - US grants

Cerebrovascular disease, or stroke is the third leading cause of death in the United States and a major cause of disability. At the present time there is no treatment of proven efficacy in treating or ameliorating strokes. Recent evidence has accumulated suggesting that specific antagonism of postsynaptic glutamate receptors, especially the N-methyl-D-aspartate (NMDA) receptor protects neurons from hypoxic or ischemic injury. Over the last three years my laboratory has shown that dextromethorphan (DM), an NMDA antagonist, and its major metabolite, dextrorphan (DX), have a significant protective effect against focal cerebral ischemia in our rabbit model when given prior to the onset of ischemia and also when administered in a delayed fashion one hour after the onset of ischemia. The purpose of this research is to further elucidate the precise conditions of focal cerebral ischemia that will respond to treatment with DM or DX and to delineate the mechanism of protection. Focal cerebral ischemia will be produced in anesthetized rabbits by transorbital, clip occlusion of the internal carotid artery, anterior cerebral artery and middle cerebral artery. The degree of cerebral ischemia will be determined using electrophysiological (somatosensory evoked potentials), radiological (magnetic resonance imaging), histological and behavioral criteria. Experiments will characterize the dose response curve for both drugs regarding cerebral protection and toxicity, will determine the relative roles of loading versus maintenance drug therapy, and will define the maximal delay after ischemia that these drugs can be administered and still demonstrate neuroprotection. Regional cerebral blood flow will be measured using two techniques (laser Doppler flowmetry and radioactive microspheres to determine whether the drugs' neuroprotective effects and regional selectivity are due to changes in blood flow. The efficacy of these agents will also be examined over a longer period of time (24 hours) and brain levels will be correlated with plasma levels. DM and DX have already been clinically tested and are known to be relatively free of side effects. If the studies outlined in this proposal confirm the benefit of DM or DX in protecting against cerebral ischemia, the implications for treatment of humans with stroke could be profound in terms of reducing neurologic morbidity or mortality. The results from the proposed project will expedite the planning and execution of a clinical study.

DESCRIPTION: (Applicant's Abstract) In recent years there has been a resurgence of interest in mild hypothermia (30-35 degrees C) as a method of cerebral protection. This project aims at defining optimal parameters in which mild hypothermia can be applied in animal models of focal stroke and explores the mechanisms of neuroprotection by which temperature manipulations and pharmacological intervention may work. Using an intraluminal suture occlusion model of focal cerebral ischemia in the rat, a series of experiments will be performed to: 1) determine the optimal depth and duration of hypothermia which provides maximal reduction in infarct size while avoiding systemic complications; 2) address the extent to which hypothermia can be delayed and still be neuroprotective as well as whether the protective effects are sustained over time; 3) determine if mild hypothermia protects against both permanent and transient focal ischemia; 4) characterize the physiological changes resulting from mild hypothermia under ischemic conditions including its effects on metabolism, excitatory amino acid release, cerebral blood flow, and blood-brain-barrier integrity; and 5) determine whether the neuroprotective effects of mild hypothermia and glutamate receptor antagonists are additive. In order to examine the feasibility of hypothermia in a potential clinical setting, hypothermia will be used in conjunction with a thrombolytic agent in a rabbit model of thromboembolic stroke. The time course of ischemia and response to treatment with hypothermia and thrombolysis will be monitored using sequential diffusion and perfusion MRIs. This project will also investigate the use of diffusion-weighted MRI (DWI) in characterizing ischemic stroke in response to alterations in brain temperature. The time course of diffusion changes and infarct size will be studied as well as the potential of DWI as a predictor of infarct severity. The knowledge gained from this project will have direct implications for the treatment of ischemic stroke in humans including its use in both medical and surgical patients with cerebrovascular disease.

The integrating theme of this project is that three classes of gene products are intimately involved with modulating injury of neurons and glia following hypoxia/ischemia and, more specifically, that altering expression of these gene products can attenuate such hypoxic/ischemic damage. The program aims to define more precisely the roles of SOD, bcl-2 and hsp70 in modifying the detrimental effects of oxidative stress on cerebral neurons/glia and determining the ultimate survival or death (necrotic versus apoptotic) of these cells. The selection of these three genes for intense study reflects their potential therapeutic value, as well as the important information their investigation will yield regarding basic biologic mechanisms of ischemic cell death in central nervous tissue. The experimental results of this project could potentially lead to novel strategies for treating clinical stroke, as well as other traumatic and degenerative neurologic disorders showing a similar pathogenesis. The Program includes three interrelated projects utilizing advanced molecular biology techniques, including gene transfer therapy and transgenic technology in several paradigms of both in vitro and in vivo neuronal and glial injury. The protective potential of the three different genes will be examined using gene transfer in neuronal and glial cultures, and in transgenic and knockout cultures under anoxic and aglycemic conditions (Project 1). The neuroprotective benefit of the same three classes of genes will be studied in several models of ischemia using gene transfer in genetically normal rats (Project 2), as well as investigating protection against ischemia in transgenic or knockout rodents (Project 3). The Vector Core 9Core C) will prepare viral vectors to be used in the gene transfer experiments of Projects 1 and 2. The Transgenic Core (Core B) will produce transgenic mice and rats, as well as knockout mutants to be used in Projects 1 and 3. The Administrative Core (Core A) will provide grant management, financial administration, statistical consultation, centralized purchase, seminar arrangements, clerical assistance, and scientific consultation through Advisory Committees.

