In this procedure, we show a Quantitative intravital microscopy approach to assess the dynamics of parasite passage through the spleen. In a rodent malaria model, mice infected with GFP transgenic parasites are prepared for intravital microscopy of the spleen vein cannulation will allow injection of intravital dyes. During the experiment, fluorescent cells are image moving in different areas of the spleen.
We provide the mythology to describe parasite mobility in the three dimensional slow compartment of the red pulp and calculate vessel blood flow. This parameters were used to compare spleen dynamics of two PULE strains. Hello everybody.
My name is Nando El Portillo and one of the main research questions in my laboratory is to try to understand the role of the spleen in malaria. The S splenomegaly is one of the landmarks of this disease, but because of technical and ethical issues to try to address studies of this organ is very difficult. In order to do that, we have enabled technologies that allow us to see the dynamic passage of this parasite and quantify this phenomena.
This is what Lorena and MI will explain in these protocols. This research is being in part conducted in collaborations with the groups of Dr.Ler from the University of Hamburg in Germany and Dr.Maria Kbo here at hospital Clinic and has received the general funding of found fion Cellex and ministerial CIA In Espanol infection is initiated from Donor M infected with either lethal or non-lethal GFP parasites at 10%parasite. To check parasite, take a small drop of blood from the mouse tail vein and extend it On a microscope.
Slide a chole metric staining with GIMs. A solution will allow visualization of infected red blood cells once dried, fix the blood smear with Methanol for two minutes, apply GIMs a solution and incubate for 20 minutes. Wash the slide with current tap water and dry enumerate the percentage of pbcs over total RBCs using a light microscope with a hundred x oil.Objective.
Infect mice with Lethal or non-lethal parasites obtained from the tail blood of donor mice diluted in PBS. Adjust the dose to inject half million parasites per mouse in order to reach a peripheral parasite of 1%at day three post-infection and inject Intraperitoneal. Alternatively, FITC labeled RBCs can be used in control.
Animals collect one milliliter of arterial blood from a mouse and wash the pellet by centrifugation in PBS containing EDTA re span in FITC solution and incubate for two hours in the dark with gentle agitation. After that time, remove the supernatant by centrifugation and wash extensively. Inject into a control mouse to average 1%in peripheral circulation and proceed to in vivo experiments.
Hello, I'm mija. To image the spleen in vivo, we need to perform a small synergy of the animal to expose the spleen on a obviously this technique. I learned it from Stephanie GR in the laboratory of worker Heisler, to whom I would like to thank and now I will show you how To proceed.
Imaging of infected myosis Performed at day three Post-infection when parat is 1%in both strains. Keep the mouse anesthetized and in regulated temperature throughout the procedure. Protect eyes from the hydration and verify that the mouse is completely anesthetized by ping the foot pad before proceeding in order to facilitate intravenous administration of substances.
During the course of the experiment, cannulate the mouse tail vein to construct the cannula. Insert a small needle in One end of a tube and its applicator in the other. Load the cannula with physiological saline removing bubbles, and check that the solution flows correctly through the cannula.
The lateral vein of the tail Will be cannulated clean. Enhance its visibility by doing a soft constriction at the base of the tail. Introduce the tip of the cannula inside the vein.
The appearance of blood will indicate that the needle is well positioned. If it is not, repeat the cannulation upstream the vein, then fix the cannula To the tail using glue and tape. Expose the inferior part of the spleen through a small incision in the skin and musculature at the left dorsal side of the animal.
Place a spleen where less breath movement Is observed and apply PBS on the surface exposed to keep it clean of the mouse hair and hydrated. Seal The spleen with a cover sleep to allow visualization. Hello, my name is Lorena.
The the shown protocol will let us to visualize the movement of PULI parasites through the spleen. This has been possible thanks to GFP transgenic parasites. Kindly donate by James Burnes from Drexel University.
Imaging movement of fluorescent parasites will let us to know if there are difference in the behavior between lethal and non-lethal strains. Vidal microscopy experiments Were carried out in a Leica multiphoton microscope, equipped with an incubation system with temperature control and a 63 x glycerol objective. Place the animal on the stage of the microscope with a cover sleep spleen facing down to the objective.Initially.
Visualization of the spleen at lower magnifications will provide a general view of the micro circulatory structure of the organ. Focus the spleen using autofluorescent tissue GFP parasites are observed passing through the different areas of the spleen. Hello, my name is Maria Albo from Scientific and Technological Centers from the University of Barcelona, and we will do intravital imaging of the spleen.
In order to study blood flow and mobility of infected red blood cells, we will use a confocal microscope equipped with a resonant scan that allows high speed imaging that is very useful to avoid the movement of the organ. This is a spectral microscope that allows to select the vision range. In order to image very fast, we will do simultaneous acquisition of the two fluorescein, labelings and reflection.
Select different zones to image with the help of RBC, reflection, contrast, and Vidal dyes to evidence spleen microvasculature. We'll now explain how to measure parasite mobility in vessel blood flow using Vidal microscopy and image J software to follow parasite movement in the three Dimensional meshwork of the red pulp. Acquire images through five Z depths using a high speed scanning mode.
Convert image stacks into a Z coded color movies to facilitate tracking of a single fluorescent cells. Detail information Is provided in the text track different particles over time on Depth on each video using X, Y, Z, and T coordinates mobility parameters such as directionality mean velocity, and residence time can be calculated and represented as distribution maps to enable comparative analysis between Strain populations. Vascular flow in the Spleen can be imaged by systemic injection of several fluorescent molecules described in the supportive table like dextrin.
And using endogenous erythrocyte reflection contrast scan the center line blood flow by setting vessels horizontally in the direction of laser scanning and using enhanced Line average. In these images, the streaks Resulting from moving cells will be used to calculate blood flow, take images of vessels with different diameters and over the cardiac cycle to Compensate for fluctuations. The spleen has long been considered a black Box in malaria research and here we developed a quantitative intravital microscopy approach to gain insight into the dynamics of parasite infection and blood flow in this organ, allowing us to address important biological questions such as in vivo adherence of infected red blood cells to the spleen.
Thank you and.
