Animal Model of Implant-Associated Infections in Mice

Establishing a stable animal model provides us reliable platforms for testing implant-associated infection therapies while investigating pathological stats and immune response within infection microenvironment. In the fight of PGI treatment, emerging technicals, such as fluorescence imaging, electron microscope, transcriptomics, offer powerful choice for investigating the life cycles and the futures of bacteria in PGI. However, we first need to establish a high-quality and reproducible PGI animal model.

The challenge now is constructing a stable, reproducible model that mimics the complex in vivo microenvironment, a clinical reality for implant-associated infections. Superior to subcutaneous abscess models, implant-associated infection models offer enhanced clinical relevance, sustain infections, improve the reproducibility, and the greater biosafety. This modeling method is highly safe and reliable, give us comprehensive investigation of pathophysiological mechanisms, and sparks the development of diagnostic criteria with targeted therapeutic stretches.

To begin, obtain cultures of Staphylococcus aureus. Remove a loopful of the culture with an inoculation loop, streak the suspension onto a blood agar plate, and incubate. Select a single, round, and smooth independent colony with golden yellow pigmentation and a clear hemolysis zone.

With a sterile loop, inoculate it into a 15 milliliter sterile centrifuge tube containing five milliliters of sterile tryptic soy broth. Place the tube in a shaking incubator set to 37 degrees Celsius and shake at 200 revolutions per minute for 12 hours. Dilute the bacterial suspension with tryptic soy broth at a one to 50 ratio and incubate again.

Then, use a spectrophotometer to measure and record the optical density at 600 nanometers to confirm the bacteria have reached the logarithmic growth phase. Next, pipette three milliliters of the bacterial suspension into a tube. Mix it with three milliliters of cold sterile PBS.

Centrifuge the mixture at 3000 G for 10 minutes at four degrees Celsius. Using a pipette, discard the supernatant and gently resuspend the bacterial pellet in PBS. Resuspend the washed bacterial pellet in PBS for further experimentation.

Randomly divide 20 C57BL bar 6J wild type mice into two groups of the implant-associated infection group and the subcutaneous abscess group. Apply distinct ear tags to each mouse for individual identification. After anesthetizing and preparing the skin of the animals, use sterile surgical blades to make a one centimeter incision in the dorsal region of each mouse.

Then, insert a sterile titanium implant into the subcutaneous pocket created by the incision. Now, use a sterile one milliliter syringe to inject 100 microliters of Staphylococcus aureus bacterial suspension directly onto the titanium surface. For the subcutaneous abscess group, create, disinfect, and suture the incision directly without implant insertion.

Inject 100 microliters of Staphylococcus aureus suspension into the incision site using a sterile one milliliter syringe. To evaluate the infections in peripheral tissue, place the harvested tissue sample into a sterile tube. Add an equal mass of sterile PBS and three sterile steel grinding beads to each tube.

Homogenize the tissues in three cycles at 70 hertz for 60 seconds each with a 20 second pause between cycles. After homogenization, vortex the samples for five minutes. Now, prepare serial dilutions of the tissue homogenate with PBS.

Using a micropipette, drop 15 microliters of each diluted homogenate onto designated sections of blood agar plates. Then incubate the blood agar plates at 37 degrees Celsius without shaking for 24 hours. The implant-associated infection group developed visible wound rupture by day three.

On day 10, both groups of mice showed signs of wound recovery, with the subcutaneous abscess group showing more pronounced recovery than implant-associated infection group on day 14. Bacterial cultures from infected tissue showed sustained high bacterial growth in the implant-associated infection group at all time points, whereas the subcutaneous abscess group showed a progressive reduction in bacterial colonies from day three to day 14. Scanning electron microscopy showed increasingly dense bacterial coverage on the titanium sheets in the implant-associated infection group from day three to day 14.

Giemsa staining revealed a marked decrease in bacteria in the subcutaneous abscess group by day 14, whereas the implant-associated infection group retained high bacterial presence throughout the period. Hematoxylin and eosin staining demonstrated that inflammatory cell infiltration in the subcutaneous abscess group diminished significantly by day 14, while the implant-associated infection group showed persistently dense cellular infiltration. Histological examination of heart, liver, spleen, lung, and kidney tissues showed no visible lesions or abnormalities in the implant-associated infection group compared with the control group.