The overall goal of this procedure is to study microcirculation in the mouse brain by intravital microscopy, which allows tracking dynamic changes in hemodynamic and inflammatory markers in the pile microcirculation over extended periods of time. This technique was developed at La Jolla Bioengineering Institute, where it was used for many types of studies. For instance, the follow-up of hemodynamic changes during the course of plasmodium burgi onca infection, and at the time of expression of cerebral malaria.
This is accomplished by first implanting a chronic cranial window two weeks prior to the intravital microscopy studies. The second step of the procedure is to record the overall morphology of the cranial window using a high resolution digital image at low magnification. The third step of the procedure is to acquire intravital microscopy images using conventional and fluorescent illumination, and to select specific study sites for online measurements of vessel diameter and RBC velocity.
The final step of the procedure is to perform intravital analysis of leukocyte and platelet adherence in the pile vessels via fluorescent labeled specific antibodies. Ultimately, results can be obtained that show in vivo, micro circulatory changes in cerebral mirroring models of physiological pathophysiological or diseased conditions through brain intravital microscopy to establish hemodynamic changes associated with these conditions. The main advantage of this technique is that we observe the brain in his physiological environment without exposing it to external agents or artificial medium intravital microscopy compared to image magnetic resonance and angiography has a spatial and temporal resolutions and allow us to look from the microscopic to the microscopic level.
This method can help to answer key questions in the physiology and pathophysiology of brain microcirculation, such as hemodynamic and inflammatory change during acute and chronic disease Two to three weeks prior to intravital microscopy. A craniotomy is performed in eight to 10 week old mice is previously demonstrated except that a titanium bar is not placed in the head of the animal. The chronic cranial window is a stable preparation, allowing examination of the pile microcirculation even months after being implanted on the day of the experiment.
First, check the body temperature of the animal before placing the animal in a stereotaxic frame infused previously prepared fluorescently labeled erythrocytes through the tail vein of the mouse. This will enable erythrocyte tracking, which will be demonstrated later. Then lightly anesthetize the mouse with isof fluorine.
4%for induction, one to 2%for maintenance. Next, place the animal in the prone position in a stereotaxic frame on a heating pad. Secure the head carefully using the ear bars and adjust the level using the right and left levers.
Gently clean the cover slip on the cranial window with a cotton swab moistened with mineral oil. Then using a stereo microscope connected to a digital camera, take a few panoramic photographs of the vessels under the window. After transferring the panoramic photos to a computer, select the best one to be printed, identified and dated.
This photo will be used as a map for measurements of vessel diameter and red blood cell velocities, which will be demonstrated next to begin intra vital microscopy. Transfer the mouse to a customized intravital microscope stage. Place a drop of water on the cranial window taking advantage of the well formed by the dental acrylic.
A 20 x water immersion objective with numerical aperture of 0.5 is used. Images are recorded using two cameras, a digital low light, high speed camera connected to a computer and monitor, and a low light analog camera connected to a VCR Tape, a timestamp, and a color monitor prior to selecting vessels for measurement, check the vessels to evaluate the quality of the preparation, and if blood is flowing in all vessels, then select the vessels to be measured. They should include venues and arterials of different diameters and cover different locations within the area exposed by the window.
In our experiment, we select 12 vessels for measurement as you view various fields within the cranial window. To select the vessels, annotate the precise location of each spot to be measured in the photo of the PY vasculature that was taken earlier for each spot, the vessel diameter is measured using an image shear device. Once the spot is selected, the image of the vessel is aligned in the vertical position and the image is sheared until the opposite.
Extremes are aligned and the reading is documented. For erythrocyte tracking, each spot is recorded by the digital camera for at least 30 seconds, and the video images are recorded at 150 frames per second. This rate is set to obtain one to six images of a cell on one video frame for determination of velocities up to six millimeters per second.
Once the data collection is done, remove the mouse from the stereotaxic frame and return it to its cage to recover from the anesthesia using appropriate software. The acquired video images are digitalized and XY.Coordinate data for each cell image is obtained. Cell positions are determined manually rather than by image analysis.
Given that the eye of a trained observer gives a good estimation of the location of the center of a cell, which in general corresponds to the location of maximal fluorescence observed. For most cell orientations, determinations of position and velocity are made for 15 cells in each vessel. An average to obtain mean RBC velocity once vessel diameter and RBC velocity measurements are available.
Calculation of the blood flow in each vessel can be made by using the formula Q equal D over two squared times PI times V where Q equals blood flow V equals RBC velocity, and D equals vessel diameter for profusion assessment and analysis of leukocyte adherence in pile vessels. Intravital microscopy is performed on an animal infused with a mixture of FSE labeled albumin and antibodies against the pan leukocyte marker CD 45 labeled with Texas Red. The fluorescent labeled albumin allows improved visualization of the vascular network, including penetrating vessels and is particularly useful in disease states such as cerebral malaria to check for non perfused or under perfused vessels.
The fluorescent labeled anti CD 45 antibodies make it easy to identify and quantify leukocyte rolling and adhesion to pile vessels. Quantification of leukocyte adhesion is made by counting the number of leukocytes in a 100 micron vessel length. Rolling is quantified by counting the number of leukocytes traveling at a velocity significantly slower than blood velocity in the same 100 micron length during 30 seconds.Shown.
Here is an example of microvascular red blood cell velocity measurements by cell tracking from high speed fluorescence. Video recordings pictures A to F are sequence images of the microcirculation. Each picture represents the position of a single flowing red blood cell, which is captured frame by frame by the high speed camera.
This graph from a representative experiment shows the changes in pile blood flow over time. In plasmodium, burgi, onca infected mice and in uninfected control mice, whereas in control mice, the pile blood flow is relatively stable over time. PBA infected mice show a market decrease in blood flow at a time of cerebral malaria development.
At day six, staining with anti CD 45, Texas red fluorescent antibodies reveal a large number of leukocytes adhering to pile vessels of a mouse infected with plasmodium burgi onca. This procedure has a wide range of applications besides the one job here today. Any optical technique that can be used in vivo can be adapted to this model and parameters such as tissue, oxygen level tissue, pH reactive oxygen species, and endothelial interaction with leukocytes can be studied in this model.
