Name: in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry
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Riconosciamo il finanziamento il sostegno del Consiglio Nazionale Salute e Ricerca Medica (concedere APP605524, 490.037 e 1.047.082), la Australian Research Council (concedere DP12010061), la strategia di National Infrastructure Research Collaborative d&#39;Australia e il fondo di investimento Education del Dipartimento di Innovazione, Industria , Scienza e la ricerca. PML è un destinatario di un premio post-laurea australiano.

Tutte le procedure sono state condotte in conformità con le politiche di Macquarie University e conformi al Consiglio nazionale della sanità e ricerca medica (NHMRC) Codice australiano di pratica. Il lavoro è stato svolto sotto gli Etica accordo No ARA 2012/017 approvate e ottenuto da parte del Comitato Etico degli animali presso Macquarie University. Tutti gli esperimenti sono stati eseguiti su SJL / J topi se non indicato diversamente. 1. Mouse e Sperimentale Malaria Infezione <ol><li> Mice House a temperatura controllata (21 ° C), con un ciclo luce-buio 12:12 h. </li><li> Scongelare una aliquota di crioconservati P. chabaudi adami DS con 5% parassitati globuli rossi (pRBCs) e iniettare 200 ml nella cavità intraperitoneale di C57BL / 6 del mouse donatore. </li><li> Monitorare donatore del mouse parassitemia ogni 1 - 2 giorni in citometria a flusso, come descritto nella Sezione 3 - 4 del presente protocollo. Una volta che i donatori raggiunge 5-15% parassitemia, raccogliere il sangue dalla puntura cardiaca come segue: <ol><li>Aspirare 100 ml di soluzione di eparina (300 U / ml di eparina in soluzione di Ringer topo (MTR) (154 mM NaCl, KCl 5,6 mM, 1 mM MgCl 2, 2.2 mM CaCl 2, 20 mM HEPES, glucosio 10 mM, 30 U / ml eparina, pH 7,4, 0,22 mM filtro sterilizzato)) in una siringa da 1 ml con un ago 26 G. </li><li> Anestetizzare il mouse per inalazione con il 5% isofluorane, confermando la profondità desiderata di anestesia per mancanza di risposta ritiro del pedale e riflessi corneali. </li><li> Posizionare il mouse sul suo lato e perpendicolarmente ago inserto appena sotto il gomito, attraverso le costole, e nel cuore. Estrarre lo stantuffo della siringa lentamente e ruotare ago fino a 0,5 - si ottiene 1 ml di sangue. </li><li> Eseguire l&#39;eutanasia umana da dislocazione cervicale sotto anestesia. </li></ol></li><li> Calcolare i pRBCs per volume di sangue dei donatori assumendo un emocromo di 9 x 10 6 RBC / ml (ossia, se il donatore era al 10% parassitemia quando sacrificati, il sangue conterrà 9 x 10 5pRBC / ml). Diluire il sangue parassitati in Krebs tamponata salina (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% di glucosio, pH 7,2, 0,22 mM filtro sterilizzato) in modo che la concentrazione è 1 x 10 4 pRBCs per 200 microlitri e iniettare 200 microlitri nella cavità intraperitoneale di topi necessari per l&#39;etichettatura RBC e sperimentare trasfusione. </li><li> Monitorare parassitemia ogni 1 - 2 giorni in citometria a flusso, come descritto nei paragrafi 3-4 di questo protocollo. </li></ol> 2. Etichettatura dei globuli rossi e Transfusion <ol><li> Prelevare il sangue eparinizzato da topi mediante puntura cardiaca come descritto al punto 1.3, e disporli sul ghiaccio. Mantenere il sangue a 4 ° C in ogni momento. Raccogliere circa 200 ml di sangue per il mouse da iniettare. Se necessario, dividere il sangue in due campioni e trattare un campione, se lo desideri. NOTA: In questo modo, invasione del samp trattatale può essere paragonato ad un controllo, campione non trattato (vedere Figura 1 per schematica). </li><li> Preparare una soluzione 2x