Recent progress in the area of stroke research suggests that a number of molecular mechanisms are intimately involved in the evolution of ischemic brain injury. Gene induction has been observed following ischemia, but the exact roles of many are not yet well known. Some gene products are known to be detrimental to the cell while others are felt to be neuroprotective. The goals of this project are to define more precisely the roles of three classes of genes which may play neuroprotective roles. They are: the proto oncogene, bcl-2, antioxidant genes (sod-1 and gspx) and the stress protein, hsp70. In Project 2, we will utilize genetically normal animals and study 3 different in vivo models of ischemia (2 focal and one global models of cerebral ischemia). We will alter gene expression via gene transfer using defective herpes simplex and adenoviral vectors. We will study the limits and conditions under which gene over-expression may improve neuron survival. We hypothesize that injury due to some, but not other kinds of insults will be attenuated with gene product over-expression, and that these observations will offer insight into the pathophysiology of cell death. We will examine whether gene transfer after the onset of injury is neuroprotective, and whether over-expression of Bcl-2 and antioxidant genes are protective against permanent as well as transient focal cerebral ischemia. We will also examine mechanisms underlying neuroprotection, or lack of neuroprotection, by examining the participation of other gene products such as the stress proteins, caspases and Bcl-2 family proteins in response to gene over-expression and cerebral injury. We will also study whether gene over-expression alters generation of superoxide and apoptosis. These novel approaches will hopefully add insight into the complex molecular processes involved in cerebral ischemia and may lead to the development of treatments for stroke and other degenerative disorders.

Mild hypothermia is the most effective therapy against experimental ischemic cerebral damage that currently exists. Mild hypothermia is already being successfully used in the treatment of acute traumatic brain injury and the feasibility of using it to treat stroke patients is currently being evaluated in some clinical trials. While multiple mechanisms for hypothermia-induced neuroprotection have been suggested, these mechanisms remain unclear and several key issues still need to be addressed. This project using a fresh approach using molecular biology techniques to test the hypothesis that mild hypothermia's neuroprotective benefit is due in part to a) attenuation of deleterious reactive oxygen species (ROS) and b) to inhibition of apoptotic cell death. Models of focal and global cerebral ischemia will be used and mild hypothermia will be applied to intraischemically and in a delayed fashion to mimic clinically relevant paradigms. A series of experiments will systemically examine the effects of mild hypothermia on 1) the generation of ROS in various brain regions during early reperfusion and at the peak of the inflammatory process; 2) the expression of endogenous antioxidants such as superoxide dismutases (SOD1 and SOD2), and glutathione peroxidase; 3) the expression of a death promoting (Bax) and death suppressing gene (Bcl-2); 4) the release of cytochrome c and caspase expression (ICE, CPP32); 5) DNA fragmentation characteristic of programmed cell death. This proposal will further test whether mild hypothermia can attenuate the increased neuronal damage observed in SOD-deficient mice and whether it does so by reducing ROS production, by altering the cytosolic translocation of cytochrome c, and/or by altering the regulation of Bcl-2 and Bax. The ability of post-ischemic administration of an antioxidant to extend the therapeutic window of mild hypothermia will also be tested. The knowledge gained from this project will elucidate novel cellular and molecular mechanisms underlying mild hypothermia's neuroprotective benefit, contribute to understanding the pathophysiology of ischemic cerebral injury, and may have important implications for the treatment of clinical stroke in humans.

