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Medical College of Wisconsin

23 ARTICLES PUBLISHED IN JoVE

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Biology

Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies
Vinod B. Shidham 1, George M. Varsegi 1, Krista D'Amore 1, Anjani Shidham 2
1Department of Pathology, University of Wisconsin - Milwaukee, 2BioInnovation LLC

Practicing of fine needle aspiration biopsies (FNAB) by trainees is relatively challenging, due to the lack of an easily available, appropriate lesion. Preparation of an AV phantom lesion for practicing the FNAB procedure and mastering proficiency is relatively easy.

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Medicine

Performing and Processing FNA of Anterior Fat Pad for Amyloid
Vinod B. Shidham 1,2, Bryan Hunt 1, Safwan S. Jaradeh 3, Alexandru C. Barboi 3, Sumana Devata 4, Parameswaran Hari 5
1Department of Pathology, Medical College of Wisconsin, 2Current Address: Department of Pathology, Wayne State University School of Medicine Detroit Medical Center, 3Department of Neurology, Medical College of Wisconsin, 4Department of Medicine, Medical College of Wisconsin, 5Division of Neoplastic Diseases and Related Disorders, Medical College of Wisconsin

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

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Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues
Alison J. Kriegel 1, Mingyu Liang 1
1Department of Physiology, Medical College of Wisconsin

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

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Biology

Tissue Triage and Freezing for Models of Skeletal Muscle Disease
Hui Meng 1, Paul M.L. Janssen 2, Robert W. Grange 3, Lin Yang 4, Alan H. Beggs 5, Lindsay C. Swanson 5, Stacy A. Cossette 1,6, Alison Frase 7, Martin K. Childers 8, Henk Granzier 9, Emanuela Gussoni 5, Michael W. Lawlor 1
1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

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Biology

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Subarna Bhattacharya *1, Paul W. Burridge *2, Erin M. Kropp 1, Sandra L. Chuppa 1, Wai-Meng Kwok 3, Joseph C. Wu 2, Kenneth R. Boheler 4,5, Rebekah L. Gundry 1,6
1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

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Medicine

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells
Muhammad Z. Afzal 1, Jennifer L. Strande 1
1Department of Medicine, Medical College of Wisconsin

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

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Medicine

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
Jerome D. Robin 1, Woody E. Wright 1, Yaqun Zou 2, Stacy A. Cossette 3, Michael W. Lawlor 3, Emanuela Gussoni 4
1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4Division of Genetics and Genomics, Boston Children's Hospital

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

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Neuroscience

Diffusion Imaging in the Rat Cervical Spinal Cord
Elizabeth Zakszewski 1, Brian Schmit 2, Shekar Kurpad 1, Matthew D. Budde 1
1Department of Neurosurgery, Medical College of Wisconsin, 2Department of Biomedical Engineering, Marquette University

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

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Biology

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta
Bradley T. Endres 1,2, Alexander Staruschenko 1, Marie Schulte 4, Aron M. Geurts 1,2,3, Oleg Palygin 1
1Departments of Physiology, Medical College of Wisconsin, 2Human and Molecular Genetics Center, Medical College of Wisconsin, 3Cardiovascular Center, Medical College of Wisconsin, 4Blood Research Institute of Wisconsin

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

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Biology

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli
Daria V. Ilatovskaya 1, Oleg Palygin 1, Vladislav Levchenko 1, Alexander Staruschenko 1
1Department of Physiology, Medical College of Wisconsin

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

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Biology

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium
Tengis S. Pavlov 1, Daria V. Ilatovskaya 1, Oleg Palygin 1, Vladislav Levchenko 1, Oleh Pochynyuk 2, Alexander Staruschenko 1
1Department of Physiology, Medical College of Wisconsin, 2Department of Integrative Biology & Pharmacology, University of Texas Health Science Center at Houston

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

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Biology

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney
Oleg Palygin 1, Vladislav Levchenko 1, Louise C. Evans 1, Gregory Blass 1, Allen W. Cowley Jr. 1, Alexander Staruschenko 1
1Department of Physiology, Medical College of Wisconsin

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

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Biology

A Murine Model of Experimental Necrotizing Enterocolitis Using Gavage Feeding, Lipopolysaccharide, and Systemic Hypoxia
Scott Welak 1, Rebecca M Rentea 2, Shannon M Koehler 3, David M Gourlay 3
1Pediatrics, Medical College of Wisconsin, 2Surgery, Children's Mercy Hospital, 3Pediatric Surgery, Medical College of Wisconsin

This protocol describes how to induce experimental necrotizing enterocolitis (NEC) in newborn rats and mice.

