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

Summary

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.

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

This method was used in the research reported in ⁷.

1. Animal infection with green fluorescent protein (GFP) transgenic parasites

1. P. yoelii–GFP transgenic lines of 17XL and 17X were generated using the same vectors, targeting strategy and protocols described elsewhere for P. berghei⁸. They express the mutant 3 variant of GFP ⁹ under the ubiquitous promoter of P. berghei elongation factor 1 (Pbeef1a), which directs constitutive expression of GFP to parasite cytosol during the entire intra-erythrocytic developmental cycle.
2. Inject animals intraperit....

Discussion

The implementation of intravital microscopy of the spleen in this rodent malaria model opened the possibility of investigating the dynamic passage of parasites through this organ which until now has been considered a "black-box" due to technical considerations. In here, a major effort was put to adapt a quantitative method that allows comparative analysis of different parasite lines at the single and population levels. In contrast to other tissues and cells that had been imaged before in malaria ^3,5, imaging .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Leica TCS-SP5 confocal microscope	Leica Microsystems	TCS-SP5 Serial no. 5100000419
Ketamine (Ketolar 50 mg/ml)	Pfizer Pharma GmbH	631028
Midazolam 15 mg/3 ml	Normon	838193
70,000 MW Dextran, conjugated to Texas Red	Molecular Probes, Life Technologies	D1830
Fluorescein Isothiocyanate, isomer I (FITC)	Sigma-Aldrich	F7250
H–chst 33342	Sigma-Aldrich	H1399
Giemsa stain	Sigma-Aldrich	GS1	Working solution is at 10% in distilled water
Super Glue-3 Loctite	Loctite	9975-0880