The integrating theme of this PPG is to investigate the role of oxidative stress and reactive oxygen species (ROS) in mediating ischemic brain injury, focusing on detrimental effects involving neurons, astrocytes, microglia and endothelial cells, and the interactions between these various components in contributing to cerebral damage. Our initial PPG studied the role of SOD, Bcl-2 and HSP70 in protecting against necrotic and apoptotic neuronal and astroglial death. The current PPG represents an evolution in our approach based on results obtained in our labs over the last 5 years. It has become clear that ischemia/reperfusion induced ROS are also involved in inflammatory events mediated by microglia, leukocytes and astrocytes as well as causing endothelial cell death, in addition to direct neuronal and astroglial injury. It is also apparent that downstream apoptotic events precipitated by ROS are more complex and interrelated than previously assumed. We therefore propose to explore the delayed effects of ROS mediated injury on neurons, astrocytes, microglia and endothelial cells. The development of the 3 interrelated projects has benefited greatly from our ongoing collaboration during the first 5 years of this Program, and will continue to employ advanced molecular biology techniques, including gene transfer, transgenic/knockout technology and bone marrow transplant chimeras using in vivo and in vitro models of ischemic injury. Project by Giffard will examine ROS-related mechanisms of microglial exacerbation of ischemic injury, in vivo and in vitro. Project by Steinberg will study the role of ROS in initiating key apoptotic pathways leading to neuronal death using in vivo and in vitro models. Project by Chan will explore mechanisms of endothelial cell death in ROS-mediated brain damage utilizing in vivo models. The Transgenic Core will provide transgenic and knockout mice for use in each project, and the Vector Core will prepare viral vectors for gene transfer to be used in Projects by Giffard and Steinberg. The administrative Core will provide grant management, financial administration, centralized purchasing, seminar arrangement, statistical consulting, clerical and scientific consultation through the Advisory Committees.

statistics /biometry

Reactive oxygen species (ROS) generated after cerebral ischemia and reperfusion activate both caspase-dependent and caspase-independent pathways leading to delayed neuronal death. This project will use a combination of in vivo and in vitro models of ischemia, viral-mediated gene transfer, and knockout models to address the central role of oxidative stress and ROS in initiating key apoptotic pathways. It will also explore the temporal dynamics and interactions between those pathways, and determine whether hypothermia alters those dynamics and interactions, and thus enhances neuroprotection. Additionally, preservation of neuronal function conferred by gene transfer therapy will be examined. First, we will explore the role of reactive oxygen species (ROS) in activating both caspase-dependent and -independent apoptotic events, assess whether and how they are altered by over-expression of anti-oxidant genes, whether such over-expression protects ischemia-vulnerable SOD2 knockout mice, and explore effects of deltaPKC on ROS and apoptosis post-ischemia. Second, we propose to examine the interactions between various mediators of apoptotic pathways after cerebral ischemia. We will investigate the effects of gene therapy using over-expression of the caspase inhibitors p35 and crmA, as well as pharmacologic caspase antagonists, and examine their effects on interactions between AIF- and caspase-dependent apoptotic pathways after ischemia. Finally, we will investigate whether post-ischemic hypothermia prolongs the temporal therapeutic window for gene therapy against global cerebral ischemia, and whether gene therapy with or without hypothermia spares neuronal function. Specifically, we will determine whether hypothermia blocks or delays ROS activity and apoptotic mediators, prolongs the time window for protection by over-expression of GPX, catalase, or other anti-apoptotic proteins, and permits gene therapy to spare neuronal function following global ischemia. We believe that by combining well-established in vivo and in vitro models of stroke with gene transfer and transgenic technology, we can apply unique and novel approaches to elucidate ROS-related mechanisms of neuronal death, survival, and function.

DESCRIPTION (provided by applicant): Stroke is the number one cause of disability among Americans each year. Currently there is no therapy to cure stroke patients except the thrombolytic treatments, which have limited use. Our long-term goal is to promote functional recovery from stroke using human neural progenitor cells (hNPCs) as a potential therapy. We and others have shown that neural stem/progenitor in some cases can improve neurological function in rodents. However, transplant viability and functional outcome vary widely across studies. Our overall hypothesis is that hNPCs facilitate long-term functional by enhancing endogenous repair mechanisms through secretion of trophic factors. Including a focus on the trophic factors gives a mechanistic understanding of how transplanted stems cells augment endogenous repair processes. Importantly, we do not believe that the cells enhance recovery integrating into the host brain circuitry. In Specific Aim 1, we determine the effect of the transplanted cells on several endogenous repair mechanisms as well as the trophic factors expressed by the hNPCs in vivo over time, and then correlate these phenomena with functional recovery. We then test specific factors by manipulating their expression levels in hNPCs before transplantation. In Specific Aim 2, we determine the host microenvironment that is most conducive to cell-induced repair by varying the timing of transplantation post- stroke, with the goal of finding the optimal time to transplant. We also test the interplay between the host microenvironment and hNPCs by surveying host factors that are affected by hNPCs and also modifying the hNPCs'sensitivity to signals for migration and survival from the host's microenvironment. Together these aims will help identify the optimal time to transplant human neural progenitor cells after stroke and link successful cell therapy with critical molecular and cellular mechanisms that underlie endogenous repair after stroke. Graft survival and biology, and its effect on host repair mechanisms, will be assessed using immunohistochemistry. Functional recovery will be examined using behavioral tests. Our expertise in stroke research and cellular therapies (Kelly, 2004), neural stem cell biology and culture methods (Palmer, 2001), synaptogenesis (Christopherson, 2005), imaging (Micheva, 2007) and genetic manipulation of hNPCs (Suzuki, 2007) provide an excellent opportunity to develop a cross-disciplinary effort to study cell transplants for brain injury at Stanford.

