It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.
19 Related JoVE Articles!
Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation
Institutions: Harvard Medical School, Massachusetts General Hospital, Harvard Medical School, Massachusetts General Hospital, Harvard Medical School.
Lung cancer is the leading cause of cancer-related deaths1
. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1
. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7
and esophageal intestinal metaplasia and dysplasia6,8-10
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo
imaging tool for evaluating the pulmonary airways11-23
). OCT has been assessed in pulmonary airways16,23
of animal models and in vivo
. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21
. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18
. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo
has been described19
. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo
pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo
consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo
setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo
with matched one-to-one histology, the criteria may then be applied to in vivo
imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo
lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24
. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.
Bioengineering, Issue 71, Medicine, Biomedical Engineering, Anatomy, Physiology, Cancer Biology, Pathology, Surgery, Bronchoscopic imaging, In vivo optical microscopy, Optical imaging, Optical coherence tomography, Optical frequency domain imaging, Histology correlation, animal model, histopathology, airway, lung, biopsy, imaging
Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR
Institutions: Universidad Peruana Cayetano Heredia, Johns Hopkins University, University of Concepcion,Chile, University of California San Diego School of Medicine.
Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer.
Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3
. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al.
which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5
is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.
Virology, Issue 65, Medicine, Genetics, norovirus, gastroenteritis, RNA extraction, diarrhea, stool samples, PCR, RT-PCR, TaqMan, silica
Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique
Institutions: Stanford University School of Medicine , Veterans Affairs Palo Alto Health Care System.
Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo
microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1
and gastrointestinal tracts,2-6
CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10
Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo
Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent—most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12
Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10
Recent availability of a < 1 mm imaging probe13
opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e.
photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14
that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15
and antibodies for endoscopic imaging of disease processes with molecular specificity.
Medicine, Issue 71, Anatomy, Physiology, Cancer Biology, Surgery, Basic Protocols, Confocal laser endomicroscopy, microscopy, endoscopy, cystoscopy, human bladder, bladder cancer, urology, minimally invasive, cellular imaging
Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue
Institutions: UCLA, UC Irvine.
The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro
, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.
Cellular Biology, Issue 80, Myofibroblasts, Mesenchymal Stromal Cells, Gastrointestinal Tract, stroma, colon, primary cells
Chronic Salmonella Infected Mouse Model
Institutions: University of Rochester.
The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella
typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo
. Mice with Salmonella
persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella
typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella
persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.
Microbiology, Issue 39, Salmonella, intestine, colitis, chronic infection, mouse model
Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy
Institutions: Raymond and Beverly Sackler Foundation, University of Medicine and Dentistry of New Jersey, Institute for Advanced Study, Princeton, New Jersey.
Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100, 000 individuals per year, and they account for 0.5% of all human malignancies.1
Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo
tumor tissue organization and microenvironment.2,3
The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro
3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2
, 37 °C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd
day. The spheroids become more spherical by the 6th
day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques.
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.
Cancer Biology, Issue 66, Medicine, Neuroscience, Cell Culture, Tissue Engineering, 3D model, multicellular spheroids, therapeutic drugs, neuroendocrine tumor cell lines, agarose overlay platform, paraffin embedding
The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques
There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo
sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci
infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.
Medicine, Issue 89, translational medicine, respiratory models, bovine lung, bronchoscopy, transbronchial lung biopsy, bronchoalveolar lavage, bronchial brushing, cytology brush
Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns
Institutions: University of Calgary , University of Calgary .
We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via
quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.
Medicine, Issue 78, Anatomy, Physiology, Cancer Biology, angular spread, architectural distortion, breast cancer, Computer-Assisted Diagnosis, computer-aided diagnosis (CAD), entropy, fractional Brownian motion, fractal dimension, Gabor filters, Image Processing, Medical Informatics, node map, oriented texture, Pattern Recognition, phase portraits, prior mammograms, spectral analysis
RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract
Institutions: University of Texas Southwestern Medical Center , Harvard Medical School, University of Texas Southwestern Medical Center .
(PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1
. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2
, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo
transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3
in order to identify changes in bacterial gene expression during alterations to the host’s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4
. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.
