Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.
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
Stereotactic Radiosurgery for Gynecologic Cancer
Institutions: University Hospitals Case Medical Center and Case Western Reserve University School of Medicine, University Hospitals Case Medical Center and Case Western Reserve University School of Medicine.
Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments.
Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.1-3
Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,4
patient mouthpiece active breathing coordination,5
respiration-correlated computed tomography,6
or image-guided tracking of fiducials implanted within and around a moving tumor.7-9
The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.10
Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.11-13
Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.7,8
Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[18
F-FDG) positron emission tomography (PET).8
Use of 18
F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.
Medicine, Issue 62, radiosurgery, Cyberknife stereotactic radiosurgery, radiation, ovarian cancer, cervix cancer
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
Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay
Institutions: University of Alabama-Birmingham, The Ohio State University Medical School, University of Alabama at Birmingham School of Medicine, University of Alabama-Birmingham.
A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2
. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.
Neuroscience, Issue 70, Genetics, Cellular Biology, Molecular Biology, Medicine, Cancer Biology, Anatomy, Physiology, DNA, DNA damage, double strand break, single strand break, repair, neurons, comet assay, cell culture
Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation
Institutions: The Alfred Medical Research and Education Precinct, The Alfred Medical Research and Education Precinct, University of Melbourne.
An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2
. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3
. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3
. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7
. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8
. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9
. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.
Cellular Biology, Issue 42, H2AX, radiation, euchromatin, heterochromatin, immunofluorescence, 3D-modeling
Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies
Institutions: Harvard School of Public Health, Brigham and Women's Hospital, Harvard Medical School, Pennsylvania State University.
Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.
samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.
Medicine, Issue 83, dried blood spots (DBS), Biomarkers, cardiometabolic risk, Inflammation, standard precautions, blood collection
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media
Institutions: Drexel University College of Medicine.
Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.
Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media
Genetics, Issue 76, Molecular Biology, Cellular Biology, Medicine, Biochemistry, Genomics, Pharmacology, Exosomes, RNA, MicroRNAs, Biomarkers, Pharmacological, Exosomes, microRNA, qPCR, PCR, blood, biomarker, TLDA, profiling, sequencing, cell culture
Quantitation of γH2AX Foci in Tissue Samples
Institutions: The Alfred Medical Research and Education Precinct, The Alfred Medical Research and Education Precinct, The University of Melbourne, Royal Children's Hospital, The University of Melbourne.
DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks1
. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2
. Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2
. Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3
. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo
and in vivo4,5
. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.
Cellular Biology, Issue 40, immunofluorescence, DNA double-strand breaks, histone variant, H2AX, DNA damage, ionising radiation, reactive oxygen species
Quantification of γH2AX Foci in Response to Ionising Radiation
Institutions: The Alfred Medical Research and Education Precinct, The University of Melbourne, The Alfred Medical Research and Education Precinct.
DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX1
. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2
. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3
. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete γH2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2
. The loss of γH2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8
. The disappearence of γH2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6
. Further, removal of γH2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8
. Importantly, the quantitative analysis of γH2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of γH2AX foci in γ-irradiated adherent human keratinocytes9
Medicine, Issue 38, H2AX, DNA double-strand break, DNA damage, chromatin modification, repair, ionising radiation
Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro
using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro
. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo
. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo
tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro
and in vivo
and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Institutions: California Institute of Technology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, University Of Washington Medical Center, Johns Hopkins University School of Medicine.
The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for C
omputer tomography-guided f
radiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.
Neuroscience, Issue 81, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
Institutions: Massachusetts Institute of Technology, Chulabhorn Graduate Institute, University of Minnesota.
DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The ‘CometChip’ is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.
Bioengineering, Issue 92, comet assay, electrophoresis, microarray, DNA damage, DNA repair
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development
Institutions: Consiglio Nazionale delle Ricerche.
The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.
Chemistry, Issue 74, Biochemistry, Chemical Engineering, Chemical Biology, chemical analysis techniques, chemistry (general), life sciences, radiation effects (biological, animal and plant), biomarker, biomimetic chemistry, free radicals, trans lipids, cyclopurine lesions, DNA, chromatography, spectroscopy, synthesis
Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, The Netherlands Cancer Institute, University of Pittsburgh School of Public Health.
We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2
. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET)3,4
. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.
Molecular Biology, Issue 66, Genetics, Cancer Biology, Base excision repair, DNA glycosylase, AP endonuclease, fluorescent, real-time, activity assay, molecular beacon, biomarker, DNA Damage, base lesion
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
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)
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
4D Multimodality Imaging of Citrobacter rodentium Infections in Mice
Institutions: Imperial College London, Caliper- A PerkinElmer Company.
This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated μCT (DLIT-μCT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-μCT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium,
which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium
and non-invasive monitoring of colonization by daily DLIT-μCT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and μCT scans using a single imaging platform. The low dose μCT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.
Infection, Issue 78, Immunology, Cellular Biology, Molecular Biology, Microbiology, Genetics, Biophysics, Biomedical Engineering, Medicine, Anatomy, Physiology, Infectious Diseases, Bacterial Infections, Bioluminescence, DLIT-μCT, C. rodentium, 4D imaging, in vivo imaging, multi-modality imaging, CT, imaging, tomography, animal model
Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography
Institutions: University of Tubingen, European Synchrotron Radiation Facility.
Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7µm. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.
Developmental Biology, Issue 15, Synchrotron X-ray tomography, Acari, Oribatida, micro-arthropods, non-invasive investigation
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