? DESCRIPTION (provided by applicant): The purpose of this Stanford Neurosurgery Resident Research Education Program is to provide education in basic and clinical research to neurosurgery residents with the goal of fostering their growth into clinician scientists. Its goal s to educate and train neurosurgeons capable of establishing and directing a scientific laboratory throughout their careers. The strategy is to identify residents with the potential for scientific research and place them for a two year term in the laboratories of senior, highly experienced research scientists. They will also receive extensive counseling by the neurosurgical directors of the program and participate in seminars on research skills and ethical research behavior. Their progress and the success of the overall program will be repeatedly assessed and appropriate changes in the program will be made. It is hoped that this program will produce neurosurgeons capable of making new discoveries regarding the causes of diseases of the brain and spinal cord, that new, more effective treatments will result, and, in fulfillment of the mission of the NI, that the health of persons afflicted with these diseases will be improved.

Center Core Grants; Genes; Microscopy; National Institute of Neurological Disorders and Stroke; Neurosciences Research; vector

This proposal seeks to establish essential core research facilities to meet the following identified shared needs of the Stanford Neuroscience research community: (1) gene vector and virus production, (2) advanced microscopy data acquisition and analysis, and (3) automated behavioral phenotyping. Viral vectors are used to express recombinant proteins and/or knock down expression of endogenous proteins in specific subsets of cells in brain tissue. It is even possible to express proteins that enable precise temporal control over the activity of individual neurons. Use of such viral tools is revolutionizing neuroscience research. A centralized core facility will vastly improve the efficiency and cost-effectiveness of virus production. Three dimensional image analysis and visualization are essential for quantifying information from volume imaging techniques, such as Array Tomography, and single- and two-photon confocal microscopy. Software and hardware for this type of analysis is expensive, and has a steep learning curve. This proposal will fund the Image Analysis Center, a central resource with technical expertise to assist scientists with image analysis problems, and an electrophysiological recording setup for an existing shared two-photon tissue slice rig. Standardized, replicable behavioral tests are critical to translating progress from basic neuroscience research to treatments relevant to human disease. Automated behavioral phenotyping can provide more consistent results by eliminating stress due to removal from the home cage and novelty effects from the test environment. Automated testing can also Improve throughput and reduce costs. This proposal will provide funds to expand capacity for automated behavioral testing in an existing core facility. These core facilities will be a central part of SINTN's effort to advance our understanding of normal brain and spinal cord function at the molecular, cellular, and neural circuit level, and to elucidate the pathological processes underlying malfunction of the nervous system following injury or neurologic and psychiatric diseases. Creating core services to meet these shared research needs will foster efficiency and productivity by minimizing the unnecessary duplication of equipment and creating a centralized source of expertise for shared tasks with the net effect of better solutions in less time. Moreover, the resulting formal and informal collaborations will provide the foundation for a richer, stronger, and more vibrant research community.

DESCRIPTION (provided by applicant): Stroke, a leading cause of serious long-term disability in the US, is in need of therapeutics that reduce damage and promote recovery. Post-stroke inflammation is a critical determinant of damage and recovery and is thus a promising therapeutic target. Mast cells (MCs), which play critical roles in the development of inflammatory processes in other pathologies, were recently ascribed a role in the exacerbation of post-stroke brain inflammation and damage. This suggests that MCs could be key determinants of stroke-induced inflammation and hence are an attractive therapeutic target. The primary focus of this proposal is to elucidate the mechanism of action of mast cells in exacerbating stroke pathology, with the long-term goal of identifying novel therapeutic strategies for stroke. Our overall hypothesis is that mast cells residing in the meninges are key effectors of stroke pathology. This is tested in Aim 1 using two different approaches. The gain-of-function approach (Aim 1a) uses a mast cell knock-in mouse model in which the MC-deficiency of genetically MC- deficient mice is selectively repaired by engraftment of in vitro grown mast cells. Direct engraftment of MCs into the meninges of these MC-deficient mice will determine the involvement of meningeal-located MCs in stroke pathology. This is complemented by the loss-of-function approach (Aim1b) where meningeal MCs are depleted by meningeal injection of diphtheria toxin (DT) into a novel mouse model that selectively expresses the DT receptor on MCs. Together, innovative use of these mouse models enables us to determine if meningeal MCs are necessary and sufficient for the detrimental effects of MCs after stroke. Aim 2 addresses the molecular mechanism of action of MCs using the mast cell knock-in mouse model. Through engraftment of MCs from various receptor knock-out mice we will investigate the mechanism of meningeal MC activation (Aim 2a), and by engraftment of MCs from cytokine knock-out mice we will identify mast cell-secreted factors important for the downstream effects of MCs on stroke pathology (Aim 2b). Identifying a crucial role for meningeal MCs after stroke will highlight the importance o the meninges in modulating brain pathology. As the meninges are relatively accessible (e.g., by intrathecal injection) this concept could potentially present a new strategy for stroke therapeutic that may overcome the hurdle of targeting drugs to the injured brain and reduce unwanted side effects of systemic immunomodulation. Furthermore, by establishing the mechanism of action of MCs we begin to delineate the molecular pathways involved in modulating the response to stroke, an essential step to finding novel therapeutic targets. Together, our proposed studies address significant gaps in the understanding of post-stroke inflammatory events that contribute to stroke pathology and may identify new strategies for stroke therapeutics.