di ogni etichetta RBC fluorescente. <ol><li> Diluire a / ml soluzione madre 2 mg di Atto 633-N-Hydroxysuccinimide (Atto 633-NHS) in MTR modo che la concentrazione è di 20 mg / ml. </li><li> Diluire a / ml soluzione madre 25 mg di biotina-N-Hydroxysuccinimide (biotina-NHS) in MTR modo che la concentrazione di 250 ug / ml. </li></ol></li><li> Split campioni di sangue in due provette e aggiungere un volume uguale di soluzione di 2x Atto 633-NHS ad un tubo, e un volume uguale del 2x biotina-NHS per l&#39;altro tubo. Mescolare immediatamente. Incubare sangue a 4 ° C per 1 ora con costante miscelazione lenta, o in alternativa, miscelare campione ogni 10 - 15 minuti. </li><li> Lavare etichettati sangue 3 volte con MTRC (MTR + 0,5% di siero albumina bovina (BSA), 0,22 mM filtro sterilizzato) come segue: <ol><li> Aggiungere almeno 2 volumi di MTRC, centrifugare a 750 xg per 5 min, rimuovere il supernatante e resuspend il pellet in almeno 2 volumi di MTRC. Ripetere 2 volte. </li><li> Dopo l&#39;ultimo lavaggio, centrifugare a 750 xg per 5 minuti e combinare il sangue pellet in diverse combinazioni (ad esempio, non trattata Atto 633 / trattati biotina, non trattati biotina / trattata Atto 633). Aggiungere MTR per portare a un volume di 200 ml per topo da iniettare. </li></ol></li><li> Iniettare sangue marcato per via intravascolare in roditori malaria topi infettati o controlli non infetti come segue: <ol><li> Eseguire trasfusione a 2 - 15% parassitemia al culmine di parassita schizogonia. NOTA: ciò avviene circa a metà del ciclo di buio (cioè tra 12 - 2 am il normale ciclo di luce, o 12 - 2 pm un ciclo di luce inversa). </li><li> Topi calda usando una lampada di calore per 5-10 minuti per aumentare il flusso di sangue, ma di prendere precauzioni per non surriscaldare i topi. Inserire topi in un dispositivo di ritegno e iniettare per via intravascolare 200 microlitri di sangue marcato (circa 1 - 2 x 10 9 RBCs) attraverso la vena della coda. </li></ol></li><li> Raccogliere campioni di sangue in diversi momenti dopo l&#39;iniezione, come descritto nella Sezione 3. NOTA: i tassi di invasione possono essere quantificabili in meno di 10 minuti dopo l&#39;iniezione, a seconda parassitemia del mouse destinatario. </li></ol> 3. Raccolta di campioni di sangue e di preparazione per Citometria a Flusso <ol><li> Preparare 50 ml di soluzione colorante per campione, più extra, il giorno dell&#39;esperimento come segue: <ol><li> Scongelare una aliquota di 6 JC-1 mM conservati a -20 ° C in dimetil solfossido (DMSO). </li><li> Caldo 50 ml di MTRC per campione, più extra, a 37 ° C. </li><li> Mentre continua il vortex MTRC pre-riscaldato, aggiungere JC-1 in modo che la concentrazione finale è di 12 micron. Questo viene fatto per ridurre la formazione di aggregati JC-1; se aggregati si formano, fare tubo non centrifuga in modo da evitare la colorazione. NOTA: Una piccola quantità di aggregazione non influirà risultati. </li><li> Aggiungi anti-CD45 APC eFluor 780 e anti-CD71 PerCP eFluor 710 in modo che la concentrazione finale è di 1 mg / ml. Se si esegue l&#39;esperimento RBC etichetta, aggiungere streptavidina PE-Cy7 in modo che la concentrazione finale è di 1 mg / ml. </li></ol></li><li> Eseguire sanguinamento coda da amputazione della punta della coda o lacerazione del vaso sanguigno nella coda. Mettere una goccia di sangue di coda su una piccola barca o pesare vetrino. Dopo la raccolta del sangue, assicurarsi che l&#39;emorragia si è fermata. Pipettare 3 ml di sangue coda in 50 ml di soluzione colorante pre-riscaldato a 37 ° C e incubare i campioni a 37 ° C per 20 min. </li><li> Scongelare una aliquota di 4 mM Hoechst 