Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.
22 Related JoVE Articles!
Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis
Institutions: University of California, San Francisco, University of California, San Francisco, University of California, San Francisco.
Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1
. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2
, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.
Neuroscience, Issue 57, vessel, vascular cast, capillary, cerebrovasculature, brain, blood, AVM, fistula
Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model
Institutions: University of Wurzburg.
Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro
BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro
BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1
). Human tissue samples are available only in a restricted number of laboratories or companies 2,3
. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4
(from cerebral cortex) and cerebEND 5
(from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9
and estrogen-treatment 10
as well as to pro-infammatory mediators, such as TNFalpha 5,8
. Moreover, we have studied the pathology of multiple sclerosis 11
and hypoxia 12,13
on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.
Immunology, Issue 66, Neuroscience, Blood-brain barrier, in vitro cell culture models, brain, microvascular endothelial cells, immortalization, cEND
Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window
Institutions: University of California, San Diego, La Jolla Bioengineering Institute.
This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo
fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei
ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.
Neuroscience, Issue 45, Brain hemodynamics, blood flow, microcirculation, vascular morphology, leukocyte adherence, cerebral malaria
Manual Drainage of the Zebrafish Embryonic Brain Ventricles
Institutions: Massachusetts Institute of Technology.
Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2
. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5
. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6
. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9
. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20
. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24
. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.
Neuroscience, Issue 70, Developmental Biology, Medicine, Anatomy, Physiology, Zebrafish, Danio rerio, eCSF, neuroepithelium, brain ventricular system, brain, microsurgery, animal model
Examination of the Telomere G-overhang Structure in Trypanosoma brucei
Institutions: Cleveland State University.
The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei
. It serves as the substrate for telomerase for de novo
telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei
is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei
cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei
telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei
pathogenesis. However, T. brucei
telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei
cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.
Immunology, Issue 47, Telomeres, telomeric G-overhang structure, native in-gel hybridization, ligation-mediated primer extension, Trypanosoma brucei
An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy
Institutions: Aix Marseille University, European Research Center for Medical Imaging, Campus de la Timone, KU Leuven Campus Gasthuisberg.
Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g.
Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.
Medicine, Issue 86, Glioblastoma multiforme, intravital two-photon imaging, animal model, chronic cranial window, brain tumors, neuro-oncology.
Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells
Institutions: University of Göttingen, University of Göttingen, University of Halle.
Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2
. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3
. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5
. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7
Despite these observations, however, up to now there is no suitable in vivo/in vitro
model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo
live imaging of carcinoma cells demonstrated their cerebral colonization behavior8
. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo
imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g.
qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo
approaches when testing targeted pharmacological manipulations.
Medicine, Issue 80, Brain Tissue, Cancer Cells, Nervous System, Neoplasms, Therapeutics, Organotypic brain slice, coculture, tumor invasion, cerebral colonization, brain metastasis, microglia, astrocyte, live-imaging
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison.
In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1
. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2
, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2
. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3
. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5
are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further.
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6
. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.
Medicine, Issue 58, BTSC, Tumor, cancer stem cell, cell migration, microfluidics, Glioblastoma Multiforme, GBM, chemotaxis, amoeboid, mesenchymal, haptotaxis, PDMS
Mouse Models of Periventricular Leukomalacia
Institutions: University of California, Davis.
We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.
JoVE Neuroscience, Issue 39, brain, mouse, white matter injury, oligodendrocyte, periventricular leukomalacia
RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes
Institutions: McGill University, McGill University, McGill University.
Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro
editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based “mix and measure” hammerhead ribozyme in vitro
reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro
RNA editing or high throughput screening of chemicals that can inhibit the editosome function.
Genetics, Issue 89, RNA editing, Trypanosoma brucei, Editosome, Hammerhead ribozyme (HHR), High-throughput screening, Fluorescence resonance energy transfer (FRET)
Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice
Institutions: The Walter and Eliza Hall Institute of Medical Research.
We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei
ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum
-mediated severe malaria in humans 1,2
. Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3
and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6
, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.
Immunology, Issue 71, Infection, Infectious Diseases, Pathology, Hematology, Molecular Biology, Cellular Biology, Mouse, Brain, Intravascular inflammation, leukocytes, Plasmodium berghei, parasite, malaria, animal model, flow cytometry
Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
Institutions: Hunter College, CUNY.
Efficient expression of transgenes in vivo
is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).
Genetics, Issue 87, hydrodynamic gene delivery, hydrodynamics-based transfection, mouse, gene therapy, plasmid DNA, transient gene expression, tail vein injection
Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE
Institutions: University of Cambridge, UK, University of Cambridge, UK.
Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via
functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v
.) or intracerebroventricular (i.c.v.
) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.
) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo
Here we describe the methods that we have developed for the i.v
. and i.c.v.
delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.
Immunology, Issue 86, Somatic neural stem/precursor cells, neurodegenerative disorders, regenerative medicine, multiple sclerosis, experimental autoimmune encephalomyelitis, systemic delivery, intravenous, intracerebroventricular
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
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
A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions
Institutions: Torrey Pines Institute for Molecular Studies, Cascade LifeSciences Inc..
Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro
technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2
tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo
behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.
Bioengineering, Issue 77, Cellular Biology, Biophysics, Physiology, Molecular Biology, Biomedical Engineering, Immunology, Cells, Biological Factors, Equipment and Supplies, Cell Physiological Phenomena, Natural Science Disciplines, Life Sciences (General), circulating cells, extravasation, physiological shear stress, endothelial cells, microenvironment, chemokine gradient, flow, chamber, cell culture, assay
Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia
Institutions: Technische Universität München, University Hospital of Basel, University of Saarland, University Hospital Zurich.
Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al.
have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.
Medicine, Issue 93, flap, ischemia, microcirculation, angiogenesis, skin, necrosis, inflammation, apoptosis, preconditioning, persistent ischemia, in vivo model, muscle.
An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei
Institutions: University Hospital Heidelberg, Research Center Borstel.
Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium
, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e.
aerosol and mosquito bite, respectively.
Infectious Diseases, Issue 84, coinfection, mouse, Tuberculosis, Malaria, Plasmodium berghei, Mycobacterium tuberculosis, natural transmission
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants
Institutions: University of Colima, Johns Hopkins University.
Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants. The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed. We show here a method to produce glass capillary needles with a 50-μm lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.
Neuroscience, Issue 46, Microinjections, Drug Delivery Systems, Micromanipulation, Demyelination
Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells
Institutions: University of Edinburgh.
Most human malaria deaths are caused by blood-stage Plasmodium falciparum
parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum
infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4
. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5
). Sequestration is also found in most microvascular tissues of the human body2, 3
. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro
model of the blood-brain barrier6
. However, Plasmodium falciparum
iRBC attach only poorly to HBEC-5i in vitro
, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum
strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum
strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum
parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum
strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7
Immunology, Issue 59, Plasmodium falciparum, cerebral malaria, cytoadherence, sequestration, endothelial cell, HBEC-5i