Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
22 Related JoVE Articles!
Clonogenic Assay: Adherent Cells
Institutions: The Alfred Medical Research and Education Precinct, The University of Melbourne, The Alfred Medical Research and Education Precinct, The University of Melbourne.
The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561
. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1
. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2
. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.
Cellular Biology, Issue 49, clonogenic assay, clonogenic survival, colony staining, colony counting, radiation sensitivity, radiation modification
Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation
Institutions: Lawrence Berkeley National Laboratory.
Tunable soft ionization coupled to mass spectroscopy is a powerful method to investigate isolated molecules, complexes and clusters and their spectroscopy and dynamics1-4
. Fundamental studies of photoionization processes of biomolecules provide information about the electronic structure of these systems. Furthermore determinations of ionization energies and other properties of biomolecules in the gas phase are not trivial, and these experiments provide a platform to generate these data. We have developed a thermal vaporization technique coupled with supersonic molecular beams that provides a gentle way to transport these species into the gas phase. Judicious combination of source gas and temperature allows for formation of dimers and higher clusters of the DNA bases. The focus of this particular work is on the effects of non-covalent interactions, i.e.
, hydrogen bonding, stacking, and electrostatic interactions, on the ionization energies and proton transfer of individual biomolecules, their complexes and upon micro-hydration by water1, 5-9
We have performed experimental and theoretical characterization of the photoionization dynamics of gas-phase uracil and 1,3-dimethyluracil dimers using molecular beams coupled with synchrotron radiation at the Chemical Dynamics Beamline10
located at the Advanced Light Source and the experimental details are visualized here. This allowed us to observe the proton transfer in 1,3-dimethyluracil dimers, a system with pi stacking geometry and with no hydrogen bonds1
. Molecular beams provide a very convenient and efficient way to isolate the sample of interest from environmental perturbations which in return allows accurate comparison with electronic structure calculations11, 12
. By tuning the photon energy from the synchrotron, a photoionization efficiency (PIE) curve can be plotted which informs us about the cationic electronic states. These values can then be compared to theoretical models and calculations and in turn, explain in detail the electronic structure and dynamics of the investigated species 1, 3
Physics, Issue 68, mass spectroscopy (application), physical chemistry, radiation chemistry, molecular beams, molecular physics, molecular structure, photon interactions with atoms and molecules, Molecular beam, mass spectrometry, vacuum ultraviolet, synchrotron radiation, proton transfer, DNA bases, clusters
Fabrication of the Thermoplastic Microfluidic Channels
Institutions: Boston University.
In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.
Cellular Biology, Issue 12, bioengineering, purification, microfluidics, DNA, RNA, solid phase, column
The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms
Institutions: Bar-Ilan University, Bar-Ilan University, Bar-Ilan University, Bar-Ilan University.
Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5
. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7
. There are also reports of biocidal effect of red and near infra red8
as well as green light9
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa
. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa
is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa
strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa
. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa
was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.
Microbiology, Issue 77, Infection, Infectious Diseases, Cellular Biology, Molecular Biology, Biophysics, Chemistry, Biomedical Engineering, Bacteria, Photodynamic therapy, Medical optics, Bacterial viability, Antimicrobial treatment, Laser, Gentamycin, antibiotics, reactive oxygen species, pathogens, microorganisms, cell culture
Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology
Institutions: University Medical Center Groningen, Technical University Munich, University Medical Center Groningen.
The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3
Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5
As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6
In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7
The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8
It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.
Medicine, Issue 44, Image-guided surgery, multispectral fluorescence, sentinel lymph node, gynecologic oncology
Preparation of Artificial Bilayers for Electrophysiology Experiments
Institutions: Weill Cornell Medical College of Cornell University.
Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels. Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties. We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.
Cellular Biology, Issue 20, Springer Protocols, Artificial Bilayers, Bilayer Patch Experiments, Lipid Bilayers, Bilayer Punch Electrodes, Electrophysiology
Ultrasonic Assessment of Myocardial Microstructure
Institutions: Harvard Medical School, Brigham and Women's Hospital, Harvard Medical School.
Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.
Medicine, Issue 83, echocardiography, image analysis, myocardial fibrosis, hypertension, cardiac cycle, open-access image analysis software
Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence
Institutions: University of Central Florida, University of Central Florida, University of Central Florida.
DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA.
In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e.,
“clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g.,
strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.
Basic Protocol, Issue 97, Forensic Science, Touch DNA Evidence, Micro-manipulation, Cell Isolation and Recovery, DNA Profiling, Short Tandem Repeat (STR) Analysis
Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images
Institutions: Vanderbilt University, Vanderbilt University School of Medicine, Vanderbilt University Medical Center, Vanderbilt University.
Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18
F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18
F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 °C) or cold-activated (CA) (17 °C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.
Medicine, Issue 96, magnetic resonance imaging, brown adipose tissue, cold-activation, adult human, fat water imaging, fluorodeoxyglucose, positron emission tomography, computed tomography
Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG
Institutions: Macquarie University.
Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity . The electrophysiological activity of the brain generates electric fields - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 °C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.