33342 conservati a -20 ° C in acqua distillata (se si utilizza un laser 355 nm) o 2 mM Hoechst 34580 conservati a -20 ° C in acqua distillata (se si utilizza un laser 405 nm). Preparare 500 microlitri per campione, più extra, di una soluzione di 4 micron o 2 micron di Hoechst in MTRC rispettivamente. </li><li> Aggiungere 500 ml di 4 micron Hoechst33342 o 2 mM Hoechst 34580 campioni e incubare a temperatura ambiente per 20 min. </li><li> Cellule centrifugare a 750 xg per 3 min a 4 ° C, scarto surnatante, aggiungere 700 microlitri MTRC e analizzare mediante citometria a flusso. </li></ol> 4. Citometria a flusso <ol><li> Analizzare i campioni utilizzando un laser 355/488/633 nm o 405/488/633 strumento laser nm. </li><li> Filtrare i campioni attraverso un colino cella di 35 micron immediatamente prima dell&#39;analisi. Risciacquare filtro cella con acqua distillata tra campioni e riutilizzo. </li><li> Registra abbastanza eventi in modo che la popolazione più piccola contiene almeno 500 eventi (di solito 1.000.000 - 10.000.000 eventi totali per campione). </li><li> Excite Hoechst 33342 utilizzando un laser 355 nm e rilevare eventi attraverso un filtro 460/50. Excite Hoechst 34580 utilizzando un laser 405 nm e rilevare gli eventi attraverso un filtro 460/50. Excite JC-1, anti-CD71 PerCP eFluor 710 e PE-Streptavidina Cy7 utilizzando un laser 488 nm e rilevare gli eventi attraverso una 530/40, 692/40, e 750LP filtrare rispettivoly. Excite Atto 633 e anti-CD45 APC eFluor 780 utilizzando un laser 633 nm e di rilevare, rispettivamente, gli eventi attraverso un filtro 670/30 o 750LP. </li><li> Eseguire l&#39;analisi e la compensazione con flusso adeguato citometria software di analisi come segue: <ol><li> Selezionare cellule intere ed escludere rumore, detriti e piastrine basato su FSC / proprietà SSC (Figura 2A). Selezionare singole cellule basate su entrambi larghezza di impulso di trigger (Figura 2B) o utilizzando l&#39;area del picco FSC al rapporto di altezza (Figura 2C). </li><li> Selezionare globuli rossi maturi, ed escludere progenitori e leucociti RBC, sulla base di fluorescenza negativo nei PerCP eFluor 710 e APC eFluor 780 canali (Figura 2D). </li><li> Se si esegue l&#39;esperimento RBC etichetta, selezionare ogni popolazione di globuli rossi etichettati sulla base di PE-Cy7 e Atto 633 fluorescenza e analizzare questi separatamente (Figura 3A). </li><li> Selezionare pRBCs basata su fluorescenza positiva sia JC-1 e Hoechst ( <strong&gt; Figura 2E-H). </li></ol></li></ol> 5. Calcoli e Statistiche <ol><li> Calcolare parassitemia dividendo il numero di pRBCs per il numero totale dei globuli rossi (cioè, il numero di eventi in cancello Q2 e il numero di eventi in porta G4). Nell&#39;effettuare la parassitemia dosaggio invasione dei due popolazioni etichettati può essere calcolata dividendo il numero di pRBCs etichettate per il numero di globuli rossi etichettati in quella popolazione (cioè, il numero di eventi che sono sia porte L1 e Q2, diviso per il numero di eventi in porta L1). NOTA: etichetta parassitemia varia notevolmente a seconda di fattori quali la parassitemia endogena e il numero di merozoiti circolanti. Per questo motivo, è utile per segnalare il rapporto parassitemia, che viene calcolato come parassitemia della popolazione trattata etichettato diviso per il parassitemia della popolazione di controllo etichettata in ciascun topo. Per correggere eventuali effetti tintura, rapporto risulta uns un rapporto medio parassitemia tra i due combinazioni di tintura. </li><li> Determinare la significatività statistica dei rapporti con il t-test un campione con 1 come media ipotetica. </li></ol>