DESCRIPTION (provided by applicant): Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Each year about 800,000 people are affected by stroke in the United States, and most survivors often live with long-term disability. Functional recovery can occur after stroke, and this recovery is attributed to brain remodeling and neuroplasticity, when the brain repairs and rebuilds connections between neurons. Brain stimulation techniques such as electrical stimulation or transcranial magnetic stimulation have been used in animals and humans to enhance recovery after stroke. However, the neural circuits involved and the mechanisms mediating this recovery are not well understood. In addition, these stimulation techniques non-specifically stimulate all cell types near the stimulation site, leading to undesired side effects. In this proposal, we will use the optogenetic approach to specifically stimulate neurons after stroke and examine the effects on functional recovery and the underlying mechanisms. Optogenetics is a novel strategy that utilizes light-sensitive algal proteins, such as Channelrhodopsin (ChR2), to manipulate the excitability of specific cell groups in the brain, in a fast and precise manner. Optogenetic stimulation can increase neuronal excitability, potentially leading to release of neurotrophic factors, enhancement of axonal spouting/synaptogenesis and increased cerebral blood flow, all of which are important in functional recovery after stroke. Therefore, we hypothesize that optogenetic stimulation of neurons in the primary motor cortex (M1) can augment endogenous repair/plasticity mechanisms and promote recovery after stroke. We will use a transgenic mouse line expressing ChR2 under a neuronal promoter to test our hypothesis. Our preliminary data show that optogenetic stimulation of neurons in the ipsilesional primary motor cortex of mice improved their behavioral recovery after stroke. In Aim 1, we will use sensorimotor behavior tests to evaluate functional recovery after optogenetic neuronal stimulation in various brain regions after stroke. We will start stimulation during the recovery phase of stroke and determine the most optimal brain stimulation target for promoting stroke recovery. In Aim2, we will investigate the underlying mechanisms that drive this recovery, including changes in cerebral blood flow, release of neurotrophic factors, axonal sprouting and synaptogenesis. Our study will advance the understanding of endogenous repair and plasticity mechanisms underlying recovery after stroke, as well as determine the most optimal brain stimulation target to promote recovery. Understanding the proteins and processes involved during repair and recovery could lead to novel discoveries of therapeutic drug targets able to facilitate recovery after stroke.

DESCRIPTION (provided by applicant): Stem cell therapy offers great promise in the search for an effective treatment for stroke, a leading cause of serious adult disability in the US afflicting nearly 800,000 Americans annually at a societal cost of billions of dollars. Preclinical data indicate that stem cell therapy, by targeting brain repair processes such as brain plasticity, has a therapeutic window of weeks to months implying it could benefit the majority of stroke patients. The mechanisms mediating stem cell-mediated recovery are, however, poorly understood. This will be important to dissect, as elucidating stem cell mechanisms of action will begin to delineate the molecular pathways of brain repair, and could also lead to improved efficacy and safety, and thus success, of stem cell therapy as it translates to the clinic. Our proposed study using cutting edge tools-TRAP, array tomography, and electrophysiology-will overcome existing technical barriers and enable us to address two significant gaps in our understanding of stem cell mechanisms: 1) What do transplanted stem cells express in vivo that promote brain plasticity and recovery? Based on previous work from us and others we hypothesize that human neural stem cells (hNPCs) elicit recovery by secreting paracrine factors that modulate brain plasticity. However, identifying these paracrine factors in vivo has been a challenge due to difficulties separating the hNPC and host expression profiles. We overcome this hurdle using the novel TRAP technique to separate transplanted hNPC mRNA from host brain mRNA. In Aim 1 we use TRAP plus microarray to generate the first in vivo transcriptional profile of hNPCs transplanted into the stroke brain and to identify candidate plasticity-modulating factors. We will then perform shRNA knockdown of these candidates in hNPCs and evaluate their effects on functional recovery and neurite plasticity using complementary in vitro and in vivo assays. 2) What synapse-level brain changes are elicited by stem cells that promote recovery? We reported that hNPCs enhance post-stroke plasticity by promoting axonal and dendritic sprouting. What is not known is how stroke and hNPCs affect synapse formation and function, which is ultimately where plasticity changes must occur to promote recovery. Such details were previously unattainable due to the complexity and minutia of synapses. The novel array tomography technique overcomes this hurdle enabling detailed structural analysis of individual synapses with cortical-layer specificity. Using this approach, we have identified a post- stroke increase in inhibitory GABAergic synapses and an hNPC-dependent increase in excitatory glutamatergic synapses. This has led to the hypothesis that hNPCs promote recovery by shifting the excitatory/inhibitory synaptic balance towards excitation. In Aim 2 we will use array tomography and electrophysiology, and our knockdown hNPC populations from Aim 1, to determine how hNPCs affect this balance, both in vitro and in vivo, and the resulting impact on post-stroke functional recovery.

