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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Infection
- Obtain BALB/c mice of both sexes between the ages of 10-12 weeks.
NOTE: Streptococcus pneumoniae serotype 4 strain TIGR4 is a virulent clinical isolate that has been sequenced. - Grow TIGR4 in Todd-Hewitt broth at 37 °C in 5% CO2 until the culture reaches an optical density at 620 nm (OD620) of 0.5 (corresponding to 1.0 × 108 colony-forming units [CFU]/ml). Pellet the pneumococcal culture by centrifugation for 10 min at 3,500 × g and remove the supernatant using a vacuum line. Suspend and dilute bacteria with sterile phosphate-buffered saline (PBS) to a final concentration of 1.0 × 104 CFU/ml.
- Using an induction chamber, anesthetize mice with 2.5% vaporized isoflurane in oxygen. Confirm anesthetized state by gentle toe pinch with blunt tweezers.
- Holding the anesthetized mouse by its scruff and upright with one hand, inject each mouse intraperitoneally (i.p.) with 100 µl of the S. pneumoniae suspension using a syringe with a 27-30 gauge needle (corresponding to a challenge dose of 1.0 × 103 CFU). Place the mouse back in its cage. Mice typically awaken 30-40 seconds after injection.
- Allow the infection to proceed for at least 24 (early) or 30 (advanced) hr.
- Euthanize the mice by CO2 asphyxiation. Perform cervical dislocation to ensure the mouse has deceased. Euthanasia is further confirmed by the removal of the heart in Step 1.6.
- Disinfect the tail with an alcohol swab and snip a 2-3 mm section. Collect 2 µl of blood and serially dilute the blood 10-fold in PBS containing sodium heparin 1 U/ml five times. Plate serial dilutions on a tryptic soy blood agar plate and incubate overnight at 37 °C in 5% CO2. The next day extrapolates from the colony counts the level of bacteremia.
- Immobilize the mouse in a supine position on a surgical platform. 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. Using scissors, cut the blood vessels connected to the heart and gently excise the heart with care to avoid bruising with forceps.
- Rinse the heart with PBS and then place it into tissue specimen collection cassettes such that coronal sections would be obtained during tissue sectioning. For paraffin embedding, place the cassettes in 10% buffered-formalin solution, and the next day send for paraffin embedding.
- Alternatively flash freeze using Optimal Cutting Temperature (O.C.T.) Compound within a cryomold that has been placed on dry ice.
2. Visualization of Cardiac Lesions and Pneumococci within Lesions
- Cut down paraffin-embedded heart sections such that the 4 chambers of the heart are visible in each tissue section. Cut slides at a thickness of 5 µm.
- Deparaffinize and stain slides with hematoxylin & eosin (H&E) using standard methods.
- View H&E-stained cardiac sections using a light microscope at low (100×, 200×) and high (oil immersion: 400×, 1,000×) magnifications. Characterize hearts for the presence of microlesions in the myocardium. Here, acquire H&E images using a Zeiss Axioskop 2 microscope equipped with a 100× 1.3 numerical aperture Plan-NEOFLUAR objective.
NOTE: At early stages, cardiac lesions are discriminated by their different colored appearance and in some instances the presence of immune cells (monocytes and granulocytes), which appear to be monocytes. In advanced lesions, particularly those seen at 30 hr, immune cells are absent, and large vacuole-like lesions are observed in their place. It is important to note that while immune cell influx during the early stages of cardiac lesion development can occur, it does not appear to be a requirement.
3. Immunofluorescent Microscopy for Pneumococci within Microlesions
- Section the heart in cryomolds with a microtome to a thickness of 5 μm and place sections onto positively charged glass slides. Allow frozen cardiac sections to thaw and air dry. Fix sections for 10 min in 10% neutral buffered formalin (pH 6.8-7.2 at 25 °C).
- Wash slides (3×) in PBS for 5 min, permeabilize for 15 min in 0.2% Triton X-100 in PBS, then wash again in PBS.
- Block tissue section on slides with 10% goat serum in PBS for 1 hour.
- Rinse slides with PBS, cover, and incubate cardiac sections with rabbit anti-serotype 4 pneumococcus antiserum at 1:1,000 in PBS with 10% goat serum for 2 hr at 37 °C.
NOTE: For negative controls, investigators should use naive rabbit antiserum - After washing with PBS, cover and incubate sections with 10% goat serum-PBS with goat anti-rabbit Fluorescein Isothiocyanate (FITC) conjugated antibody at 1:2,000 for 30 min at 37 °C.
- Following the manufacturer’s instruction, use DAPI (4',6- Diamidino-2-Phenylindole) at 5 mg/ml for visualization of eukaryotic nuclei. Wash and mount tissue sections with FluorSave.
- Acquire fluorescent images using a confocal microscope system at both low and high magnification. Adjust the numerical aperture accordingly to obtain optimal results. Here, acquire fluorescent images using a confocal system with a 60× 1.42 numerical aperture objective.