Immunology, Issue 55, Pseudomonas, RNA, murine, cecum, transcriptome, qPCR, RT-PCR, PCR
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging
Institutions: Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, Rice University.
The ability to differentiate benign metaplasia in Barrett’s Esophagus (BE) from neoplasia in vivo
remains difficult as both tissue types can be flat and indistinguishable with white light imaging alone. As a result, a modality that highlights glandular architecture would be useful to discriminate neoplasia from benign epithelium in the distal esophagus. VFI is a novel technique that uses an exogenous topical fluorescent contrast agent to delineate high grade dysplasia and cancer from benign epithelium. Specifically, the fluorescent images provide spatial resolution of 50 to 100 μm and a field of view up to 2.5 cm, allowing endoscopists to visualize glandular morphology. Upon excitation, classic Barrett’s metaplasia appears as continuous, evenly-spaced glands and an overall homogenous morphology; in contrast, neoplastic tissue appears crowded with complete obliteration of the glandular framework. Here we provide an overview of the instrumentation and enumerate the protocol of this new technique. While VFI affords a gastroenterologist with the glandular architecture of suspicious tissue, cellular dysplasia cannot be resolved with this modality. As such, one cannot morphologically distinguish Barrett’s metaplasia from BE with Low-Grade Dysplasia via this imaging modality. By trading off a decrease in resolution with a greater field of view, this imaging system can be used at the very least as a red-flag imaging device to target and biopsy suspicious lesions; yet, if the accuracy measures are promising, VFI may become the standard imaging technique for the diagnosis of neoplasia (defined as either high grade dysplasia or cancer) in the distal esophagus.
Bioengineering, Issue 87, fluorescence imaging, Barrett’s esophagus, esophageal adenocarcinoma
Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration
Institutions: State University of New York, Buffalo, Roswell Park Cancer Institute, State University of New York, Buffalo.
Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1
. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.
Medicine, Issue 93, substernal thyroid, retrosternal thyroid, intra-thoracic thyroid, goiter, endobronchial ultrasound, EBUS, transbronchial needle aspiration, TBNA, biopsy, needle biopsy
Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation
Institutions: University Hospital Münster, University Children's Hospital Münster.
Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo,
necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live
mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo
murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo
imaging and may significantly impact on preclinical research in various fields.
Medicine, Issue 90,
gastroenterology, in vivo imaging, murine endoscopy, diagnostic imaging, carcinogenesis, intestinal wound healing, experimental colitis
Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System
Institutions: Case Western Reserve University School of Medicine, Cleveland, Case Western Reserve University School of Medicine, Cleveland, Case Western Reserve University School of Medicine, Cleveland.
The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).
Medicine, Issue 80, Crohn's disease, ulcerative colitis, colon cancer, Clostridium difficile, SAMP mice, DSS/AOM-colitis, decimal scoring identifier
Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay
Institutions: Technische Universität München, University of Applied Sciences Kaiserslautern/Zweibrücken.
Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro
conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.
Medicine, Issue 86, Autonomic Nervous System Diseases, Digestive System Neoplasms, Gastrointestinal Diseases, Pancreatic Diseases, Pancreatic Neoplasms, Pancreatitis, Pancreatic neuroplasticity, dorsal root ganglia, myenteric plexus, Morphometry, neurite density, neurite branching, perikaryonal hypertrophy, neuronal plasticity
Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice
Institutions: Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Memorial Sloan Kettering Cancer Center, Medical Institute, The Rockefeller University.
The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3
. Loss or injury to these networks is associated with a number of motility disorders4
. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6
. Importantly, normal KIT activity is required for ICC development5,6
. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8
. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9
. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11
. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12
. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.
Developmental Biology, Issue 53, mice, fluorescence microscopy, gastrointestinal track, motility, interstital cells of cajal, kit, ano1, pgp9.5
Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique
Institutions: Appalacian State University, Appalachian State University, Carolinas Medical Center NorthEast.
The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergström needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergström needle consists of an outer cannula with a small opening (‘window’) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90° and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.
Medicine, Issue 91, percutaneous muscle biopsy, needle biopsy, suction-modified, metabolism, enzyme activity, mRNA, gene function, fiber type, histology, metabolomics, skeletal muscle function, humans