Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.
24 Related JoVE Articles!
The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Institutions: Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e.
endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
Basic Protocol, Issue 82, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication
Institutions: University of Sydney, University of Hull, Hull and East Yorkshire Hospitals, Addenbrookes Hospital.
Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual's use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual's postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.
Medicine, Issue 82, Posture, Computerized dynamic posturography, Ankle brachial pressure index, Peripheral arterial disease, Intermittent claudication, Balance, Posture, EquiTest, Sensory Organization Test, Motor Control Test
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Assessing Functional Performance in the Mdx Mouse Model
Institutions: Leiden University Medical Center.
Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx
mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g
. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.
Behavior, Issue 85, Duchenne muscular dystrophy, neuromuscular disorders, outcome measures, functional testing, mouse model, grip strength, hanging test wire, hanging test grid, rotarod running, treadmill running
Nucleoside Triphosphates - From Synthesis to Biochemical Characterization
Institutions: University of Bern.
The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.
Chemistry, Issue 86, Nucleic acid analogues, Bioorganic Chemistry, PCR, primer extension reactions, organic synthesis, PAGE, HPLC, nucleoside triphosphates
A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology
Institutions: University of Ottawa, University of Ottawa, University of Calgary, University of Ottawa, University of Ottawa.
Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.
Bioengineering, Issue 88, cell stretching, tissue mechanics, nuclear mechanics, uniaxial, biaxial, anisotropic, mechanobiology
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue
Institutions: Massachusetts General Hospital/Harvard Medical School, China Pharmaceutical University, Perkin Elmer.
Brown adipose tissue (BAT), widely known as a “good fat” plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18
F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18
F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18
F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.
Medicine, Issue 92, Cerenkov luminescence imaging, brown adipose tissue, 18F-FDG, optical imaging, in vivo imaging, spectral unmixing
Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Institutions: Louisiana State University Health Sciences Center.
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.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
Analysis of Cell Migration within a Three-dimensional Collagen Matrix
Institutions: Witten/Herdecke University.
The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.
Bioengineering, Issue 92, cell migration, 3D collagen matrix, cell tracking
A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat
Institutions: Université de Sherbrooke, Université de Sherbrooke, Université de Sherbrooke, Université de Sherbrooke.
We present a method for comparing the uptake of the brain's two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which 11
C-AcAc and 18
F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11
C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11
C-AcAc and 18
F-FDG brain uptake (cerebral metabolic rate; μmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases.
Neuroscience, Issue 82, positron emission tomography (PET), 18F-fluorodeoxyglucose, 11C-acetoacetate, magnetic resonance imaging (MRI), kinetic modeling, cerebral metabolic rate, rat
Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering
Institutions: The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, VA Medical Center, Providence, RI, University of Texas Southwestern Medical Center .
We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.
Bioengineering, Issue 74, Biomedical Engineering, Biophysics, Cellular Biology, Medicine, Anatomy, Physiology, Cell Engineering, Bioreactors, Culture Techniques, Cell Engineering, Tissue Engineering, compression loads, shear loads, Tissues, bioreactor, mechanical loading, compression, shear, musculoskeletal, cartilage, bone, transplantation, cell culture
Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Institutions: Yale University, Yale University, Yale University, Yale University, Massachusetts General Hospital, University of California, Irvine.
We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7
. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8
that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11
C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7
to a conventional model 9
. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.
Behavior, Issue 78, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Image Processing, Computer-Assisted, Receptors, Dopamine, Dopamine, Functional Neuroimaging, Binding, Competitive, mathematical modeling (systems analysis), Neurotransmission, transient, dopamine release, PET, modeling, linear, time-invariant, smoking, F-test, ventral-striatum, clinical techniques
Vascular Occlusion Training for Inclusion Body Myositis: A Novel Therapeutic Approach
Institutions: University of São Paulo, University of São Paulo.
Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise function and quality of life. Moreover, clinical practice suggests that, unlike other inflammatory myopathies, the majority of IBM patients are not responsive to treatment with immunosuppressive or immunomodulatory drugs to counteract disease progression1
. Additionally, conventional resistance training programs have been proven ineffective in restoring muscle function and muscle mass in these patients2,3
. Nevertheless, we have recently observed that restricting muscle blood flow using tourniquet cuffs in association with moderate intensity resistance training in an IBM patient produced a significant gain in muscle mass and function, along with substantial benefits in quality of life4
. Thus, a new non-pharmacological approach for IBM patients has been proposed. Herein, we describe the details of a proposed protocol for vascular occlusion associated with a resistance training program for this population.
