प्लीहा के intravital माइक्रोस्कोपी: परजीवी गतिशीलता और रक्त प्रवाह के मात्रात्मक विश्लेषण
Summary
हम तिल्ली के intravital माइक्रोस्कोपी GFP ट्रांसजेनिक मलेरिया परजीवी और इस अंग के भीतर परजीवी गतिशीलता और रक्त प्रवाह की मात्रा का ठहराव का उपयोग प्रदर्शन के लिए विधि दिखा.
Abstract
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.
Protocol
Discussion
इस कृंतक मलेरिया मॉडल में तिल्ली के intravital माइक्रोस्कोपी के कार्यान्वयन इस अंग है जो अब तक एक "ब्लैक बॉक्स" तकनीकी कारणों की वजह से माना गया है के माध्यम से परजीवी के गतिशील यात्रा की जांच की संभावना ख?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम विशेष रूप से प्रारंभिक प्रशिक्षण के लिए और मलेरिया परजीवी के intravital माइक्रोस्कोपी में सतत इनपुट एस Graewe और वी. Heussler के लिए आभारी हैं, GFP ट्रांसजेनिक परजीवी दान ए बॉश (Confocal यूनिट, CCiT यूबी, IDIBAPS) के लिए जे बर्न्स छवि विश्लेषण और और तकनीकी सहायता के लिए मात्रा का ठहराव पी. Astola में सहायता के लिए. हम आर Tous और वीडियो उत्पादन के लिए मैं Caralt धन्यवाद. म्यूचुअल फंड कातालुन्या की व्यापकता से एक स्नातक फेलोशिप के प्राप्तकर्ता है. पड़ना एक ICREA अनुसंधान प्रोफेसर है. पड़ना की प्रयोगशाला में काम अनुदान समझौता एन के तहत यूरोपीय समुदाय सातवें फ्रेमवर्क कार्यक्रम (FP7/2007-2013) ° 242095, निजी फाउंडेशन CELLEX (Catalonia, स्पेन), और विज्ञान और नवाचार के स्पेनिश मंत्रालय (द्वारा वित्त पोषित है SAF2009 07,760).
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Leica TCS-SP5 confocal microscope | Leica Microsystems, Heidelberg, Germany | TCS-SP5 Serial no. 5100000419 | |
| Ketamine (Ketolar 50 mg/ml) | Pfizer | 631028 | |
| Midazolam 15 mg/3 ml | Normon | 838193 | |
| 70,000 MW Dextran, conjugated to Texas Red | Molecular Probes | D1830 | |
| Fluorescein Isothiocyanate, isomer I (FITC) | Sigma | F7250 | |
| Hoechst 33342 | Sigma | H1399 | |
| Giemsa stain | Sigma | GS1 | Working solution is at 10% in distilled water |
| Super Glue-3 Loctite | Loctite | 9975-0880 |
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