There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
21 Related JoVE Articles!
Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data
Institutions: The Feinstein Institute for Medical Research.
The scaled subprofile model (SSM)1-4
is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1
). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6
. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8
. Using logistic regression analysis of subject scores (i.e.
pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e.
composite networks with improved discrimination of patients from healthy control subjects5,6
. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9
. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10
. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11
. These standardized values can in turn be used to assist in differential diagnosis12,13
and to assess disease progression and treatment effects at the network level7,14-16
. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.
Medicine, Issue 76, Neurobiology, Neuroscience, Anatomy, Physiology, Molecular Biology, Basal Ganglia Diseases, Parkinsonian Disorders, Parkinson Disease, Movement Disorders, Neurodegenerative Diseases, PCA, SSM, PET, imaging biomarkers, functional brain imaging, multivariate spatial covariance analysis, global normalization, differential diagnosis, PD, brain, imaging, clinical techniques
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool
Institutions: Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation.
Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the Multiple Sclerosis Performance Test (MSPT), which represents a new approach to quantifying MS related disability. The MSPT takes advantage of advances in computer technology, information technology, biomechanics, and clinical measurement science. The resulting MSPT represents a computer-based platform for precise, valid measurement of MS severity. Based on, but extending the Multiple Sclerosis Functional Composite (MSFC), the MSPT provides precise, quantitative data on walking speed, balance, manual dexterity, visual function, and cognitive processing speed. The MSPT was tested by 51 MS patients and 49 healthy controls (HC). MSPT scores were highly reproducible, correlated strongly with technician-administered test scores, discriminated MS from HC and severe from mild MS, and correlated with patient reported outcomes. Measures of reliability, sensitivity, and clinical meaning for MSPT scores were favorable compared with technician-based testing. The MSPT is a potentially transformative approach for collecting MS disability outcome data for patient care and research. Because the testing is computer-based, test performance can be analyzed in traditional or novel ways and data can be directly entered into research or clinical databases. The MSPT could be widely disseminated to clinicians in practice settings who are not connected to clinical trial performance sites or who are practicing in rural settings, drastically improving access to clinical trials for clinicians and patients. The MSPT could be adapted to out of clinic settings, like the patient’s home, thereby providing more meaningful real world data. The MSPT represents a new paradigm for neuroperformance testing. This method could have the same transformative effect on clinical care and research in MS as standardized computer-adapted testing has had in the education field, with clear potential to accelerate progress in clinical care and research.
Medicine, Issue 88, Multiple Sclerosis, Multiple Sclerosis Functional Composite, computer-based testing, 25-foot walk test, 9-hole peg test, Symbol Digit Modalities Test, Low Contrast Visual Acuity, Clinical Outcome Measure
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
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
Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity
Institutions: Alpert Medical School, Brown University, University of Georgia.
Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of psychiatric illness severity and provide added validity to the clinical diagnostic process. Recent neuroimaging research suggests that DMN processes may be disrupted in a number of stress-related psychiatric illnesses, such as posttraumatic stress disorder (PTSD).
Although specific DMN functions remain under investigation, it is generally thought to be involved in introspection and self-processing. In healthy individuals it exhibits greatest activity during periods of rest, with less activity, observed as deactivation, during cognitive tasks, e.g.
, working memory. This network consists of the medial prefrontal cortex, posterior cingulate cortex/precuneus, lateral parietal cortices and medial temporal regions.
Multiple functional and structural imaging approaches have been developed to study the DMN. These have unprecedented potential to further the understanding of the function and dysfunction of this network. Functional approaches, such as the evaluation of resting state connectivity and task-induced deactivation, have excellent potential to identify targeted neurocognitive and neuroaffective (functional) diagnostic markers and may indicate illness severity and prognosis with increased accuracy or specificity. Structural approaches, such as evaluation of morphometry and connectivity, may provide unique markers of etiology and long-term outcomes. Combined, functional and structural methods provide strong multimodal, complementary and synergistic approaches to develop valid DMN-based imaging phenotypes in stress-related psychiatric conditions. This protocol aims to integrate these methods to investigate DMN structure and function in PTSD, relating findings to illness severity and relevant clinical factors.
Medicine, Issue 89, default mode network, neuroimaging, functional magnetic resonance imaging, diffusion tensor imaging, structural connectivity, functional connectivity, posttraumatic stress disorder
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
Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients
Institutions: Hadassah Hebrew-University Medical Center.
In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.
Medicine, Issue 86, Optic neuritis, visual impairment, dynamic visual functions, motion perception, stereopsis, demyelination, remyelination
Nest Building as an Indicator of Health and Welfare in Laboratory Mice
Institutions: Charles River, Tufts University, Stanford University, Stanford University.
The minimization and alleviation of suffering has moral and scientific implications. In order to mitigate this negative experience one must be able to identify when an animal is actually in distress. Pain, illness, or distress cannot be managed if unrecognized. Evaluation of pain or illness typically involves the measurement of physiologic and behavioral indicators which are either invasive or not suitable for large scale assessment. The observation of nesting behavior shows promise as the basis of a species appropriate cage-side assessment tool for recognizing distress in mice. Here we demonstrate the utility of nest building behavior in laboratory mice as an ethologically relevant indicator of welfare. The methods presented can be successfully used to identify thermal stressors, aggressive cages, sickness, and pain. Observation of nest building behavior in mouse colonies provides a refinement to health and well-being assessment on a day to day basis.