Medicine, Issue 40, exercise training, therapeutical, myositis, vascular occlusion
Isolating Stem Cells from Soft Musculoskeletal Tissues
Institutions: Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, University of Pittsburgh, University of Pittsburgh, University of Pittsburgh, University of Pittsburgh.
Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st
enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.
Cellular Biology, Issue 41, Adult stem cells, isolation, softy tissue, adhesion
Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells
Institutions: University of Toronto, University of Toronto.
This protocol describes the fabrication of a type of micro-tissues called modules. The module approach generates uniform, scalable and vascularized tissues. The modules can be made of collagen as well as other gelable or crosslinkable materials. They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells. The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable. These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues. There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.
Bioengineering, Issue 46, Tissue engineering, micro-tissue, endothelial cells, collagen gels, modules, 3D tissue culture.
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
Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context
Institutions: University of Glasgow, University of Glasgow.
Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1
. Furthermore, interaction with other cell types, including stromal fibroblasts 2
and immune cells 3
, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro
and in vivo 4
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo
environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5
. As such, they can serve an important role in reducing the need for experiments on living animals.
Bioengineering, Issue 56, Organotypic culture, cell migration, invasion, 3-dimensional matrix, Collagen I, second harmonic generation, host-tumor interaction, microenvironment
Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Institutions: University Medical Center Utrecht.
Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1
. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2
. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4
. These soluble factors can be studied in vitro
by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro
. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro
studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.
Neuroscience, Issue 61, Axon guidance, collagen matrix, development, dissection, dopamine, medium spiny neuron, rat tail collagen, striatum, striatonigral, mesostriatal
Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
Institutions: University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Perelman School of Medicine.
Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1
and carotid intimal medial thickness (C-IMT)2
provide information about the burden of disease. However, despite multiple validation studies of CAC3-5
, and C-IMT2,6
, these modalities do not accurately assess plaque characteristics7,8
, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13
F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15
. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16
, an important source of cellular inflammation in vessel walls. More recently, we17,18
and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20
. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo
imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22
and is also highly associated with overall burden of atherosclerosis23
. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24
as well as longer term therapeutic lifestyle changes (16 months)25
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26
. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28
Medicine, Issue 63, FDG-PET/CT, atherosclerosis, vascular inflammation, quantitative radiology, imaging
A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation
Institutions: New York University School of Medicine, New York University School of Medicine, New York University School of Medicine, New York University School of Medicine, New York University School of Medicine, New York University School of Medicine, New York University School of Medicine.
Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo
studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo
characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro
model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1
) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+
neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1
. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.
Immunology, Issue 62, neuron cell culture, Herpes Simplex Virus (HSV), molecular biology, virology
Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry
Institutions: Sanford-Burnham Medical Research Institute, University of California, San Diego , VA San Diego Healthcare Center, University of California, San Diego .
Although human saliva proteome and peptidome have been revealed 1-2
they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4
may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6
. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7
and pre-digestion with trypsin, which makes it difficult for clinical use.
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11
. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo
from various microenvironments in animals in a dynamic and minimally invasive manner 8-11
. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11
. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.
Medicine, Issue 66, Molecular Biology, Genetics, Sampling, Saliva, Peptidome, Ultrafiltration, Mass spectrometry
In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
Institutions: University of Toronto, University of Toronto.
Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo
through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro
, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.
Bioengineering, Issue 67, Chemistry, Biochemistry, Tissue Engineering, Collagen, Self-assembly, Native, Fibrous long spacing, Segmental long spacing, AFM, atomic force microscopy
Methods to Assess Subcellular Compartments of Muscle in C. elegans
Institutions: University of Nottingham.
Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans
is an established model for understanding the genomic regulation of biological processes of interest. This worm’s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans
is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo
. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo
. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans
provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo
in any other organism.
Developmental Biology, Issue 93, Physiology, C. elegans, muscle, mitochondria, sarcomeres, ageing