Wir erkennen die Finanzierung der Unterstützung des National Health und Medical Research Council (gewähren APP605524, 490.037 und 1.047.082), der Australian Research Council (gewähren DP12010061), der National Collaborative Research Infrastructure Strategy of Australia und der Bildung Investmentfonds von der Abteilung Innovation, Industrie , Wissenschaft und Forschung. PML ist ein Empfänger einer australischen Postgraduate Award.

Alle Verfahren wurden in Übereinstimmung mit den Richtlinien der Macquarie University durchgeführt und entsprach der nationalen Gesundheits-und Medical Research Council (NHMRC) Australian Verhaltenskodex. Die Arbeit wurde im Rahmen des Abkommens Ethik Kein ARA 2012/017 genehmigt und von der Tierethikkommission an der Macquarie Universität erhalten durchgeführt. Alle Experimente wurden an SJL / J-Mäusen durchgeführt, sofern nicht anders angegeben. 1. Mäuse und experimentelle Malariainfektion <ol><li> Hausmäuse bei kontrollierter Temperatur (21 ° C) mit einem 12:12 Stunden Licht-Dunkel-Zyklus. </li><li> Tauwetter ein Aliquot von kryokonservierten P. chabaudi adami DS mit 5% parasitierten Erythrozyten (PRBCs) und injizieren 200 ul in die Bauchhöhle von C57BL / 6 Spendermaus. </li><li> Überwachen Spendermaus Parasitämie alle 1 - 2 Tage mittels Durchflusszytometrie, wie in Abschnitt 3 beschrieben - 4 dieses Protokolls. Sobald Spendern erreicht 5 - 15% Parasitämie, sammeln Blut durch Herzpunktion wie folgt: <ol><li>Aspirat 100 ul Heparin (300 U / ml Heparin in der Maus Ringer-Lösung (MTR) (154 mM NaCl, 5,6 mM KCl, 1 mM MgCl 2, 2,2 mM CaCl 2, 20 mM HEPES, 10 mM Glucose, 30 U / ml Heparin, pH 7,4, 0,22 um Filter sterilisiert)) in eine 1 ml Spritze mit einer 26 G-Nadel. </li><li> Anesthetize die Maus durch Inhalation mit 5% Isofluoran bestätigte die gewünschte Tiefe der Anästhesie durch Fehlen Pedalrückzugsreaktion und Hornhautreflexe. </li><li> Platzieren der Maus auf die Seite und senkrecht Einstichkanüle gerade unterhalb des Ellenbogens, durch die Rippen und in das Herz. Langsam herausziehen Spritzenkolben und drehen Nadel bis 0,5-1 ml Blut erhalten wird. </li><li> Führen humane Sterbehilfe durch Genickbruch unter Narkose. </li></ol></li><li> Berechne die prbcS pro Volumen von Spenderblut Annahme einer Blutbild von 9 x 10 6 RBC / ul (dh, wenn der Spender mit 10% Parasitämie bei geopfert Blut 9 x 10 5 enthältpRBC / ul). Verdünne parasitierten Blut in Krebs-gepufferter Salzlösung (126 mM NaCl, 2,5 mM KCl, 25 mM NaHCO 3, 1,2 mM NaH 2 PO 4, 1,2 mM MgCl 2, 2,5 mM CaCl 2, 0,2% Glucose, pH 7,2, 0,22 um Filter sterilisiert) so dass die Konzentration 1 x 10 4 prbcS pro 200 &amp; mgr; l und 200 &amp; mgr; l Injektion in die Bauchhöhle von Mäusen für die RBC Kennzeichnung und Transfusionsexperiment erforderlich. </li><li> Überwachen Parasitämie alle 1 - 2 Tage mittels Durchflusszytometrie als in den Abschnitten 3 beschrieben - 4 dieses Protokolls. </li></ol> 2. Markierung der Erythrozyten und Transfusions <ol><li> Sammeln heparinisiertem Blut von Mäusen durch Herzpunktion, wie in Schritt 1.3 beschrieben, und legen Sie auf dem Eis. Halten Blut bei 4 ° C zu allen Zeiten. Sammeln etwa 200 ul Blut pro Maus injiziert werden. Bei Bedarf aufgeteilt Blut in zwei Proben und Behandlung einer Probe, wie gewünscht. HINWEIS: Auf diese Weise Invasion der behandelten sample kann zu einer Kontrolle verglichen werden, unbehandelten Probe (siehe Abbildung 1 schematisch). </li><li> Bereiten Sie