Behavior, Issue 82, Animal Structures, Surgical Procedures, Life Sciences (General), Behavioral Sciences, Mouse, Welfare assessment, Nest building
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
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Institutions: Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City.
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g.
drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2
. RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4
in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Genetics, Issue 72, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale
Institutions: Brown University, Women & Infants Hospital of Rhode Island, University of Massachusetts, Boston.
There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.
Behavior, Issue 90, NICU Network Neurobehavioral Scale, NNNS, High risk infant, Assessment, Evaluation, Prediction, Long term outcome
Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence
Institutions: University of Edinburgh, HistoRx Inc..
Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2
, and some of these sub-clones give rise to metastatic (and therefore lethal) disease.
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3
, or on macrodissection4
. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5
, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6
. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.
Medicine, Issue 56, quantitative immunofluorescence, heterogeneity, cancer, biomarker, targeted therapy, immunohistochemistry, proteomics, histopathology
Novel Apparatus and Method for Drug Reinforcement
Institutions: University of Texas at Austin.
Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.
Neuroscience, Issue 42, conditioned place preference (CPP), self-administration, rat, behavioral neuroscience, drug reinforcement, cocaine, animal models
Basics of Multivariate Analysis in Neuroimaging Data
Institutions: Columbia University.
Multivariate analysis techniques for neuroimaging data have recently received increasing attention as they have many attractive features that cannot be easily realized by the more commonly used univariate, voxel-wise, techniques1,5,6,7,8,9
. Multivariate approaches evaluate correlation/covariance of activation across brain regions, rather than proceeding on a voxel-by-voxel basis. Thus, their results can be more easily interpreted as a signature of neural networks. Univariate approaches, on the other hand, cannot directly address interregional correlation in the brain. Multivariate approaches can also result in greater statistical power when compared with univariate techniques, which are forced to employ very stringent corrections for voxel-wise multiple comparisons. Further, multivariate techniques also lend themselves much better to prospective application of results from the analysis of one dataset to entirely new datasets. Multivariate techniques are thus well placed to provide information about mean differences and correlations with behavior, similarly to univariate approaches, with potentially greater statistical power and better reproducibility checks. In contrast to these advantages is the high barrier of entry to the use of multivariate approaches, preventing more widespread application in the community. To the neuroscientist becoming familiar with multivariate analysis techniques, an initial survey of the field might present a bewildering variety of approaches that, although algorithmically similar, are presented with different emphases, typically by people with mathematics backgrounds. We believe that multivariate analysis techniques have sufficient potential to warrant better dissemination. Researchers should be able to employ them in an informed and accessible manner. The current article is an attempt at a didactic introduction of multivariate techniques for the novice. A conceptual introduction is followed with a very simple application to a diagnostic data set from the Alzheimer s Disease Neuroimaging Initiative (ADNI), clearly demonstrating the superior performance of the multivariate approach.
JoVE Neuroscience, Issue 41, fMRI, PET, multivariate analysis, cognitive neuroscience, clinical neuroscience
Using Learning Outcome Measures to assess Doctoral Nursing Education
Institutions: Harris College of Nursing and Health Sciences, Texas Christian University.
Education programs at all levels must be able to demonstrate successful program outcomes. Grades alone do not represent a comprehensive measurement methodology for assessing student learning outcomes at either the course or program level. The development and application of assessment rubrics provides an unequivocal measurement methodology to ensure a quality learning experience by providing a foundation for improvement based on qualitative and quantitatively measurable, aggregate course and program outcomes. Learning outcomes are the embodiment of the total learning experience and should incorporate assessment of both qualitative and quantitative program outcomes. The assessment of qualitative measures represents a challenge for educators in any level of a learning program. Nursing provides a unique challenge and opportunity as it is the application of science through the art of caring. Quantification of desired student learning outcomes may be enhanced through the development of assessment rubrics designed to measure quantitative and qualitative aspects of the nursing education and learning process. They provide a mechanism for uniform assessment by nursing faculty of concepts and constructs that are otherwise difficult to describe and measure. A protocol is presented and applied to a doctoral nursing education program with recommendations for application and transformation of the assessment rubric to other education programs. Through application of these specially designed rubrics, all aspects of an education program can be adequately assessed to provide information for program assessment that facilitates the closure of the gap between desired and actual student learning outcomes for any desired educational competency.
Medicine, Issue 40, learning, outcomes, measurement, program, assessment, rubric
Manual Muscle Testing: A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients
Institutions: Johns Hopkins University, Johns Hopkins Hospital , Johns Hopkins University, University of Maryland Medical System.
Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6
Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8
This video demonstrates a protocol for MMT, which has been taught to ≥43 research staff who have performed >800 assessments on >280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11
Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2
Medicine, Issue 50, Muscle Strength, Critical illness, Intensive Care Units, Reproducibility of Results, Clinical Protocols.
Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat
Institutions: University of Lethbridge.
High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.
Neuroscience, Issue 22, Rat walking, gait analysis, rotorod, rat forelimb, Parkinson disease model, dopamine depletion