2012 CBIS Seed Grant Recipients

Sandip Biswal, MD

Radiology
Project: PET/MRI Image-guided Therapy of Peripheral Neuropathic Pain using a Sigma-1 Receptor Antagonist

Summary
Current methods used to diagnose and treat pain generators in chronic pain sufferers are inadequate and a paradigm-shifting approach is desperately needed. The accurate identification of sites of nerve injury and ensuing neuroinflammation has tremendous clinical value in the management of neuropathic pain. The sigma-1 receptor (S1R), an intracellular receptor chaperone known to play an important role in signaling and neurotransmitter systems, is a potential biomarker of neuroinflammation. Preliminary studies in our lab have demonstrated increased S1R expression in the neuroma caused by nerve injury in the sciatic nerve of a rat model of neuropathic pain. Additionally, S1R antagonism has been under close scrutiny for potential treatment of chronic pain. It is thus increasingly apparent that specific therapy targeted at selectively blocking S1R activity in chronic neuropathic pain conditions, especially at sites of increased S1R expression, can provide significant pain relief. Using a radiolabeled S1R antagonist, [18F]FTC-146, we have been able to identify sites of nerve injury in intact living models of neuropathic pain using positron emission tomography/magnetic resonance imaging (PET/MRI) (Please see Figure). This permits localization of pain generators and facilitates image-guided treatments at the site of nerve injury. The overarching goal of this proposal is to establish the utility of S1R imaging with [18F]FTC-146 PET/MRI in image-guided therapy of peripheral neuropathic pain generators with novel S1R antagonists. Successful demonstration of this approach will potentially open up a new way to diagnose and treat pain generators.

The biodistribution of [18F]FTC-146 is highly specific to regions of known high S1R expression. PET/MRI scans show increased specific uptake of [18F]FTC-146 in the neuroma in the sciatic nerve of a rat neuropathic pain model (Spared Nerve Injury (SNI)). The increased uptake in PET/MRI images has been verified by ex vivo autoradiography of the sciatic nerves (Figs. A and B, respectively). Control and sham models do not show any increase in tracer uptake in the nerves.

Vinicio A. de Jesus, MD

Pulmonary/CCM
Project: Optical Coherence Tomography as a Novel Tool in the Evaluation of the Pulmonary Circulation

Summary
Pulmonary arterial hypertension (PAH) is a disorder characterized by loss and narrowing of small distal arteries resulting in abnormal elevation of pulmonary pressures. Currently, the gold standard for PAH diagnosis is right heart catheterization (RHC) but this technique does not provide any information on the state of vascular wall pathology. We propose that use of optical coherence tomography (OCT), an innovative intravascular imaging technique recently approved by the FDA for use in the assessment of coronary artery disease (CAD), is a safe diagnostic procedure that can be done as part of the RHC in PAH patients to characterize the extent of vascular remodeling in pulmonary vessels at diagnosis and following institution of therapy. We believe that this information could be used alongside established clinical and hemodynamic predictors when determining a patient's chances for progression, survival and response to therapy at any given point in the natural history of the disease.

Audrey Ellerbee, PhD

Electrial Engineering
Project: 3D Localization of Cell Positioning for Early Prediction of Embryonic Development to Blastocyst Stage

Summary
Despite known adverse outcomes, the low success rate of post-implantation human embryos drives the current clinical practice of transferring multiple embryos during in vitro fertilization (IVF) procedures. Recently, it has been shown that automated 2D image analysis can successfully predict progression of the zygote to the blastocyst stage with > 93% sensitivity and specificity, which could lead to better and more informed IVF outcomes. The goal of this project is to use optical coherence microscopy to investigate embryonic development in 3D, with the hope that additional information provided by the 3D images will produce more salient markers for blastocyst development and extend knowledge of the process of human development.

Other Key Personnel:
Tom Baer; Barry Behr, PhD, HCLD;

Cell boundaries identified with automated analysis and images of blastocyst formation. [Wong et al., Nature Biotech 28 (2010).]

Label-free, en face images obtained with OCT from three planes of a non-viable human embryo showing 3D localization of cells in different positions and planes.

