Gli autori desiderano ringraziare la signorina Liana Mackiewicz per l&#39;assistenza tecnica. Questo lavoro è stato reso possibile grazie vittoriana sostegno governativo Infrastrutture Stato operativo e del governo australiano National Health and Medical Research Council IRIISS e Progetto di sovvenzione 1.031.212.

1. L&#39;infezione di topi con P. berghei-ANKA <ol><li> Sbrinamento un&#39;aliquota di crioconservati P. berghei ANKA PRBC. </li><li> Trattenere una malaria cerebrale resistente topo BALB / c donatore (8-12 settimane di età) con la tecnica a due mani di ritenuta. Iniettare il mouse con 100-200 ml di PRBC utilizzando un insulina siringa da 1 ml (28G ago). Di routine, 1-2 topi donatori vengono iniettati. </li><li> In 4-5 giorni post-infezione (pi) rimuovere topo donatore da gabbia e collocarla su una workstation o un tampone monouso di lavoro. </li><li> Delicatamente frenare il mouse alla fine della coda e con le forbici piccole tagliare la punta della coda (circa 1 mm). In alternativa, una foratura piccola coda usando un ago 25 G può essere eseguita. </li><li> Una goccia di sangue dalla vena della coda del topo in prossimità della fine di un frosted-end vetrino da microscopio. </li><li> Posizionare una seconda slitta (spreader) su un angolo di 45 ° e sostenerlo nella goccia di sangue. Una volta che il sangue si estende lungo il bordo, premere il divaricatore-slide vigiliaolo attraverso il vetrino da microscopio per rendere la striscio di sangue. Lasciare lo striscio asciugare all&#39;aria. </li><li> Fissare strisci di sangue in metanolo 100% per 30 sec e poi colorare i vetrini in Giemsa appena preparato per 10 min. </li><li> Sciacquare striscio di sangue con acqua corrente per 1 minuto, permette di asciugare vetrino ed esaminare al microscopio (100x, immersione in olio). Enumerare PRBC nel 1000 eritrociti e calcolare parassitemia per cento. Topi donatori dovrebbero raggiungere livelli parassitemia tra il 2,5-5% prima di poter essere sanguinato per infezione di animali da laboratorio. </li><li> Raccogliere il sangue dal topo donatore da retro-orbitale sanguinamento usando un micro-ematocrito eparinizzata tubo capillare. Tutti gli esperimenti sono eseguiti in conformità con la Sala Walter &amp; Eliza Istituto animali previste dal Comitato Etico. A seconda delle esigenze locali animali Ethic Comitatologia sanguinamento altre potrebbero essere utilizzate. Sciogliere quantità di sangue in mezzo RPMI ad una concentrazione finale di 1x10 6 pRBC/0.2 ml. Si consideri cheun ematocrito normale mouse è ~ 6x10 9 cellule rosse del sangue / 1 ml. </li><li> Infect C57BL sperimentale / 6 topi (8-12 settimane) iniettando per via intraperitoneale (ip) 1x10 6 pRBC/0.2 ml. </li><li> Per monitorare i livelli di parassitemia di topi infettati seguire i passaggi 1,3-1,8. Parassitemia deve essere determinato ogni 2-3 giorni a partire dal giorno 2 o 3 pi </li><li> Insorgenza di malaria grave in C57BL / 6 topi potrebbe manifestarsi come aspetto ingobbito, ruffed pelliccia e bassa attività. Alcuni topi possono recuperare in questa fase, ma la progressione della perdita di auto-raddrizzante riflesso indica malattia irreversibile e gli animali devono essere sottoposti a eutanasia. </li></ol> 2. Perfusione Intracardial di topi e di estrazione del cervello <ol><li> Assemblare un sistema manuale di perfusione gravità assicurando un serbatoio di 500 ml di una colonna di supporto fissato alla parte superiore di una colonna di supporto 60 cm. Collegare tubo di plastica per l&#39;estremità inferiore del serbatoio e fissare un morsetto del tubo per controllare il flusso del buffer. Collegare il tubo di un ago 23 G. Fill il serbatoio con PBS (mantenuto a 22 ° C), aprire la fascetta e permettono di eseguire tampone attraverso il tubo per rimuovere tutte le bolle d&#39;aria. </li><li> Eutanasia P. berghei ANKA-infetto mouse CO 2 inalazione. Confermare la morte per mancanza di pedale, orbitale e le risposte respiratorie. </li><li> Pin del mouse eutanasia dalle zampe posteriori e anteriori dorsalmente su una tavola di dissezione di polistirolo contenute in un vassoio di plastica. A seconda del locale Comitato di Etica degli animali anestesia requisiti possono essere somministrati prima di inizio pefusion intracardial. </li><li> Pulire lato ventrale con il 70% di etanolo. </li><li> Usare le forbici grandi e pinze per aprire la pelle lungo la linea mediana per esporre la cavità toracica. Piegare la pelle e il pin ai lati. </li><li> Tenere sterno con pinza sottile e tagliare il diaframma