In Vivo Imaging of Leishmania infantum-infected Hamsters by Gingival Inoculation of Axenic Amastigotes Expressing Luciferase

We aimed to standardize a methodology for the intravenous injection of Leishmania infantum into the gingival vein of hamsters to evaluate its potential as an alternative route for infection. Additionally, we investigated whether axenic amastigotes would be a better option for infection than promastigotes, utilizing the bioluminescent imaging system for follow up.

The in vivo imaging system is a refinement of the infection evaluation. Bioluminescent sites can be followed up in real time in the same animal, while the other most used assessments would be the parasite load in blood and tissues, histopathological analysis and PCR, which may require euthanasia.

The gingival vein is minimally invasive. allows intravenous access without local injuries when compared to other routes of intravenous infection. Another important point is that axenic amastigotes can be obtained in vitro in contrast to amastigotes obtain from tissues.

For Leishmania infection via the gingival vein, a smaller gauge was sanctioned due to vein perforation, bleeding, and inocculus leakage. Bioluminescent imaging system revealed that infection decline a long time and was only detectable by PCR, a characteristic of the reference strain whose infection progresses slowly and displays low parasite burden.

We have standardized another infection route for the L. infantum on golden hamsters. By performing intravenous infections into the gingival vein, we aim to achieve a stable and reproducible infection rate for the preclinical tests.

[Interviewer] To begin, restrain the anesthetized hamster in the supine position using both hands. Gently pull down the lower lip with the thumbs to expose the gum and the gingival vein. Using a one-milliliter syringe coupled with a thinner needle, position the needle below the lower incisors, along the midline between the pair of teeth, at a 25 degree angle. Insert the needle two to four millimeters into the mandibular labial vein. Aspirate blood into the syringe to confirm that the needle is in the mandibular labial vein. Ensure the blood reaches the barrel of the needle. Slowly inject 50 microliters of the inoculum containing 100 million firefly luciferase expressing Leishmania in phantom amastigotes or promastigotes in PBS. Before removing the needle, apply light pressure with a cotton swab. Simultaneously remove the needle from the vessel while maintaining pressure with the cotton swab to promote hemostasis. Restrain the hamster infected with Leishmania and phantom in hand. Inject 150 milligrams per kilogram deluciferan intraperitoneally into the hamster. Five minutes after anesthetizing, place the animal in a ventral position for bioluminescence imaging. under the CCD camera. Obtain images of the hamster two hours and 24 hours after infection, as well as eight and 22 days post-infection. Based on the bioluminescence emission levels, set the exposure time between 30 seconds and five minutes. Choose Binning as medium or large, set F/Stop to 1, and select the Field of View to D. Use the manual measurement tool to identify the regions of interest or ROI in the head and body of the animal. Quantify the bioluminescence in the selected ROI from the CCD camera. To measure the background signal, calculate the average ROI and subtract it from the bioluminescent emission obtained from the manual ROI. Express the bioluminescence emission in radiance as photons per second per square centimeter per steradian. In the maxillary region of hamsters infected intragingivally with Leishmania and phantom, amastigote infected animals displayed higher bioluminescence than promastigote infected ones two hours and at one day post infection. By eight days post infection, a 36% drop in bioluminescence was observed in promastigote-infected animals and 90% in amastigote-infected animals in the maxillary region. At 22 days post-infection, both promastygote and amastigote infected animals showed similar low levels of bioluminescence in the maxillary region. Eight months after infection with amastigotes via the gingival route, parasites could be detected by PCR in the liver and also displayed moderate piloerection, orbital tightness, and arched posture.