PROJECT SUMMARY Stroke is the leading cause of death with very limited treatment options. This devastating neurological disease is increasingly viewed as a disease of brain connectivity as a damaged stroke area can affect both local and connected brain regions, causing disruptions in neuronal activity and metabolism network-wide. Recovery of lost function can occur after stroke and is attributed to brain remodeling in areas adjacent to or connected to the infarct. In this proposal, we aim to investigate the role of key brain circuits in post-stroke recovery at the functional, cellular and molecular level, using optogenetics, advanced live imaging and high throughput RNA sequencing techniques. Previously our lab has demonstrated that selective optogenetic neuronal stimulation in the ipsilesional motor cortex (iM1) can activate plasticity mechanisms and promote recovery. Recently we have employed the optogenetic functional MRI technique to systematically map brain-wide changes in neural circuits after stroke. We have identified key circuits altered by stroke and demonstrated two key circuits restored by iM1 stimulations. Our map data also revealed two candidate circuits that were not restored by iM1 stimulations, suggesting that greater recovery could be achieved if we can rescue these circuits by directly stimulating them. In this proposal we aim to investigate key neural circuits we identified from our activation maps and elucidate their role in post-stroke recovery. In Aim1 we will use circuit-specific optogenetic tools and functional behavior tests to interrogate the role of key circuits in post-stroke recovery. This aim will address whether these circuits have beneficial or maladaptive role during post-stroke recovery. In Aim2 we will examine cellular resolution of real-time neuronal activity dynamics in key circuits after stroke using a portable live calcium imaging system. This will elucidate the neural activity dynamics (excitatory and inhibitory) of key circuits at the cellular level, allowing us to identify the temporal profile and the key neuronal populations altered by stroke, and how iM1 stimulations affect these characteristics to enhance recovery. In Aim3 we will investigate the transcriptome of key circuit areas using RNAseq, in order to identify key molecular targets and pathways altered by stroke and by iM1 stimulations. Preliminary RNAseq analysis revealed distinct pathways altered by iM1 stimulations. We aim to perform RNAseq in multiple regions including iM1 (stimulation site) and ipsilesional thalamus (iM1- connected region) to elucidate whether similar pathways are involved, and if we can identify a common molecular signature that drive recovery. We will also perform RNAseq in both sexes in order to ascertain any sex-specific differences that may be present in post-stroke recovery. Together these results will 1) advance the understanding of neural circuit dynamics during post-stroke recovery; and 2) identify key neural circuits/cell types/molecular targets and optimal time window for designing brain stimulation strategies and other therapeutic interventions in future clinical studies.