e lungo i due lati dello sterno tranciamento delle costole. Fare attenzione a non danneggiare i grandi vasi sanguigni. </li><li> Pin la gabbia toracica dalla sterno liberamente accanto alla testa. </li><li> Tenere spirito ventricolih pinza sottile e accuratamente incise atrio destro con le forbici sottili. </li><li> Inserire l&#39;ago 23G collegato al sistema di perfusione gravità in ventricolo sinistro verso l&#39;aorta ascendente, mentre è in esecuzione PBS. Inserire solo 0,5 cm dalla punta dell&#39;ago. </li><li> Perfusione del mouse per 5 minuti o fino a quando effluenti è chiara. </li><li> Sblocca il mouse e lo posi sul suo addome. Pulire la testa con il 70% di etanolo. </li><li> Utilizzando le forbici grandi, fare un taglio appena sopra la zona cervicale del midollo spinale. </li><li> Utilizzare le forbici pregiati per fare una mediana caudale-rostrale taglio a partire dalla base del cranio e la pelle a buccia per scoprire l&#39;cranio. </li><li> Tenendo la testa, posizionare le lame di una forbice piccole in ciascuna cavità orbitale ed effettuare un taglio tra le orbite. </li><li> Praticare un taglio longitudinale lungo la sutura sagittale e con attenzione buccia il teschio su ogni emisfero del cervello verso l&#39;esterno. </li><li> Con una spatola, sollevare delicatamente il cervello e metterlo in una provetta da centrifuga da 10 ml contenente mezzo RPMI. </li></ol>3. Isolamento di Brain-sequestrati leucociti (BSL) <ol><li> Lavorare in una cappa di sicurezza, posto appena prelevato cervello sopra un filtro in acciaio inossidabile cella (40-60 maglia) in una piastra Petri contenente 3-5 ml di mezzo di coltura RPMI tessuto. </li><li> Tagliare il tessuto cerebrale in piccoli pezzi. </li><li> Spingere piccoli pezzi di tessuto cerebrale attraverso il filtro cella utilizzando uno stantuffo cristallo. </li><li> Trasferire omogenato cerebrale in una provetta da 10 ml e centrifugare a 250 xg per 10 min a 4 ° C. </li><li> Sciogliere pellet in 3 ml di mezzo RPMI, contenente 0,05% Collagenase D e 2U/ml DNAsi I. </li><li> Ruotare miscela per 30 min a temperatura ambiente. Rimuovere i detriti cellulari spingendo la miscela attraverso un filtro 70 micron cella nylon e / o mediante incubazione in ghiaccio per 5 min. </li><li> Lay omogenato cerebrale su un cuscino 7 ml di Percoll 30% e centrifugare a 400 xg (senza freni) per 20 min a temperatura ambiente. </li><li> Risospendere il pellet in 1 ml di tampone di lisi cellulare rosso sangue e incubare in ghiaccio per 5 min alisare PRBC aderente, che non può essere rimosso dal cervello mediante perfusione intracardial. </li><li> Aggiungere 9 ml di terreno RPMI, lavare le cellule e centrifugare a 250 xg per 10 min a 4 ° C. </li><li> Risospendere BSL contenenti pellet in 50-100 ml di mezzo RPMI e trasferire le cellule su un tubo Eppendorf. </li><li> Diluire un&#39;aliquota 5-10 microlitri del campione cellulare 1:2 in Trypan blu per l&#39;identificazione di cellule vitali. </li><li> Contare le cellule vitali al microscopio utilizzando un emocitometro. Dal giorno 6 in poi, quando pi P. berghei ANKA topi infettati mostrare segni neurologici, 20,000-100,000 BSLs possono essere recuperati. </li></ol> 4. Tipizzazione di BSL di Citometria a Flusso Multicolor <ol><li> Centrifugare le cellule a 250 xg per 10 min a 4 ° C, aspirare il surnatante e risospendere pellet in 50 pl di tampone colorazione (PBS, 1% di siero fetale bovino (FCS), 2 mM EDTA). </li><li> Aggiungere il 1 ml di anticorpo purificato anti-CD16/32 (Fcblock). </li><li> Incubare le cellule per 10 min in ghiaccio. </li><li> Lavare le cellule con 1 ml di tampone di colorazione. Centrifugare le cellule a 250 xg per 10 min a 4 ° C e aspirare il surnatante. </li><li> Risospendere le cellule in 50 pl di tampone contenente colorazione diluizioni ottimali predeterminate di richieste anticorpi fluorescenti, ovvero anti-CD4, anti-CD8, anti-TCR e anti-NK1.1. </li><li> Incubare per 1 ora su ghiaccio. </li><li> Lavare le cellule con 1 ml di tampone di colorazione. Centrifugare le cellule a 250 xg per 10 min a 4 ° C e aspirare il surnatante. </li><li> Risospendere le cellule in 100 microlitri di PBS. </li><li> Trasferire le cellule a un tubo di plastica di citometria a flusso. </li><li> Utilizzando un citofluorimetro, acquisire almeno 5.000-10.000 eventi. Adeguati controlli e campioni di cellule non marcate con fluorocromi di compensazione dovrebbe essere incluso come richiesto. </li><li> Analizzare i risultati utilizzando il software appropriato citometria a flusso. </li></ol>