Neuroscience, Issue 36, Magnetoencephalography, Pediatrics, Brain Mapping, Language, Brain Development, Cognitive Neuroscience, Language Acquisition, Linguistics
Minimal Erythema Dose (MED) Testing
Institutions: Fox Chase Cancer Center , University of Pennsylvania , Drexel University , Fox Chase Cancer Center , The Cancer Institute of New Jersey.
Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual's skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual's skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.
Medicine, Issue 75, Anatomy, Physiology, Dermatology, Analytical, Diagnostic, Therapeutic Techniques, Equipment, Health Care, Minimal erythema dose (MED) testing, skin sensitivity, ultraviolet radiation, spectrophotometry, UV exposure, psoriasis, acne, eczema, clinical techniques
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Sealable Femtoliter Chamber Arrays for Cell-free Biology
Institutions: University of Tennessee, Knoxville, Oak Ridge National Laboratory, University of Tennessee, Knoxville.
Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g.,
global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.
Bioengineering, Issue 97, Cell-free, synthetic biology, microfluidics, noise biology, soft lithography, femtoliter volumes
Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
Institutions: University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine.
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs
-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf
transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf
animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1
. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf
fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Medicine, Issue 79, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment
Institutions: West Virginia University, West Virginia University, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, GRECC, VA Medical Center, West Virginia University.
The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer’s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer’s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.
Behavior, Issue 100, Spatial learning, spatial reference memory, Morris water maze, Alzheimer’s disease, behavior, tau, hippocampal-dependent learning, rTg4510, Tg2576, strain background, transgenic mouse models
Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses
Institutions: Saint Louis University.
Currently, most of the in vitro
cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 – 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn’t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.
Bioengineering, Issue 97, Multiwell, substrate stiffness, drug screening, polyacrylamide, Young’s modulus, high-throughput
Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract
Institutions: Karolinska Institutet, Uppsala University .
Cataract is the leading cause of blindness in the world 1
. The World Health Organization defines cataract as a clouding of the lens of the eye which impedes the transfer of light. Cataract is a multi-factorial disease associated with diabetes, smoking, ultraviolet radiation (UVR), alcohol, ionizing radiation, steroids and hypertension. There is strong experimental 2-4
and epidemiological evidence 5,6
that UVR causes cataract. We developed an animal model for UVR B induced cataract in both anesthetized 7
and non-anesthetized animals 8
The only cure for cataract is surgery but this treatment is not accessible to all. It has been estimated that a delay of onset of cataract for 10 years could reduce the need for cataract surgery by 50% 9
. To delay the incidence of cataract, it is needed to understand the mechanisms of cataract formation and find effective prevention strategies. Among the mechanisms for cataract development, apoptosis plays a crucial role in initiation of cataract in humans and animals 10
. Our focus has recently been apoptosis in the lens as the mechanism for cataract development 8,11,12
. It is anticipated that a better understanding of the effect of UVR on the apoptosis pathway will provide possibilities for discovery of new pharmaceuticals to prevent cataract.
In this article, we describe how cataract can be experimentally induced by in vivo
exposure to UVR-B. Further RT-PCR and immunohistochemistry are presented as tools to study molecular mechanisms of UVR-B induced cataract.
Medicine, Issue 69, Neuroscience, Molecular Biology, Ophthalmology, Immunology, UVR-B, lens, cataract, qRT-PCR, PCR, immunohistochemistry, rat restrainer, animal model
Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area
Institutions: Mississippi State University College of Veterinary Medicine.
Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.
Neuroscience, Issue 96, Laser capture microdissection, dopamine neuron, Immunohistochemistry, Tyrosine hydroxylase, Ventral tegmental area, PEN membrane glass slide.
Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact
Institutions: University of Southampton, University of Southampton.
The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to clearly describe the acromion marker cluster (AMC) method of determining scapular kinematics when using a passive marker motion capture system, with consideration for the sources of error which could affect the validity and reliability of measurements. The AMC method involves placing a cluster of markers over the posterior acromion, and through calibration of anatomical landmarks with respect to the marker cluster it is possible to obtain valid measurements of scapular kinematics. The reliability of the method was examined between two days in a group of 15 healthy individuals (aged 19-38 years, eight males) as they performed arm elevation, to 120°, and lowering in the frontal, scapular and sagittal planes. Results showed that between-day reliability was good for upward scapular rotation (Coefficient of Multiple Correlation; CMC = 0.92) and posterior tilt (CMC = 0.70) but fair for internal rotation (CMC = 0.53) during the arm elevation phase. The waveform error was lower for upward rotation (2.7° to 4.4°) and posterior tilt (1.3° to 2.8°), compared to internal rotation (5.4° to 7.3°). The reliability during the lowering phase was comparable to results observed during the elevation phase. If the protocol outlined in this study is adhered to, the AMC provides a reliable measurement of upward rotation and posterior tilt during the elevation and lowering phases of arm movement.
Medicine, Issue 96, Scapula, kinematics, reliability, acromion marker cluster
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
Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding
Institutions: University Hospital Würzburg, Würzburg Branch of the Fraunhofer-Institute Interfacial Engineering and Biotechnology, IGB.
models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.
Bioengineering, Issue 96, Tissue engineering, 3D in vitro models, test system, alternative to animal testing, full thickness, skin equivalent, skin injury, wound model, automation, wounding device