eine 2x Lösung von jeder Leuchtstoff RBC-Label. <ol><li> Verdünne eine 2 mg / ml Stammlösung von Atto 633-N-Hydroxysuccinimid (Atto 633-NHS) in MTR so daß die Konzentration 20 ug / ml. </li><li> Verdünne mit 25 mg / ml Vorratslösung von Biotin-N-hydroxysuccinimidester (Biotin-NHS) in MTR so daß die Konzentration 250 ug / ml. </li></ol></li><li> Aufgeteilt Blutproben in zwei Röhrchen entnommen und das gleiche Volumen von 2x Atto 633-NHS-Lösung auf ein Rohr, und ein gleiches Volumen von 2x-Biotin-NHS zur anderen Röhre. Mischen Sie sofort. Inkubieren Blut bei 4 ° C für 1 h mit konstantem langsamem Mischen oder alternativ mischen Probe jedes 10-15 min. </li><li> Blut beschriftet 3 Mal Waschen mit MTRC (MTR + 0,5% Rinderserumalbumin (BSA), 0,22 um Filter sterilisiert) wie folgt: <ol><li> Fügen Sie mindestens 2 Volumina MTRC, Zentrifuge bei 750 xg für 5 min, entfernen Sie den Überstand und resuspend die Pellets in mindestens 2 Volumina MTRC. Wiederholen Sie 2 weitere Male. </li><li> Nach dem letzten Waschen, Zentrifugieren bei 750 × g für 5 min pelletiert und verbinden das Blut in verschiedenen Kombinationen (dh unbehandelten Atto 633 / Biotin behandelten, unbehandelten Biotin / behandelt Atto 633). Hinzufügen MTR bis zu einem Volumen von 200 ul pro Maus zu machen, eingespritzt werden. </li></ol></li><li> Injizieren beschriftet Blut intravaskulär in Nagetier Malaria infizierten Mäusen oder infizierten Kontrollen wie folgt: <ol><li> Führen Trans bei 2 - 15% Parasitämie am Gipfel des Parasiten Schizonten. HINWEIS: In diesem Fall etwa in der Mitte durch die Dunkel-Zyklus (dh zwischen 12 bis 2 Uhr morgens an einem normalen Lichtzyklus oder 12-2 Uhr an einem umgekehrten Lichtzyklus). </li><li> Warm-Mäuse mit einer Wärmelampe für 5-10 Minuten, um den Blutfluss zu erhöhen, aber keine Vorkehrungen, um die Mäuse überhitzen. Zeigen Mäuse in einem Rückhalteeinrichtung und intravaskulär injizieren 200 ul markierte Blut (ca. 1 - 2 x 10 9 RBCs) über die Schwanzvene. </li></ol></li><li> Sammeln von Blutproben zu verschiedenen Zeitpunkten nach der Injektion, wie in Abschnitt 3 beschrieben. HINWEIS: Invasion Raten von weniger als 10 min nach der Injektion quantitativ bestimmt werden, abhängig von Parasitämie der Empfängermaus. </li></ol> 3. Entnahme von Blutproben und Vorbereitung für die Durchflusszytometrie <ol><li> Vorbereitung 50 ul Farblösung pro Probe plus zusätzliche, am Tag des Experiments wie folgt: <ol><li> Abtauen eines Aliquots von 6 mM JC-1 bei -20 ° C in Dimethylsulfoxid (DMSO) gelagert. </li><li> Warm 50 ul MTRC pro Probe, plus extra, auf 37 ° C. </li><li> Während kontinuierlich Vortexen des vorgewärmten MTRC Fügen JC-1, so dass die Endkonzentration 12 &amp; mgr; M. Dies wird gemacht, um die Bildung von JC-1-Aggregate zu reduzieren; wenn Sie bilden Aggregate, nicht Zentrifugenröhrchen, da dies Färbung zu verhindern. ANMERKUNG: Eine kleine Menge von Aggregation hat keinen Einfluss auf Ergebnisse. </li><li> Anti hinzufügenCD45 APC eFluor 780 und Anti-CD71 PerCP eFluor 710, so dass die Endkonzentration 1 ug / ml. Bei Durchführung des markierten RBC Experiment hinzuzufügen Streptavidin PE-Cy7, so daß die Endkonzentration 1 ug / ml. </li></ol></li><li> Führen Schwanz Blutungen durch Amputation der Schwanzspitze oder Platzwunde des Blutgefäßes in den Schwanz. Geben Sie einen Tropfen Blut Schwanz auf eine kleine wiegen Boot oder Glasträger. Nach der Blutentnahme, sicherzustellen, dass die Blutung