<ol>
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Nessun conflitto di interessi dichiarati.

L&#39;isolamento e l&#39;analisi di BSL è un metodo che permette la caratterizzazione e la quantificazione delle cellule infiammatorie migrazione al cervello in risposta al danno tissutale o infezione in modelli murini sperimentali. L&#39;introduzione di una fase di perfusione intracardial per la rimozione del comparto sangue prima dell&#39;estrazione organi e isolamento cellulare successiva è utile per prevenire la contaminazione di cellule infiammatorie con leucociti circolanti non-infiammatorie. Questo potrebbe non...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td></td><td></td><td></td><td> Soluzioni e tamponi </td></tr><tr><td> Giemsa azur eosina blu metilene soluzione </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> Diluizione 1:10 in acqua distillata </td></tr><tr><td> RPMI media </td><td></td><td></td><td> Mouse tonicità </td></tr><tr><td> Mouse tonicità PBS </td><td></td><td></td><td> 20 mM fosfato di sodio, 0,149 NaCl, pH 7,3 </td></tr><tr><td> 0,4% Trypan Blue </td><td> Sigma Aldrich </td><td> T-8154 </td><td> 1:2 </td></tr><tr><td> Collagenasi D </td><td> Biochimica Worthington </td><td></td><td></td></tr><tr><td> Deoxyribonuclease (DNAsi) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> Da pancreas bovino </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> Soluzione al 30% in PBS </td></tr><tr><td> Ultrapura Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Cloruro di ammonio (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Red Cell Lysis Buffer </td><td></td><td></td><td> 17 mM Tris, 14nM NH 4 Cl, pH 7,2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA sale disodico </td><td> Merck </td><td> 10093.5V </td><td> 0,1 M, pH 7,2 </td></tr><tr><td></td><td></td><td></td><td> Anticorpi e coniugati </td></tr><tr><td> Anti-mouse CD16/CD32 (Fc Block), clone 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553142 </td><td> 1 ml in 50 microlitri tampone di colorazione (0.5mg/50 ml) </td></tr><tr><td> FITC-anti-topo CD4, clone H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-anti-topo NK1.1, clone PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5-anti-topo CD8, clone 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC-anti-topo TCR-β, clone H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-anti-topo CXCR3, clone 220803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinilato anti-topo-CCR5, clone C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Steptavidin-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzature e materiali </td></tr><tr><td> SuperFrost vetrino da microscopio </td><td> Lomb Menzel-Glaser </td><td></td><td></td></tr><tr><td> Pinze, forbici dissezione </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml PBS serbatoio </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Tubi in gomma vulcanizzata </td><td></td><td></td><td></td></tr><tr><td> Ago 23G </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> Cella dissociazione kit contenenti metalli setaccio </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 micron nylon filtro cella </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Emocitometro </td><td> Neubauer GmbH </td><td> 717810 </td><td></td></tr><tr><td> Citometria a Flusso tubi </td><td> BD Falcon </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Si descrive un metodo per l&#39;isolamento e la caratterizzazione di cellule infiammatorie aderenti da vasi sanguigni cerebrali di P. berghei ANKA topi infettati. L&#39;infezione di ceppi sensibili del mouse con questo risultati di deformazione parassita nella induzione di sperimentale malaria cerebrale, una sindrome neurologica che riassume alcuni aspetti importanti del Plasmodium falciparum mediata da malaria grave in 1,2 esseri umani. Forme mature di sangue stadi malaria esprimere proteine ​​parassite sulla superficie del eritrociti infetti, che consente loro di legarsi a cellule endoteliali vascolari. Questo processo induce ostruzioni del flusso sanguigno, causando ipossia e 3 emorragie e stimola anche il reclutamento di leucociti infiammatori al sito di sequestro parassita. A differenza di altre infezioni, ad esempio virus neutrotopic 4-6, sia la malaria-parassitati globuli rossi (PRBC), così come associati inflammleucociti stre rimangono sequestrati all&#39;interno dei vasi sanguigni e non infiltrante il parenchima cerebrale. Perciò, per evitare la contaminazione di leucociti sequestrati con cellule circolanti non-infiammatorie, ampia perfusione intracardial di topi infetti-prima dell&#39;estrazione di organi e lavorazione dei tessuti è necessario in questa procedura per rimuovere il comparto sangue. Dopo la perfusione, i cervelli vengono raccolte e sezionati in piccoli pezzi. La struttura del tessuto è ulteriormente disturbato da trattamento enzimatico con collagenasi D e DNAsi I. L&#39;omogenato cerebrale risultante viene poi centrifugato su un gradiente di Percoll che permette la separazione dei cervelli sequestrati leucociti (BSL) mielina e detriti da altri tessuti. Cellule isolate vengono quindi lavate, contati utilizzando un emocitometro e colorate con anticorpi fluorescenti per la successiva analisi mediante citometria a flusso. Questa procedura consente una completa caratterizzazione fenotipica dei leucociti infiammatori migrazione al cervello in response a vari stimoli, compreso il colpo così come le infezioni virali o parassitarie. Il metodo prevede anche un utile strumento per la valutazione di nuovi trattamenti antinfiammatori in pre-clinici modelli animali.