Edward E. Graves, PhD
Donna M. Bouley, PhD

Radiation Oncology and Comparative Medicine
Project: New Course on Imaging Anatomy in Animal Models

Summary
Stanford offers a wide variety of excellent undergraduate and graduate courses on imaging, training students in subjects including imaging physics and technology, signal processing, and data analysis. In addition, a number of courses are available to expose students to biomedical applications of these tools. A critical element of this comprehensive education in imaging is providing an understanding of the biological source of imaging signals when these technologies are used in biomedical and clinical research. This involves learning anatomy, with a special focus on cross-sectional and projection anatomy as encountered in imaging studies. While a course dedicated to imaging anatomy in humans is offered yearly (Bioengineering 220), there is no counterpart available that focuses on imaging anatomy in common laboratory animals. Stanford's current course offerings therefore teach students how imaging works and how it may be used in research, but not how to interpret imaging data they encounter in their studies and/or research. With the support of CBIS we will create a one quarter, 3 unit course entitled "Imaging Anatomy in Animal Models". The initial offering of this course would be in the fall quarter, 2013. The course will include both didactic lectures as well as laboratories focusing on anatomy of mice, rats, dogs, and pigs. In the laboratories students will aid in the dissection of these animals as well as imaging of mice in the Stanford Center for Innovation in In Vivo Imaging. We anticipate that this course will synergize with existing imaging courses at Stanford and will enhance both the educational and research environments on campus.

CBIS Courses

Jan Skotheim, PhD

Biology
Project: A Single-Molecule Approach to Cell Size Control

Summary
Cell size is an important physiological trait that is primarily regulated during the G1 phase of the cell division cycle, prior to DNA replication. Although the traditional tools of biochemistry and cell biology have indentified key regulatory proteins effecting size control, the underlying molecular mechanism remains unclear. Here, I propose to address this fundamental question in cell biology using a novel single-molecule imaging approach to quantify the key aspects of cell size control. Of the cyclin-CDK complexes that control progression through the cell cycle, several are known to phosphorylate protein targets that are pre-bound to DNA. By analogy to site-specific DNA binding proteins (including, e.g., transcription factors and DNA repair enzymes), we predict that these kinase complexes will undergo combined 3D and 1D diffusion during their target search. To test this hypothesis, we will employ a single-molecule approach in which binding to and 1D diffusion along flow-stretched DNA by individual protein complexes can be observed using TIRF microscopy. Our proposed TIRF assay will allow us to test the hypothesis that that cells measure their size by counting the number of Cln3 molecules by directly assaying the interaction between cyclins and DNA at single molecule resolution.

λ DNA molecules are attached by one end to a glass coverslip. Elongation of DNA is then induced using flow through a microfluidic device. This allows observation of 1D diffusion by DNA binding proteins.

Justin L. Sonnenburg, PhD
Kerwyn Casey Huang, PhD

Microbiology & Immunology
Project: Development of Anaerobe--Compatible Fluorescent Microscopy Techniques to Study Gut--Resident Symbionts

Summary
Despite recent advances in bacterial cell biology using time-lapse fluorescence microscopy, methods have primarily relied on fluorescent proteins that require oxidation prior to imaging. One of the main limitations in studying commensals from the intestinal microbiota is the paucity of fluorescent proteins that are compatible with anaerobic conditions. Here we propose to identify a fluorescent labeling scheme for studying the genetically tractable anaerobic gut resident, Bacteroides thetaiotaomicron. Specifically, we plan to use fluorescence microscopy to study the subcellular distribution of a novel class of signaling molecules known as the hybrid two-component systems (HTCS) before and during activation. This project will provide important tools for imaging molecules within diverse anaerobic bacteria and provide insight into the biology of a recently identified signaling protein that is abundant in the human microbiota.

Principal Investigators:
Justin L. Sonnenburg, PhD; Kerwyn Casey Huang, PhD

Other Key Personnel:
Jonathan B. Lynch, PhD

Figure. Schematic of project overview. Using fluorescence microscopy, we will optimize techniques to image the spatial relationships between HTCSs and their target loci. These experiments will allow us to determine a) whether ligand (black hexagon) binding to the HTCS leads to a change in colocalization between an HTCS and its DNA target, b) the distance between known target loci and the cell membrane where HTCSs presumably localize, c) the spatial pattern of HTCS proteins, and d) and spatial organization of HTCS genomic targets.

Michael Zeineh, MD

Radiolgy
Project: MRI to Assess for Longitudinal Brain Atrophy Associated with Concussive and Sub-concussive Forces

Summary
Mild traumatic brain injury (mTBI), synonymous with concussion, occurs at high rates in contact-sport athletes. The cumulative effects may result in significant functional impairment. High-school and college football players in particular experience concussions at relatively high rates,and there is growing concern for the long-term impact to society given the enormous numbers of players nationwide. Understanding the relationship between traumatic forces and brain injury ultimately may enable the prevention of permanent cognitive deficits. Quantifying brain injury presents a great challenge because routine clinical brain imaging often does not demonstrate significant abnormalities in mTBI. In this longitudinal study, we will be performing a quantitative analysis of changes in cortical thickness in athletes that endure concussive and sub-concussive impacts relative to a control population. We expect this study to enhance our understanding of potentially permanent changes that occur secondary to mild trauma in contact sports.