This method can help answer key questions in the pathogenesis of infective endocarditis, and pathobiograms through fields such as the interplay between bacteria, blood cells, and endothelial cells lining musculature. The main advantage of this technique is that the tissue grafts might be directly exposed to mutual interactions with various targets, such as bacteria, platelets, and proteins in standardized flow conditions. The implication of this technique extend to our therapy of infective endocarditis, because it can unravel important molecular interactions and factors driving high infectiveness of certain tissues.
This method provides insight into pathogenesis of vegetation formation. It can also be applicable in studies investigating vascular permeability, cell mobility, and gene cell expression. To begin the protocol, place a previously prepared tissue biopsy 10 millimeters in diameter, and the same thickness between a microscope slide and an eight millimeter circular perforation, and a rubber gasket with the inner surface facing up, to contact the bacterial suspension.
Focus on the tissue graft orientation while placed in the holder. Make sure the inner surface of the tissue is already exposed to the bloodstream faces up to contact your experimental medium. Next, insert your holder with the tissue into the gasket sheet that is embedded in the bottom metal frame of the chamber.
Attach the upper metal frame with the corresponding gasket sheet onto the bottom of the chamber with the previously inserted tissue holder. Then mount the entire chamber with eight screws and screw nuts. Make sure that the chamber height is always the same across grafts by using a caliper or ruler.
Next connect the flow chamber with a peristaltic pump. Then connect the 400 milliliter fluid reservoir with the tubes. Perfuse the tissues with 100 milliliter suspensions of ten to the seven fluorescently-labeled colony forming units in phosphate buffered saline with the sheer stress of three dines per square centimeter and corresponding flow rate of four milliliters per minute for one hour using the peristaltic pump and the bacterial reservoir.
Recirculate continuously the 100 milliliter bacterial suspension using the same collection reservoir. After perfusion, dismantle the chamber to release the graft. Wash the tissue piece two times with four milliliters of PBS in a 12-well plate using an orbital shaker for three minutes.
Then cut the inner part of the graft using a skin biopsy punch with a smaller diameter. Place each tissue biopsy into a separate 14 milliliter tube containing one milliliter of sterile 0.9%sodium chloride. Detach the bacteria from the tissue using a sonication bath for 10 minutes.
Prepare a single 14 milliliter tube with 10 milliliters of sterile 0.9%sodium chloride to make serial dilutions of the bacterial suspension obtained after sonication. Label the tube number two. Prepare three 14 milliliter tubes with 10 milliliters of sterile 0.9%sodium chloride for serial dilutions of the initial bacterial suspension taken to the profusion experiment.
Label the tubes number three, number four, and number five. Next, secure three agar plates, one for the bacterial perfusion, one for the control perfusion of PBS, and the third one for the initial bacterial suspension used for perfusions. Label three sectors per plate for the tissue experiment in the following manner:10-1, 10-3, and 10-4.
To count the number of colony-forming units in the bacterial perfusing, label the plate as follows:10-1, 10-3, 10-5, and 10-7. Vortex the tubes containing the tissue biopsy for 15 seconds each, to make serial dilutions. To prepare the serial dilutions, transfer 100 microliters of tube number one to tube number two, and vortex the tube to thoroughly mix the solution.
Spread 100 microliters of the contents of tube number one and number two onto the corresponding sectors, 10-1 and 10-3 of the agar plate. Then spread 10 microliters of tube number two on sector 10 four. Repeat this step four times to obtain four separate growths from each volume of 10 microliters.
To prepare serial dilutions of the initial culture, first vortex the dilution for 15 seconds and transfer 100 microliters of bacterial suspension to tube number three, and mix vigorously by vortexing. Add 100 microliters of tube number three to tube number four and mix again. Repeat the procedure for tube number five from tube number four.
Spread 100 microliters of the contents of tubes number three, number four, number five, and the labeled initial culture respectively onto sectors 10-3, 10-5, 10-7, and 10-1 of the blood agar plate. Leave the blood agar plates under the laminar hood to air dry the bacterial spreads, typically for 10 minutes. Afterwards, place the plates at 37 degrees Celsius for overnight incubation.
After overnight incubation, count the bacterial colonies to obtain the number of CFUs. Under sheer stress conditions, similar bacterial attachment across the various graft tissues was observed for both staphylococcus aureus and staphylococcus epidermidis infection. Following the procedures, streptococcus sanguinis displayed significant reduction of adherence to the bovine jugular vein wall, when compared to the bovine pericardium patch.
When comparing the three species of bacteria, streptococcus sanguinis presents significantly lower adhesion to the bovine jugular vein wall in relation to staphylococcus aureus and staphylococcus epidermidis. While attempting this procedure, it's important to remember to have prepared correct tissues of the same head. It will ensure similar conditions across all experiment specimens and minimize data variability between different series of experiments.
After its development, this technique allows researchers to explore the complex disorder systems in a step by step manner by enriching their in vitro model approaches with different elements to build up full complexity close to medical status.