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Immunology and Infection

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice
Dorothee Weihrauch 1, John G. Krolikowski 1,2, Deron W. Jones 3, Tahniyath Zaman 3, Omoshalewa Bamkole 1, Janine Struve 4, Paul S. Pagel 5, Nicole L. Lohr 6, Kirkwood A. Pritchard, Jr. 3
1Department of Anesthesiology, Medical College of Wisconsin, 2Clement J. Zablocki Veterans Affairs Medical Center, 3Department of Surgery, Division of Pediatric Surgery, Children's Research Institute, 4Department of Orthopedic Surgery, Medical College of Wisconsin, 5Deptarment of Anesthesiology, Clement J Zblocki Veteran Affairs Medical Center, 6Department of Medicine, Division of Cardiology, Medical College of Wisconsin

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

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Medicine

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats
Kathleen M. Lukaszewicz 1, Matthew J. Durand 2, Jessica R.C. Priestley 3, James R. Schmidt 4, L. Adrienne Allen 5, Aron M. Geurts 3, Julian H. Lombard 3
1Department of Physical Therapy, Marquette University, 2Medical College of Wisconsin, 3Department of Physiology, Medical College of Wisconsin, 4Graduate Programs of Nurse Anesthesia, Texas Wesleyan University, 5Office of Research, Medical College of Wisconsin

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

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Medicine

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery
Paul S. Pagel 1, Lonnie Dye III 2, Graham E.D. Hill 2, Juan L. Vega 2, Justin N. Tawil 2, Derek J. De Vry 2, Kiran Chandrashekarappa 1, Zafar Iqbal 1, Brent T. Boettcher 1, Julie K. Freed 2
1Anesthesia Service, Clement J. Zablocki Veterans Affairs Medical Center, 2Department of Anesthesiology, Medical College of Wisconsin

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

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Biochemistry

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo
Patrick Moran 1,2, Yichen Guo 3,4, Rong Yuan 1,3, Nicholas Barnekow 1, Jordan Palmer 2, Adam Beck 3, Bin Ren 3,4,5
1Blood Research Institute, Blood Center of Wisconsin, 2Department of Medicine, Medical College of Wisconsin, 3Department of Surgery, The University of Alabama at Birmingham, 4Department of Biomedical Engineering, The University of Alabama at Birmingham, 5GBS Program, Graduate School, The University of Alabama at Birmingham

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

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Medicine

Inverse Probability of Treatment Weighting (Propensity Score) using the Military Health System Data Repository and National Death Index
Joshua D. Mitchell 1, Brian F. Gage 2, Nicole Fergestrom 3, Eric Novak 1, Todd C. Villines 4
1Cardiovascular Division, Washington University School of Medicine, 2General Medical Sciences, Washington University School of Medicine, 3Center for Advancing Population Science, Medical College of Wisconsin, 4Cardiology Service, Department of Medicine, Walter Reed National Military Medical Center

When randomized controlled trials are not feasible, a comprehensive health care data source like the Military Health System Data Repository provides an attractive alternative for retrospective analyses. Incorporating mortality data from the national death index and balancing differences between groups using propensity weighting helps reduce biases inherent in retrospective designs.

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Medicine

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry
Linda A. Allen *1, Maia Terashvili *1, Alison Gifford *1, Julian H. Lombard 1
1Department of Physiology, Medical College of Wisconsin

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

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Biology

Non-thermal Infrared Light Treatment of Ischemia/Reperfusion Injury and Subsequent Analysis of Macrophage Differentiation
Tyler Compton 1, Nicholas Poellinger 2, Janine Struve 3, John G. Krolikowski 1, James T. Ninomiya 4, Dorothee Weihrauch 1
1Department of Anesthesiology, Medical College of Wisconsin, 2Department of Internal Medicine, Loyola University Medical Center, 3Department of Orthopedic Surgery, Medical College of Wisconsin, 4Department of Orthopedic Surgery, University of New Mexico

We describe the reduction of reperfusion injury by 670 nm irradiation in a mouse model of ischemia and reperfusion by tourniquet placement. This 670 nm irradiation reduced the inflammatory response, decreased the number of proinflammatory macrophages, and increased the protective macrophages.

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Developmental Biology

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development
Zain Zaki Zakaria 1,2, Shahram Eisa-Beygi 3, Fatiha M. Benslimane 1, Ramani Ramchandran 4, Huseyin C. Yalcin 1
1Biomedical Research Center, Qatar University, Doha, Qatar, 2Department of Biological and Environmental Sciences, College of Arts and Science, Qatar University, Doha, Qatar, 3Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 4Department of Pediatrics, Medical College of Wisconsin

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

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Medicine

Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats
Satoshi Shimada 1, Allen W. Cowley, Jr. 1
1Department of Physiology, Medical College of Wisconsin

The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry).

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Medicine

Augmenting Large Language Models via Vector Embeddings to Improve Domain-specific Responsiveness
Nathan M. Wolfrath 1,2, Nathaniel B. Verhagen 1, Bradley H. Crotty 2, Melek Somai 2, Anai N. Kothari 1
1Department of Surgery, Division of Surgical Oncology, Medical College of Wisconsin, 2Inception Health Labs, Medical College of Wisconsin

In this protocol, foundation large language model response quality is improved via augmentation with peer-reviewed, domain-specific scientific articles through a vector embedding mechanism. Additionally, code is provided to aid in performance comparison across large language models.

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