I risultati in Fig. 2 percentuali di spettacoli e di numeri assoluti delle diverse popolazioni BSL recuperati da cervelli di perfusi o unperfused topi di controllo della malaria-infettati e ingenuo. Isolato BSL sono state colorate con PE-anti-NK1.1 e APC-anti-TCR β-anticorpi, come indicato nel testo del protocollo. In linea con i risultati precedenti 7-9, αβTCR + cellule T comprende una percentuale elevata della piscina BSL nel cervello di perfusi malaria topi infettati (giorno 6 pi). Questa pop...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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

Un metodo per l&#39;isolamento di aderenti leucociti infiammatori dai vasi sanguigni cerebrali dei<em&gt; Plasmodium berghei</em&gt; ANKA topi infettati è descritto. Il metodo consente la quantificazione e caratterizzazione fenotipica di leucociti isolati dopo colorazione con anticorpi fluorescenti e successiva analisi mediante citometria a flusso.

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. ...

isolation-analysis-brain-sequestered-leukocytes-from-plasmodium

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

12.7K Views.  The Walter and Eliza Hall Institute of Medical Research. Un metodo per l'isolamento di aderenti leucociti infiammatori dai vasi sanguigni cerebrali dei Plasmodium berghei ANKA topi infettati è descritto. Il metodo consente la quantificazione e caratterizzazione fenotipica di leucociti isolati dopo colorazione con anticorpi fluorescenti e successiva analisi mediante citometria a flusso.

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

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, ...

Ricerca

Neuroscienze

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

Isolamento di Brain-infiltrazione Leucociti

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

Immunologia e infezioni

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Un modello sperimentale per studiare la tubercolosi, la malaria coinfezione sulla trasmissione naturale di Mycobacterium tuberculosis E Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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.

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

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of 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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Mieloide isolamento delle cellule da topo pelle e drenante linfonodi seguito intradermica immunizzazione con vivo attenuato<em&gt; Plasmodium</em&gt; sporozoiti

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Isolamento concomitante di astrociti primari e microglia per l'infezione da parassiti di Protozoi

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...

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 ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Generazione di sporozoiti geneticamente modificati di Plasmodium berghei

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

Isolamento e analisi di Brain-sequestrati da leucociti

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