Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (<10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2>0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.
28 Related JoVE Articles!
Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats
Institutions: University Hospital RWTH Aachen.
Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5
The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8
The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11
This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al.
as well as others.12-14
Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8±3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.
Medicine, Issue 69, Anatomy, Physiology, Immunology, Surgery, microsurgery, neointimal hyperplasia, venous interposition graft, external jugular vein, common carotid artery, rat
Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye
Institutions: Leiden University Medical Center, Leiden University Medical Center, Leiden University Medical Center.
The presence of placental vascular anastomoses is a conditio sine qua non
for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2
. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1
. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2
. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5
. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.
Medicine, Issue 55, monochorionic twin placenta, vascular anastomoses, twin-to-twin transfusion syndrome, twin anemia polycythemia sequence, colored dye injection, fetoscopic laser surgery
Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis
Institutions: Western Kentucky University, University of Houston.
Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.
Immunology, Issue 48, Leukocyte phenotyping, programmed cell death, muscular activity, technique development
Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples
Institutions: University of Manitoba, University of Manitoba.
Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo
flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.
Medicine, Issue 89, mucosal, immunology, FGT, lavage, cervical, CMC
A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes
Institutions: Université Libre de Bruxelles, Université Libre de Bruxelles, Université Libre de Bruxelles, Université Libre de Bruxelles.
The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+
cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.
) or molecular (DNA, RNA and/or protein) approaches. CD45+
cells can also be used for subpopulation purification, in vitro
expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.
Immunology, Issue 94, Tumor immunology, tumor infiltrating lymphocytes, CD45+, breast cancer, fresh tissue homogenate, non-enzymatic dissociation, primary tissue supernatant
Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes
Institutions: Roswell Park Cancer Institute, University of Pennsylvania , SciGro, Inc., University of Pennsylvania .
Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro
and in vivo
Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
stem and progenitor cell quiescence, proliferation and/or differentiation6-8
antigen-driven membrane transfer9
and/or precursor cell proliferation3,4,10-18
immune regulatory and effector cell function1,18-21
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11
. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18
. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
Failure to achieve bright, uniform, reproducible labeling
. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.
Suboptimal fluorochrome combinations and/or failure to include critical compensation controls
. Tracking dye fluorescence is typically 102
times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.
Failure to obtain a good fit with peak modeling software
. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.
Cellular Biology, Issue 70, Molecular Biology, Cell tracking, PKH26, CFSE, membrane dyes, dye dilution, proliferation modeling, lymphocytes
Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR
Institutions: Spedali Civili di Brescia.
T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106
cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.
Immunology, Issue 94, B lymphocytes, primary immunodeficiency, real-time PCR, immune recovery, T-cell homeostasis, T lymphocytes, thymic output, bone marrow output
Femoral Bone Marrow Aspiration in Live Mice
Institutions: Memorial Sloan-Kettering Cancer Center.
Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.
Medicine, Issue 89, Bone marrow, Leukemia, Hematopoiesis, Aspiration, Mouse Model, Hematopoietic Stem Cell
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Isolation of Murine Lymph Node Stromal Cells
Institutions: University of Basel and University Hospital Basel.
Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host.
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.
Immunology, Issue 90, lymph node, lymph node stromal cells, digestion, isolation, enzymes, fibroblastic reticular cell, lymphatic endothelial cell, blood endothelial cell
Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology
Institutions: San Diego State University, University of California San Diego.
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
Environmental Sciences, Issue 93, dissolved organic carbon, particulate organic matter, nutrients, DAPI, SYBR, microbial metagenomics, viral metagenomics, marine environment
Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain
Institutions: University of Montreal, University of Montreal, Stereology Resource Center.
The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2
, generates reliable structural data for biological features in the mammalian brain3
. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4
. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus
), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer
systems, this study provides a detailed protocol for the Stereologer
Neuroscience, Issue 27, Stereology, brain bank, systematic sampling, non-human primate, cryostat, antigen preserve
Heterotopic Heart Transplantation in Mice
Institutions: University of California, San Francisco - UCSF.
The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.
Developmental Biology, Issue 6, Microsurgical Techniques, Heart Transplant, Allograft Rejection Model
Non-Terminal Blood Sampling Techniques in Guinea Pigs
Institutions: University of Copenhagen.