aufgehört hat. Pipette 3 ul Schwanzblut in 50 &amp; mgr; l Färbelösung auf 37 ° C vorgewärmt und Heizen von Proben bei 37 ° C für 20 min. </li><li> Abtauen eines Aliquots 4 mM Hoechst 33342 bei -20 ° C in destilliertem Wasser (bei Verwendung eines 355 nm Laser) gespeichert oder 2 mM Hoechst 34580 bei -20 ° C in destilliertem Wasser (bei Verwendung eines 405 nm Laser) gespeichert. Bereiten Sie 500 ul pro Probe, plus extra, einer 4 &amp; mgr; M oder 2 uM Lösung von Hoechst in MTRC auf. </li><li> Fügen Sie 500 ul von 4 uM Hoechst33.342 oder 2 uM Hoechst 34.580 Proben und Inkubation bei Raumtemperatur für 20 min. </li><li> Zentrifuge Zellen bei 750 g für 3 min bei 4 ° C, Überstand verwerfen, fügen 700 ul MTRC und durch Strömungs analysieren Zytometrie. </li></ol> 4. Durchflusszytometrie <ol><li> Analysieren Sie die Proben mit einem 355/488/633 nm Laser oder 405/488/633 nm Lasergerät. </li><li> Filter Proben durch einen 35 um Zellsieb unmittelbar vor der Analyse. Spülen Zellsieb mit destilliertem Wasser zwischen den Proben und Wiederverwendung. </li><li> Nehmen Sie genug Ereignisse, so dass die kleinste Bevölkerung mindestens 500 Ereignisse (in der Regel eine Million - 10 Millionen Gesamt Ereignisse pro Probe). </li><li> Excite Hoechst 33342 mit einem 355-nm-Laser und erkennt Ereignisse durch eine 460/50-Filter. Excite Hoechst 34580 mit einem 405-nm-Laser und erkennt Ereignisse durch eine 460/50-Filter. Excite JC-1, anti-CD71 PerCP eFluor 710 und Streptavidin PE-Cy7 mit einem 488-nm-Laser und erkennt Ereignisse durch ein 530/40, 692/40 und 750LP filtern jeweiligenly. Excite Atto 633 und Anti-CD45 APC eFluor 780 mit einem 633-nm-Laser und erkennt Ereignisse durch ein 670/30 oder 750LP-Filter auf. </li><li> Führen Sie die Analyse und Kompensation mit geeigneten Durchflusszytometrie-Analyse-Software wie folgt: <ol><li> Wählen ganze Zellen und verstehen sich inklusive Lärm, Schmutz und Blutplättchen basierend auf FSC / SSC Eigenschaften (Abbildung 2A). Auswahl einzelner Zellen, die entweder auf Triggerimpulsbreite (2B) oder mit dem FSC-Peakfläche zu Höhe Verhältnis (2C). </li><li> Wählen reifen Erythrozyten und schließen RBC Vorläuferzellen und Leukozyten, basierend auf negativen Fluoreszenz in den PerCP eFluor 710 und APC eFluor 780 Kanäle (2D). </li><li> Beim Arbeiten mit der Bezeichnung RBC Experiment, wählen Sie die einzelnen Population markierter Erythrozyten auf Basis von PE-Cy7 und Atto 633 Fluoreszenz und analysieren diese separat (3A). </li><li> Wählen Sie basierend auf positive Fluoreszenz sowohl für JC-1 und Hoechst PRBCs ( <strong&gt; 2E-H). </li></ol></li></ol> 5. Berechnungen und Statistik <ol><li> Berechnen Parasitämie durch Dividieren der Anzahl von prbcS durch die Gesamtzahl von Erythrozyten (dh die Anzahl von Ereignissen in Gate Q2 in Gatters G4 geteilt durch die Anzahl von Ereignissen). Bei der Durchführung des Invasionsassay Parasitämie der beiden markierten Populationen, indem die Anzahl der markierten prbcS durch die Anzahl der markierten RBCs in der Population (berechnet, dh, die Anzahl der Ereignisse, die in beide Tore L1 und Q2 sind, dividiert durch die Anzahl der Ereignisse, im Tor L1). HINWEIS: Labeled Parasitämie variiert beträchtlich in Abhängigkeit von Faktoren wie beispielsweise die endogene Parasitämie und Anzahl zirkulierender Merozoiten. Aus diesem Grund ist es hilfreich, die Parasitämie Verhältnis, das als das Parasitämie des behandelten markierten Population in jeder einzelnen Maus, geteilt durch die Parasitämie der Steuer markierten Population berechnete melden. Um mögliche Farbeffekte zu korrigieren, führt zu einem Berichts eine durchschnittliche Parasitämie Verhältnis zwischen den beiden Farbstoffkombinationen. </li><li> Bestimmen Sie eine statistische Signifikanz von Verhältnissen mit dem eine Probe t-Test mit 1 als hypothetische Mittelwert. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Wir haben ein Verfahren für die gleichzeitige Messung von Parasitämie und Merozoiten Invasion in vivo Proben beschrieben. In Bezug auf die Parasitämie Messung bietet dieses Verfahren einen Vorteil gegenüber bisherigen Methoden 10-13 in diesem HJ-Erythrozyten aus PRBCs unterschieden werden, wodurch die Anzahl der falsch-positive Ereignisse zu reduzieren. Während HJ-RBCs sind gewöhnlich beim Menschen selten einige Studien berichten hohen Niveaus in Mäusen 15,16 die Unterscheidung zwis...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table><tbody><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Hoechst 33342. Store a 4 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>Hoechst 34580</td><td>Sigma-Aldrich</td><td>63493</td><td>Store a 2 mM stock solution at -20 &amp;deg;C in distilled water</td></tr><tr><td>JC-1 Dye</td><td>Life Technologies</td><td>T-3168</td><td>Store small aliquots of 6 mM stock solution at -20 &amp;deg;C in DMSO</td></tr><tr><td>Anti-Mouse CD45 APC-eFluor 780</td><td>eBioscience</td><td>47-0451-80</td><td>Clone 30-F11</td></tr><tr><td>Anti-Mouse CD71 PerCP-eFluor 710</td><td>eBioscience</td><td>46-0711-80</td><td>Clone R17217</td></tr><tr><td>Atto 633 NHS ester</td><td>Sigma-Aldrich</td><td>1464</td><td>Atto 633-NHS. Store a 2 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>EZ-Link Sulfo-NHS-LC-Biotin</td><td>Thermo Fisher Scientific</td><td>21335</td><td>Biotin-NHS. Store a 25 mg/ml stock solution at -20 &amp;deg;C in DMF</td></tr><tr><td>Streptavidin PE-Cyanine7</td><td>eBioscience</td><td>25-4317-82</td><td>Streptavidin PE-Cy7</td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H478</td><td></td></tr><tr><td>35 &amp;micro;M filter cap tubes</td><td>Becton Dickinson</td><td>352235</td><td></td></tr><tr><td>Flow cytometer: BD LSRFortessa</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD FACSAria II</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: BD Influx</td><td>Becton Dickinson</td><td></td><td></td></tr><tr><td>Flow cytometer: CyAn ADP Analyzer</td><td>Beckman Coulter</td><td></td><td></td></tr></tbody></table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Die Messung der Parasitämie. Für die Messung der Parasitämie sollte Blutkörperchen zuerst ausgewählt werden, und Lärm, Schmutz und Plättchen ausgeschlossen, bezogen auf FSC / SSC-Eigenschaften (2A). Abhängig von der Zytometer verwendet wird, sollten einzelne Zellen dann basierend auf jeder Triggerimpulsbreite (2B) oder FSC Peakhöhe auf Flächenverhältnis (2C) ausgewählt werden. Verbleibende Ereignisse sollten von Leukozyten bestehen,...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Die Malaria-Parasiten dringt und Wiederholungen in den roten Blutkörperchen. Die genaue Beurteilung der Merozoiten Invasion und Parasitämie ist daher entscheidend für die Beurteilung der Verlauf der Malaria-Infektion. Hier beschreiben wir eine Durchflusszytometrie basiertes Protokoll für die Messung dieser Parameter in einem Maus-Modell der Malaria.

