In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

The overall goal of the following experiment is to investigate the interaction between bacteria and the endothelium and sub endothelium under sheer stress. This is achieved by fluorescently labeling bacteria to visualize them in real time using video microscopy as a second step. An in vitro flow chamber is used to study the different players involved with adhesion of flowing bacteria to cellular or matrix components.

Next, the interactions identified in the in vitro model are studied in the in vivo mesenteric perfusion model to test their relevance in a complex organism, the results show that staphylococcus reus is able to adhere to activated endothelial cells and to the sub endothelium under shear stress. The main advantage of this technique over existing methods is that it includes both an in vitro and an in vivo model, which are complimentary to study the pathogenesis of endovascular infections Under sheer stress. These methods can help to answer key questions in the field of infectious diseases and vascular biology, such as how infective endocarditis, and metastatic infections are established under physiological sheer stress, demonstrating a procedure through be Malin looks a technician specializing in animal work from our laboratory Begin by preparing the fluorescent dye used to track the bacteria.

Add 150 microliters from a stock carboxy fluorescent solution into 850 microliters of laboratory grade water and mix. Store the solution protected from light at minus 20 degrees Celsius until it is needed. Next pellet, a five milliliter culture of S reus.

Bacteria grown overnight in tryptic soy broth wash once with PBS and then resuspend. The new pellet in 800 microliters of PBS add 200 microliters of the dye solution to the resuspended cells for in vitro perfusion experiments, or 400 microliters of the dye solution. For in vivo experiments, cover the tubes with aluminum foil to prevent photobleaching of the dye and incubate on a shaker for 30 minutes at room temperature.

Next, add 6%bovine serum albumin solution in PBS to the mixture to block non-specific binding. Check the concentration of cells using optical cytometry and dilute bacteria into PBS at various concentrations depending on their application. Once diluted, protect the tubes from light and place them on ice.

Dilute von Vand factor in laboratory grade water to a final concentration of 50 micrograms per milliliter. Also dilute collagen in isotonic glucose solution to a final concentration of 160 micrograms per milliliter. Then drop 200 microliters of each coating onto a strip of paraform.

Place the cover slips on top of the droplets to coat them with the proteins. Incubate the cover slip in a humidified container for four hours at room temperature. Then carefully lift the cover slips from the paraform with a blunt needle and mount the cover slip in the bottom part of a flow chamber for experiments with endothelial cells, coat plastic slips with one milliliter of a 1%gelatin solution in PBS and incubate them for 30 minutes at 37 degrees Celsius.

Then seed human umbilical vein endothelial cells on the gelatin coated plastic slips and grow them to 70 to 80%co fluency. Mount the protein coated cover slip or the endothelial cell coated plastic slip in the bottom part of the flow chamber. Then connect the inlet and outlet tubing to the upper part of the flow chamber and connect the outlet tubing to a waste container.

Gently place the upper part of the flow chamber on the bottom part and assemble the flow chamber. While avoiding the formation of air bubbles. Inject one milliliter of medium through the chamber to make sure that the chamber is not leaking and to remove excess coating solution.

Next, set up the infusion pump for a one milliliter syringe so that it flows at 75 microliters per minute. Working in a dark room, fill a one milliliter syringe with one milliliter of fluorescently labeled bacteria and place it into the infusion pump. Connect the outlet of the syringe to the inlet of the flow chamber.

Taking care to avoid air bubbles, start the infusion pump for 10 minutes at 75 microliters per minute. After 10 minutes, remove the first syringe and connect a one milliliter syringe containing PBS or DMEM to the inlet tube. Start the infusion, pump and rinse for 10 minutes to wash away any unbound bacteria.

Once rinsed, leave the infusion pump on and take at least 15 black and white images using an exposure time of 1.5 seconds in random locations, spread over the coated surface of the flow chamber. Using an image analysis program like Image J.Subtract the background to remove smooth continuous backgrounds from the image and define the threshold to set the lower and upper threshold values, which segments the gray scale images into features of interest. Then measure the area limited to the newly created threshold and save this value known as the fluorescent area for each sample.

Tested fast to eight week old mice the night before the experiment to reduce bowel movement. The day of the experiment begin by injecting 0.1 milligrams of buprenorphine per kilogram of body weight as an analgesic. Next, anesthetize the mouse and apply ointment to the eyes.

Place the mouse on the thermo controlled heating pad under the dissecting scope and test that the mouse shows no petal reflex. Make a one centimeter incision parallel to the jugular vein. Remove the right side of the cervical muscle and then isolate the jugular vein from the surrounding tissue.

Insert a two French intravenous catheter into the right jugular vein and secure it in place using stitches. Then make a midline abdominal incision to open the peritoneal cavity. Place the mouse on its right side on a transparent plate and secure the cannula with tape.

Gently pull out the intestines using cotton swabs and spread the mesenterium. To visualize the mesenteric arteriolar and ular circulation. Place a hot pack over the mouse to prevent hypothermia and drop 500 microliters of 0.9%NACL on the intestines every 30 minutes to prevent dehydration of the tissue.

Use cotton swabs to immobilize the vessels and visualize them under an inverted microscope. Next topically, apply five microliters of a 10 millimolar solution of calcium iono four dissolved in DMSO. This will activate the release of Von Villa Brandt, factor from the endothelial cells in the vessels.

After 10 seconds, inject 100 microliters of the fluorescently labeled bacteria through the jugular catheter. At this time, begin taking time-lapse images, acquire time-lapse images using the acquisition tool in the toolbar using 40 cycles of 1000 images per second. Once image acquisition is complete, euthanize the mouse according to institutional guidelines.

Save the images in an appropriate image file format, and then process the images using image J analysis software as previously described to emphasize the role of sheer stress on bacterial adhesion, perfusions of staphylococcus aureus were performed at different shear rates on Von Vbr factor coated cover slips. As the sheer rates increase, so does bacterial adhesion indicating that high shear forces do not inhibit, but reinforce the adhesion of the bacteria to the protein. In the absence of Von Vand factor adhesion of staphylococcus aureus to collagen decreased with increasing shear rates.

However, when Von Vand factor was present in the medium, the adhesion of the bacteria increased with increasing shear rates, endothelial cells activated with a calcium iono four to release vand factor showed significantly more bacterial adhesion than nonactivated cells. Additionally, the bacteria formed typical string like patterns of fluorescently labeled bacterial clusters that were aligned in the direction of the sheer force, suggesting the binding of bacteria along a linear stretched VWF molecule to test the effect of Von Vbr factor in vivo endothelial cells of the mesenteric circulation were activated to release von Vbr factor, and then fluorescently labeled bacteria cells were introduced into the bloodstream. Bacteria locally adhered to the activated vessel wall and formed aggregates.

After watching this video, you should have a better understanding on how to investigate the interaction to bacteria and the vessel wall on the shear stress by fluorescently labeling the bacteria and by visualizing them both in an in vitro flow chamber and in an in vivo intra vital mesenteric profusion model. This technique paves the way for researchers in the field of cardiovascular infections to explore the interaction between pathogens and the vessel wall in infective endocarditis and metastatic infections.