Guinea pigs possess several biological similarities to humans and are validated experimental animal models1-3
. However, the use of guinea pigs currently represents a relatively narrow area of research and descriptive data on specific methodology is correspondingly scarce. The anatomical features of guinea pigs are slightly different from other rodent models, hence modulation of sampling techniques to accommodate for species-specific differences, e.g
., compared to mice and rats, are necessary to obtain sufficient and high quality samples. As both long and short term in vivo
studies often require repeated blood sampling the choice of technique should be well considered in order to reduce stress and discomfort in the animals but also to ensure survival as well as compliance with requirements of sample size and accessibility. Venous blood samples can be obtained at a number of sites in guinea pigs e.g
., the saphenous and jugular veins, each technique containing both advantages and disadvantages4,5
. Here, we present four different blood sampling techniques for either conscious or anaesthetized guinea pigs. The procedures are all non-terminal procedures provided that sample volumes and number of samples do not exceed guidelines for blood collection in laboratory animals6
. All the described methods have been thoroughly tested and applied for repeated in vivo
blood sampling in studies within our research facility.
Medicine, Issue 92, guinea pig, animal model, blood sampling, non-terminal, saphenous, tarsal, jugular
Assessment of Vascular Function in Patients With Chronic Kidney Disease
Institutions: University of Colorado, Denver, University of Colorado, Boulder.
Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMDBA
) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMDBA
, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMDBA
, aPWV, and vascular endothelial cell protein expression.
Medicine, Issue 88, chronic kidney disease, endothelial cells, flow-mediated dilation, immunofluorescence, oxidative stress, pulse-wave velocity
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model
Institutions: University Medical Center Utrecht, Vascular Connect b.v., University Medical Center Utrecht, University Medical Center Utrecht.
To simplify and facilitate beating heart (i.e.,
off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e.,
occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.
Medicine, Issue 93, Anastomosis, coronary, anastomotic connector, anastomotic coupler, excimer laser-assisted nonocclusive anastomosis (ELANA), coronary artery bypass graft (CABG), off-pump coronary artery bypass (OPCAB), beating heart surgery, excimer laser, porcine model, experimental, medical device
Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement
Institutions: Brigham and Women's Hospital/Harvard Medical School, CHUV University Hospital, University of Bordeaux, CHUV University Hospital.
The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex vivo
perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex vivo
conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.
Medicine, Issue 92, vein, human, intimal hyperplasia, neointima, perfusion, mesh, pressure, ex vivo
Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+
release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound
Institutions: Johns Hopkins University, Johns Hopkins University, Johns Hopkins University, Macquarie University.
We present a protocol for measuring in vivo
aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo
approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.
Medicine, Issue 94, Aortic stiffness, ultrasound, in vivo, aortic compliance, elastic modulus, mouse model, cardiovascular disease
Manual Restraint and Common Compound Administration Routes in Mice and Rats
Institutions: Charles River , Charles River.
Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.
Basic Protocols, Issue 67, Anatomy, Medicine, Rodents, training, handling, restraint, injections, oral gavage
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
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
Small Bowel Transplantation In Mice
Institutions: University of California, San Francisco - UCSF.
Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.
Issue 7, Immunology, Transplantation, Transplant Rejection, Small Bowel
Pulse Wave Velocity Testing in the Baltimore Longitudinal Study of Aging
Institutions: National Institute of Aging.
Carotid-femoral pulse wave velocity is considered the gold standard for measurements of central arterial stiffness obtained through noninvasive methods1
. Subjects are placed in the supine position and allowed to rest quietly for at least 10 min prior to the start of the exam. The proper cuff size is selected and a blood pressure is obtained using an oscillometric device. Once a resting blood pressure has been obtained, pressure waveforms are acquired from the right femoral and right common carotid arteries. The system then automatically calculates the pulse transit time between these two sites (using the carotid artery as a surrogate for the descending aorta). Body surface measurements are used to determine the distance traveled by the pulse wave between the two sampling sites. This distance is then divided by the pulse transit time resulting in the pulse wave velocity. The measurements are performed in triplicate and the average is used for analysis.
Medicine, Issue 84, Pulse Wave Velocity (PWV), Pulse Wave Analysis (PWA), Arterial stiffness, Aging, Cardiovascular, Carotid-femoral pulse
Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas
Institutions: University of California, San Francisco, University of Alberta, University of California, San Francisco, University of California, San Francisco.
Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1
. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3
. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero
obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4
. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5
. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS.
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6
. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.
Medicine, Issue 55, placenta, monochorionic twins, vascular mapping, twin-to-twin transfusion syndrome (TTTS), obstetrics, fetal surgery
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
Institutions: Cornell University, Cornell University.
Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.
Plant Biology, Issue 15, Annual Review, Aphids, Plant Virus, Potato Leaf Roll Virus, Microinjection Technique