Gary Steinberg, MD - US grants

? DESCRIPTION (provided by applicant): Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Recovery of lost function can occur after stroke and is attributed to brain reorganization and neuroplasticity. In this proposal, we will use optogenetics and imaging techniques to study brain circuit dynamics and axonal plasticity during post-stroke recovery. Optogenetics allows activation or inhibition of specific cell groups and circuits in the brain with millisecond-scale precision, thus is a valuable tool for studying neural circuits involved in post-stroke recovery. We recently demonstrated that selective neuronal stimulation in the ipsilesional motor cortex (iM1) using optogenetics can activate plasticity mechanisms and promote recovery. In Aim 1 we will use optogenetic functional MRI (ofMRI) to study global changes in brain circuit activation evoked by selective optogenetic stimulations during post-stroke recovery in non-stimulated stroke mice and repeatedly-stimulated stroke mice. In Aim 2A we will examine brain circuit activation at the cellular level in 3-D whole brain using two lines of activity reporter mice: neuronal activation reporter mice (FosTRAP) and synaptic activity reporter mice (ArcTRAP). We will determine the cell type of these activated neurons (excitatory vs inhibitory). In Aim 2B we will investigate how stimulations alter structural plasticity through axonal sprouting using the anterograde tracer (biotinylated-labeled dextranamine). The CLARITY technique will be used for Aim 2 to visualize cellular resolution of circuit activities and axonal sprouting in 3-D whole brains. In Aim 3 we will use the optogenetics technique to determine the role of the contralesional cortex (side opposite to stroke) during post-stroke recovery. Some studies indicate that contralesional cortex activation is necessary for recovery, whereas other studies suggest that contralesional cortex activation may be maladaptive and worsen recovery. To determine whether the contralesional cortex exerts beneficial or deleterious effects during recovery and whether there is a time-dependent role, we will manipulate specific neural circuits in the contralesional cortex at different post- stroke phases (early vs late) and examine their effects on functional recovery. Functional recovery will be evaluated by a panel of sensorimotor behavior tests that are well established in our lab. Activity-dependent neurotrophin expression and structural plasticity (axonal sprouting) will be examined in the groups that exhibit functional recovery. Our findings will advance the understanding of neural circuits and brain reorganization during post-stroke recovery.

? 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.

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): 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.

? 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.