? DESCRIPTION (provided by applicant): Stanford Neuroscience Cores for Behavior, Microscopy, and Gene Vectors Today our ability to study how the nervous system evolves during development, aging, learning, life experiences, or during disease progression is fundamentally dependent on our ability to make genetic constructs and viral products for genetic manipulation, and carry out functional validation in the whole animal, tissue slices or neuronal cells followed by post mortem and in-life imaging. The Stanford Neurosciences Cores for Gene Vectors, Behavior, and Microscopy were created in 2008-2010 to develop and centralize the underlying techniques that are fundamental to the research of the vast majority of Stanford University's neuroscience community. Towards this end, our interdisciplinary community of scholars deliberated on what would best aid them in achieving the Stanford Neurosciences Institute's (SNI) ultimate mission of using discovery to revolutionize our understanding of brain function. Careful assessment of the community's needs highlighted the necessity of a trio of state-of-the-art research cores that would be strategically equipped with resources that would provide critical support to the widest range of research projects. These cores would provide services and techniques that would not otherwise be attainable to each individual researcher, and the cores would be headed by knowledgeable leaders who operate at the frontiers of their respective specialty and are eager to provide guidance and consultation to their peers. From these efforts were born the Gene Vector and Virus Core (GVVC), the Neuroscience Microscopy Services (NMS), and the Behavioral and Functional Neuroscience Laboratory (BFNL). Invaluable support from Stanford University and the NINDS helped to make this creation a success. So far, nearly 200 Stanford laboratories and at least 75 publications have been supported by these cores, demonstrating that they have become an integral part of the neuroscience research effort at Stanford. The majority of the core users are NIH-funded neuroscience labs. In line with our mission, it has remained a central concern to support NINDS-funded projects. During the last 4 years the cores supported 23 NINDS investigators and 71 NIH investigators, holding grants from various NIH institutes and centers. The unique capabilities of these cores are not only a resource for users on our campus, but have also supported many users from different institutions regionally, nationally, and internationally. These external institutions include but are not limited to UC Berkeley, UCSF, the Gladstone Institute, UCSD, Yale University, Harvard University, Princeton University, Columbia University, University of Pennsylvania, University of Texas (Austin, El Paso), and MIT. We are already an established national resource and considering the pace of our growth, we expect our cores to become a significant resource for all NINDS-funded researchers in need of viral constructs and neurobehavioral and imaging services. Under the proposed grant we will continue to provide the most up-to date and cutting edge services in each of the three domains. We will also expand our services as we are moving forward. GVVC will be setting up production of canine adenovirus type-2 (CAV-2) and aims to provide larger scale AAV production using double CsCl gradients. NMS will continue with all of its microscopy and image analysis services, including efforts to expand the use of its super-resolution microscope and two photon microscopes. NMS also plans to replace an aging confocal microscope, and aims to expand services when the move to the new SNI research center makes more space available. SBFNL will allocate additional resources to address more actively some of the rising concerns in the field about the predictive value and inherent limitations of animal models in drug discovery and also innovate and expand upon its automated testing capabilities. All of these advances will be shared with the neuroscience community via our newly improved resource sharing websites. The Stanford Neuroscience Cores provide much-needed infrastructure at Stanford, and they have also supported researchers from across the country. Their activities, as demonstrated by the number of investigators and publications supported, have clearly made significant contributions to the NINDS mission to seek fundamental knowledge about the brain and nervous system, and to reduce the burden of neurological disease. Strong support from both NINDS and Stanford's Neuroscience Institute has been essential to the success of these cores. With continued support we will build on this success.

Project Summary (Administrative Core) The adminstrative Core was successfully created 4 years ago to oversee the strategic development of the Stanford neuroscience cores and to assist in the daily operation of the scientific cores, providing a solid administrative, financial, IT, and web infrastructure. The support and oversight of the administrative core has made the operation of these 3 cores a success. Going forward the administrative core will continue to oversee the strategic development of the Stanford Neuroscience Cores. The Administrative Core Steering Committee will provide overall strategic direction to the cores during their biannual or as needed meetings, which will bring together the leadership of the Stanford Neuroscience Institute and key NINDS-funded investigators to critically review and guide its operation. The Steering Committee will insure close coordination of cross-disciplinary projects conducted in the cores and will make sure the tools, material, and technology is widely and openly disseminated to the neuroscience community both at Stanford and internationally. In addition to strategic direction, the Administrative Core has and will continue to provide centralized support services that will enable the scientific cores to do a better job of delivering research services. With dedicated personnel, the Administrative Core has been assisting with internal advertising, soliciting and analyzing user feedback, and creating online communication tools for scientific core user groups. The Administrative Core will also provide the following essential support functions for the neuroscience cores with dedicated time from expert staff, 1) End-user communication and web infrastructure. 2) Information technology services, 3) Accounting and 4) General administrative support. This centralized administrative support has provided many benefits to the scientific cores to enhance their ability to deliver research support.

Human neural stem cell (hNSC) therapy for stroke is showing promise as it moves from the bench into early clinical trials to treat the long term disabilities resulting from stroke. This provides hope for the millions of Americans living with the chronic, debilitating effects of stroke. Major questions remain, however, about how stem cells injected into the brain drive stroke recovery. A clue to stem cell mechanism of action is the recent discovery of the positive correlation between stroke recovery and a brain MRI signal ? T2-FLAIR signal? in stroke patients treated with stem cells. We have successfully reproduced this stem cell-induced FLAIR signal in stroke-injured rats, and shown that its associated with inflammation. This led to our central hypothesis: stem cell transplantation drives recovery by inducing a regenerative inflammatory response. The objective of this grant is to use a rat model of subcortical stroke to investigate the immunomodulatory effects of hNSC transplanted at the chronic stage of stroke, at the regional, cellular and molecular levels using a multimodal approach. In Aim1 we use MRI and PET imaging to identify which brain regions show inflammatory changes after hNSC transplantation, and which inflammatory regions best correlate with recovery. This will also test the utility of these clinically relevant imaging modalities as biomarkers for stroke recovery. In Aim 2 we identify the immune changes induced by hNSC treatment using multiple tools to characterize the types and molecular signatures of the immune cells present, and their spatial interactions. In Aim 3 we determine which hNSC-secreted factors modulate the immune response by testing several candidates, in vitro and in vivo, using CRISPR tools to modulate expression of key candidate factors. We will study the impact of manipulating the levels of these proteins on stroke recovery and immunomodulation. Upon conclusion of the study, we will have made significant advancements in understanding how hNSC-induced immunomodulation affects brain repair. This contribution is significant because it will: a) identify potential biomarkers, both pre- and post-treatment, for hNSC-induced recovery; b) begin to delineate the molecular pathways involved in brain repair; and c) ultimately lead to identification of novel therapies for stroke.