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Abbiamo descritto un metodo per la misura sia parassitemia e merozoite invasione campioni in vivo. In termini di misurazione parassitemia, questo metodo offre un vantaggio rispetto ai metodi precedenti 10-13 in quella HJ-globuli rossi possono essere distinti da pRBCs, riducendo così il numero di eventi positivi falsi. Mentre HJ-globuli rossi sono di solito rare negli esseri umani, alcuni studi riportano livelli elevati nei topi 15,16 la distinzione tra queste cellule e pRBCs importanti pe...

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.

Misura della parassitemia. Per la misura di parassitemia, cellule del sangue devono prima essere selezionati, e il rumore, detriti e piastrine esclusi, sulla base di FSC / SSC proprietà (Figura 2A). A seconda del citometro utilizzato, singole cellule devono poi essere selezionati sulla base sia di larghezza di impulso di trigger (Figura 2B), o altezza del picco FSC rapporto dell&#39;area (Figura 2C). Rimanendo eventi dovrebbero consistere di l...

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

Gli invade parassita della malaria e replicati all&#39;interno delle cellule rosse del sangue. La valutazione accurata di invasione merozoite e parassitemia è quindi fondamentale per la valutazione nel corso di infezione malarica. Qui si descrive un protocollo basato citometria a flusso per la misurazione di questi parametri in un modello murino di malaria.

In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso

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 Views.  Macquarie University.  Gli invade parassita della malaria e replicati all'interno delle cellule rosse del sangue. La valutazione accurata di invasione merozoite e parassitemia è quindi fondamentale per la valutazione nel corso di infezione malarica. Qui si descrive un protocollo basato citometria a flusso per la misurazione di questi parametri in un modello murino di malaria. 

Video: 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.

Protocollo per la produzione di un incrocio fra i roditori parassiti della malaria

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

Ricerca

Immunologia e infezioni

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.

Microscopia intravitale della microcircolazione cervello di topo usando una finestra chiusa del cranio

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

Neuroscienze

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.

Microscopia intravitale della Milza: analisi quantitativa della mobilità Parasite e del flusso sanguigno

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.

Isolamento e analisi di Brain-sequestrati da leucociti<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati

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 Purificazione di<em&gt; Plasmodium falciparum</em&gt; Merozoiti Per l&#39;uso in opsonizzante anticorpo saggi

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.

Un<em&gt; In Vitro</em&gt; Modello per la misurazione Risposte immunitarie Malaria nel contesto della coinfezione da HIV

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.

Phenotypic Analisi del roditore Malaria Parassita Sessuale e Sessuale Fasi Sangue e Mosquito Fasi

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.

Tracciamento in vivo di sviluppo dell&#39;edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l&#39;utilizzo di immagini a risonanza magnetica

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 ...

Medicina

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).

Misurazione di anticorpi che inducono la fagonenza acquisita naturalmente ai parassiti Plasmodium falciparum mediante un'analisi basata sulla citometria del flusso

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 Saggio dell'uccisione dei globuli rossi infetti da plasmodio da parte di linfociti citotossici

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

In Vivo Valutazione del roditore Plasmodium parassitemia e merozoite Invasion di Citometria a flusso

Riepilogo

Esplora Altri Video

Capitoli in questo video

Protocollo per la produzione di un incrocio fra i roditori parassiti della malaria

Microscopia intravitale della microcircolazione cervello di topo usando una finestra chiusa del cranio

Microscopia intravitale della Milza: analisi quantitativa della mobilità Parasite e del flusso sanguigno

Isolamento e analisi di Brain-sequestrati da leucociti

High Yield Purificazione di

Un

Phenotypic Analisi del roditore Malaria Parassita Sessuale e Sessuale Fasi Sangue e Mosquito Fasi

Tracciamento in vivo di sviluppo dell'edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l'utilizzo di immagini a risonanza magnetica

Misurazione di anticorpi che inducono la fagonenza acquisita naturalmente ai parassiti Plasmodium falciparum mediante un'analisi basata sulla citometria del flusso

In vitro Saggio dell'uccisione dei globuli rossi infetti da plasmodio da parte di linfociti citotossici