Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil β2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-α-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.
20 Related JoVE Articles!
A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
Institutions: Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Liverpool School of Tropical Medicine, New York University School of Medicine.
causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum
-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum
has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1
. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2
and restrict their environment to a more favorable low oxygen pressure 3
. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro
, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5
. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6
. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8
has also been associated with severe disease 9
One of the most recently described P. falciparum
cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro
. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10
. Whether clumping occurs in vivo
remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11
. In addition, the ability of clinical isolate cultures to clump in vitro
was directly linked to the severity of disease in Malawian 12
and Mozambican patients 13
, (although not in Malian 14
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15
. Here, we present a method for in vitro
platelet-mediated clumping of P. falciparum
with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.
Infection, Issue 75, Infectious Diseases, Immunology, Medicine, Microbiology, Molecular Biology, Cellular Biology, Parasitology, Clumping, platelets, Plasmodium falciparum, CD36, malaria, malarial infections, parasites, red blood cells, plasma, limited resources, clinical techniques, assay
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Institutions: University of Ontario Institute of Technology , University of Ontario Institute of Technology , University of Ontario Institute of Technology .
In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2
. Manual FV assays have been described 3, 4
, but they are time consuming and subjective. Automated FV assays have been reported 5-7
, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9
. Microplate assays have been reported for clot lysis 10
, platelet aggregation 11
, and coagulation Factors 12
, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1
. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14
. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15
. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.
Immunology, Issue 67, Factor V, Microplate, Coagulation assay, Human plasma, Disseminated intravascular coagulation (DIC), blood clotting
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System
Institutions: Örebro University Hospital.
Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.
Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (≥ 2.0 x1011
platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.
Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g.
pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.
Medicine, Issue 70, Immunology, Hematology, Infectious Disease, Pathology, pathogen inactivation, pathogen reduction, double-dose platelets, INTERCEPT Blood System, amotosalen, UVA, platelet, blood processing, buffy coat, IBS, transfusion
PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP
Institutions: West Virginia University , University of Pittsburgh, WVNano Initiative, Mary Babb Randolph Cancer Center.
Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro
antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro
antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus,
Group A Streptococcus
, and Neisseria gonorrhoeae
. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.
Infection, Issue 74, Infectious Diseases, Immunology, Microbiology, Medicine, Cellular Biology, Molecular Biology, Bacterial Infections and Mycoses, Musculoskeletal Diseases, Biological Factors, Platelet-rich plasma, bacterial infection, antimicrobial, kill curve assay, Staphylococcus aureus, clinical isolate, blood, cells, clinical techniques
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry: a Novel Use for a Cell Purification Method
Institutions: University of California, University of California, University of California, University of California, University of California, University of California.
Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck & Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47
), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.
Immunology, Issue 50, spinal cord injury, cellular inflammation, neuroinflammation, OptiPrep, central nervous system, neutrophils, macrophages, microglia, T-cells, flow cytometry
Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis
Institutions: University of California, Irvine (UCI).
Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence).
Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs.
The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.
Immunology, Issue 5, Autoimmune Disease, Animal Model, EAE, Experimental Allergic Encephalomyelitis, Multiple Sclerosis, Immunology, Clinical Scoring, Disease Model, Inflammation, Central Nervous System
In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers
Institutions: University of Nebraska, Lincoln, University of Nebraska, Lincoln, University of Nebraska, Lincoln.
This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ
in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-α (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+
cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs
/PLP 139-151 dextramers (specific)/anti-CD4 and IAs
/Theiler’s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk
/Myhc 334-352 dextramers/anti-CD4 and IAk
/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+
cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the ‘Z’ serial images.
Immunology, Issue 90, dextramers; MHC class II; in situ; EAE; brain; EAM; heart; confocal microscopy.
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
Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice
Institutions: University of Münster, Interdisciplinary Center for Clinical Research (IZKF), Münster, University of Münster.
Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55
is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.
Immunology, Issue 86, experimental autoimmune encephalomyelitis, EAE, multiple sclerosis, MS, animal model, Autoimmunity, neuroinflammation, central nervous system, pertussis
Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis
Institutions: Aix Marseille University, European Research Center for Medical Imaging (CERIMED).
Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.
Medicine, Issue 82, Spinal cord, two-photon microscopy, In vivo, intravital microscopy, EAE, Multiple Sclerosis, transgenic mouse
Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge
Institutions: St. John-Providence Health System, Wayne State University School of Medicine.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b
) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 μg/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 μg/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.
Immunology, Issue 62, Autoimmune diseases, experimental autoimmune encephalomyelitis, immunization, myelin basic protein, adoptive transfer, paralysis
Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation
Institutions: Harvard Medical School.
Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1
. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2
. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3
. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4
and pathologies such as inflammation5
. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6
. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ
hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ
allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.
Immunology, Issue 65, Neuroscience, Genetics, microglia, macrophages, microRNA, brain, mouse, real-time PCR, neuroinflammation
Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes
Institutions: The University of Florida.
Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-β. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.
Immunology, Issue 79, Cellular Biology, Molecular Biology, Medicine, Infection, Th17 cells, IL-17, Th17 differentiation, T cells, autoimmunity, cell, isolation, culture
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),
Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets
Institutions: University of Alabama at Birmingham.
Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.
Immunology, Issue 85, bioenergetics, translational, mitochondria, oxidative stress, reserve capacity, leukocytes
Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)
Institutions: University of Wuerzburg, Max Planck Institute of Neurobiology, Martinsried, Ludwig-Maximilians University of Munich.
Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+
indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+
indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro
. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+
indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+
indicator and a hydrophilic fluorescent dye/Ca2+
complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0
Cellular Biology, Issue 75, Neurobiology, Neuroscience, Molecular Biology, Biochemistry, Biomedical Engineering, Bioengineering, Virology, Medicine, Anatomy, Physiology, Surgery, Endoplasmic Reticulum, ER, Calcium Signaling, calcium store, calcium imaging, calcium indicator, metabotropic signaling, Ca2+, neurons, cells, mouse, animal model, cell culture, targeted esterase induced dye loading, imaging
Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients
Institutions: University of Texas at San Antonio - UTSA.
Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.
Immunology, Issue 48, Microglia, monocytes/macrophages, CNS, inflammation, EAE, chemokines, mouse, flow cytometry
Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE
Institutions: University of California, Irvine (UCI).
Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure.
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.
Neuroscience, Issue 10, Immunology, brain, spinal cord, lymphocyte, infiltrate, experimental autoimmune encephalomyelitis, CNS, inflammation, mouse