Aging; animal tissue; Behavior; Behavioral; Cells; Communities; Development; Disease Progression; Genes; Genetic; genetic manipulation; Image; Learning; Life; Life Experience; Microscopy; Nervous system structure; Neurons; Neurosciences; Neurosciences Research; Research; Slice; Techniques; tool; Validation; vector; Viral;

? DESCRIPTION (provided by applicant): Stroke is a leading cause of death in the United States, and those who survive stroke often live with serious long-term disabilities. With no neuroprotectants available to treat stroke, there is an urgent unmet need for new therapeutics that can recover lost function. The major type of stroke is focal cerebral ischemia, which is caused by a blocked artery in the brain. Primary brain injury occurs immediately after ischemic onset, and secondary injury is caused by reperfusion, when flow is restored to the blocked artery. Reperfusion injury is characterized by inflammation that involves the recruitment of macrophages (M?s) in the ischemic brain. Ischemic postconditioning (IPostC), or the mechanical interruption of reperfusion, is an emerging and promising neuroprotective strategy, but the underlying protective mechanisms of IPostC are largely unknown. Because reperfusion injury involves M?s, and IPostC interrupts reperfusion, the investigators hypothesize that M?s could be involved the protective effects of IPostC. In addition, M?s in the ischemic brain comprise both resident microglia-derived M?s (MiM?s) and blood monocyte-derived M?s (MoM?s), but their potential unique roles in IPostC have not been studied. Moreover, M?s are polarized into a pro-inflammatory M1 form and an anti-inflammatory M2 form, but how M1 and M2-polarized M?s are involved in IPostC has not been studied. The investigators will therefore use novel approaches to study how the inhibition of M?s and the alteration of M1/M2 polarization contribute to IPostC's protective effects. First, they have established a new IPostC model in mice that enable them to study M?s using various genetically-modified mouse strains and antibodies. Second, they will use the fluorescence-activated cell sorting (FACS) technique to identify, quantify and sort MoM?s from MiM?s. Third, they will use the cutting-edge high-throughput Fluidigm(r) BioMark HD system, a real-time PCR technique, to measure the gene expression of purified MoM?s and MiM?s. Pilot data have already shown that: (1) IPostC inhibited the accumulation of MoM?s, but had less effect on MiM?s; (2) exogenous MoM?s, but not resident MiM?s, had the strongest M1 and M2 gene expression; (3) MoM? depletion resulted in smaller infarctions in wild-type (WT) mice; (4) inhibition of MoM? recruitment in the ischemic brain by CCR2/CCL2 inhibitors or by CCR2 gene deficiency attenuated brain injury in WT and CCR2 gene knockout (KO) mice, respectively; (5) M1 polarization enlarged while M2 polarization inhibited infarct sizes; and (6) impairment of M2, specifically, resulted in larger infarctions in M? conditional IL4R? gene KO mice. Based on these preliminary findings, the investigators therefore propose 3 Specific Aims: (1) to study the effects of IPostC on MoM? and MiM? accumulation and polarization in acute ischemic brain injury; (2) to study whether inhibition of MoM? accumulation is critical for the protective effect of IPostC against stroke; an (3) to study the role of M? polarization in IPostC-mediated protective effects against stroke. The long-term goal is to advance the clinical translation of IPostC and M? strategies for stroke patients.

Project Summary / Abstract The Stanford Neurosurgery and Neurology Resident Research Education Program provides training in basic and clinical research, with the goal of fostering resident and fellow growth into independent physician scientists. Through this education participants are then capable of establishing and directing an independent scientific program and obtaining individual research funding. We identify residents with the potential for and commitment to scientific research, and place them for a one to two year term in the laboratories of highly experienced and productive research scientists. Participants also receive extensive counseling and participate in seminars on research skills and ethical research behavior. Their progress and the success of the overall program is repeatedly assessed, including obtaining individual research funding, and appropriate changes in the program will be made. This program will produce neurosurgeons and neurologists making new discoveries regarding the causes of diseases of the brain and spinal cord, and enabling more effective treatments of these diseases.