In-vivo-Bewertung der Nager Plasmodium Parasitämie und Merozoite Invasion durch Durchflusszytometrie

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.9K Aufrufe.  Macquarie University.  Die Malaria-Parasiten dringt und Wiederholungen in den roten Blutkörperchen. Die genaue Beurteilung der Merozoiten Invasion und Parasitämie ist daher entscheidend für die Beurteilung der Verlauf der Malaria-Infektion. Hier beschreiben wir eine Durchflusszytometrie basiertes Protokoll für die Messung dieser Parameter in einem Maus-Modell der Malaria. 

Serie: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Protokoll für die Herstellung eines genetischen Kreuz der Nager Malaria-Erregern

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Forschung

Immunologie und Infektion

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

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.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Intravitalmikroskopie von Gehirn der Maus Mikrozirkulation mit einem Closed Cranial Fenster

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neurowissenschaften

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Intravitalmikroskopie der Milz: Quantitative Analyse von Parasite Mobility und die Durchblutung

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

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.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolierung und Analyse von Brain-sequestered Leukozyten aus<em&gt; Plasmodium berghei</em&gt; ANKA-infizierte Mäuse

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

High Yield Reinigung von<em&gt; Plasmodium falciparum</em&gt; Merozoiten Zur Verwendung in opsonierende Antikörper-Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Ein<em&gt; In-vitro-</em&gt; Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Phänotypische Analyse von Nagetier Malaria Parasiten Asexual und Sexuelle Blutstadien und Mückenstadien

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

In vivo Verfolgung von Ödementwicklung und mikrovaskulärer Pathologie in einem Modell der experimentellen zerebralen Malaria mit Magnetresonanztomographie

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Medizin

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Messung natürlich erworbener Phagozytose-induzierender Antikörper gegen Plasmodium falciparum Parasiten durch einen Flow Cytometry-based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

In vitro Untersuchung der Abtötung von Plasmodium-infizierten roten Blutkörperchen durch zytotoxische Lymphozyten

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

In-vivo-Bewertung der Nager Plasmodium Parasitämie und Merozoite Invasion durch Durchflusszytometrie

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