Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

The overall goal of the following experiment is to demonstrate a reproducible protocol for ESMO cardiac micro lesion formation within the heart of infected mice. This is achieved by infecting mice with ESMO and allowing the development of bacteremia, thus giving bacteria access to the cardiac vasculature where they can invade the myocardium and form microscopic lesions. Then the hearts are either paraffin, embedded and stained conventionally or fixed into OCT compound for immunofluorescent staining.

Evaluation of the stained sections can show cardiac micro lesions randomly distributed across the myocardium collagen depositions and convalescent my heart sections and pneumococcal capsules within the cardiac micro lesion Demonstrating the procedure. Is Armand Brown a graduate student in my lab? Though this method can provide insight into the cardiac damaging effects of S pneumonia during invasive pneumococcal disease in mice.

It can also be applied to cardiac samples from other systems such as non-human primates or humans. Begin with growing up the ESMO Serotype four strain Tiger four in Todd Hewitt broth to an optical density of 0.5 at 620 nanometers. When at the correct density pellet the culture at 3, 500 G for 10 minutes.

Then remove the supernatant using a vacuum line and resuspend the bacteria with sterile PBS at 10, 000 colony forming units per milliliter. Now anesthetized 10 to 12 week old valve C mice and confirm their anesthetized state with a gentle toe pinch from a pair of blunt tweezers, lotus syringe with the bacterial suspension and attach a 27 to 30 gauge needle. Then take hold of an anesthetized mouse by its scruff with one hand while intraperitoneal injecting 100 microliters of suspension using the other hand, place each mouse back into its cage.

The mice typically awaken 30 to 40 seconds after the injection. Getting the dose of S pneumonia correct is critical, or the mice can recover from infection or comes to invasive pneumococcal disease too quickly for lesions to develop. Since each bacterial strain has different levels of ear lens, it may take trial and error to identify the correct dose.

Allow the infection to proceed for at least 24 hours to an early stage or to 30 hours for an advanced stage infection. Later euthanize each infected mouse. First, take a blood sample, disinfect the tail with an alcohol swab and snip off a two to three millimeter section.

Collect two microliters of blood from the snip site. Serially dilute the blood tenfold in PBS containing sodium heparin at one unit per milliliter. Plate five levels of dilutions on triptych soy blood agar plates, and incubate them overnight.

The next day take colony counts. Proceed with harvesting the heart as described in the next section. Alternatively, 30 hours post-infection.

Attempt to rescue the mouse by administering ampicillin interperitoneal in 100 microliters of saline. Continue providing ampicillin every 12 hours for 36 hours and collect hearts at the desired time points. First, harvest the heart.

Immobilize the euthanized mouse in a supine position on a surgical platform, and spray the chest with 70%ethanol and pat dry. Using surgical scissors and forceps, open the chest cavity. Remove the rib cage and transect the diaphragm to expose the heart and lungs.

Then cut the blood vessels connected to the heart and use forceps to gently excise the heart without bruising. Rinse the heart with PBS and place it into a tissue specimen collection Cassette for coronal sectioning. Then transfer the cassettes to 10%buffered formalin solution, and on the following day, send for paraffin embedding.

At early stages, cardiac lesions can be identified by their change in color appearance, and in some instances the presence of immune cells. In advanced lesions, immune cells will be absent and large. VA like lesions are found in their place.

Using a five micron cardiac section on positively charged glass slides first freeze the sections, then thaw them and allow them to air dry. Fix the slides in 10%neutral buffered formin for 10 minutes at 25 degrees Celsius. Then remove the fixative with three washes and PBS for five minutes per wash.

Follow by permeable the tissue with 0.2%tritton X 100 in PBS for 15 minutes. Remove the detergent with three more washes in straight PBS. Then block the tissue with 10%coat serum in PBS for an hour after a rinse in PBS, cover the section with antiserum for two hours at 37 degrees Celsius.

Treat negative controls with naive rabbit antiserum Because cardiac tissue readily absorbs the XI Immunofluorescent stain leading to high background levels, the concentration in incubation time for different types of antibodies will likely need to be optimized Two hours later, wash the antis serum away with PBS. Then replace the solution with a secondary antibody and let the slides incubate at 37 degrees Celsius for 30 minutes. Now apply DPI at five milligrams per milliliter and wash the slides with PBS.

Finally, mount the slides in floor, save and image them with confocal microscopy. Begin by removing the paraffin and hydrating the tissue sections. Use xylene washes followed by a graded series of alcohol washes.

Then bathe the sections in 0.2%phospho metic acid for five minutes, followed by a five minute water rinse next stain with 0.1%serious red and ric acid for two hours. Then wash the sections in 0.01 normal hydrochloric acid for three minutes and rinse them in 70%ethanol for a minute. Now dehydrate the sections by reversing the graded alcohol bath series, followed by xylene washes using light microscopy mount and evaluate the slides.

Mice developed invasive pneumococcal disease following a challenge with the tiger. Four strain cardiac micro lesions were observed in the ventricles and an increase in lesion size and number was noted between 24 and 30 hours post-infection to check that s pneumonia were indeed within the lesions. Sections were stained for the serotype four capsular polysaccharide produced by Tiger four.

This confirmed the presence of tightly packed and dense aggregates within the microlesion sites. Piro serious red staining was performed on cardiac sections from mice that had been rescued with ampicillin and uninfected controls. In rescues.

There was a dramatic increase in the presence and intensity of the stain with bands streaming from what appear to be former lesion sites in the ventricles. While attempting this procedure, it's important to remember to infect mice with sufficiency of use of S pneumonia to allow the infection to proceed for at least 28 to 30 hours so that bacteremia and cardiac micro lesions have enough time to form.