The overall goal of this procedure is to demonstrate specific procedural issues associated with using the experimental animal model of vaginal candidiasis as a means to quantify vaginal fungal burden, and also to use vaginal or lymphoid cells in specific assays such as immunological and histological assays. This will be achieved by first demonstrating the vaginal inoculation technique on estrogen treated mice. Next, after euthanizing the mouse, a vaginal lavage will be performed to recover candida.
Then the vaginal tissue and or the vaginal draining lymph nodes will be extracted. Lavage fluid can be viewed microscopically to show candida associated with vaginal epithelial cells or plated on agar to quantify vaginal fungal burden. A smear of the cellular fraction can also be used to stain for epithelial cells and lymphoid cells.
So the main advantage of demonstrating this technique is so that the users of the animal model of vaginitis can do it most efficiently and effectively. And the key questions that are being answered via the technique are in the field of vaginitis and ecology and answer key questions such as host immune responses, cellular distribution, efficacy of drugs, immunotherapeutic efficacy, and also effects of immunization and demonstrating the technique. Today will be a graduate student in my laboratory, junco yano, To infect a mouse with C albicans three days after administering an injection of beta estradiol.
Begin by stabilizing the mouse on a flat gRED surface for it to grip. Then hold the base of the tail with two fingers and lift the hip upward so that the vaginal opening faces towards you. Insert the pipette tip about five millimeters deep into the vaginal lumen and pipette up to 20 microliters of the inoculum suspension.
Complete this step as quickly and gently as possible to minimize distress in the mouse. After the desired incubation period, euthanize the animal according to the protocol for your facility. Then with two fingers, hold the mouse downward by the base of the tail so that the vaginal opening becomes exposed with a pipette.
Introduce 100 microliters of sterile PBS into the vaginal lumen, and with repeated aspiration and agitation, perform lavage. If the pipette tip becomes clogged with cells, dispense the obstructing cells and continue ravaging with the remaining PBS. Collect the lavage fluid in a micro centrifuge tube.
Following the vaginal lavage procedure, lay the mouse on its back and saturate the groin area with 70%ethanol. Using a pair of forceps, lift the urinary orifice upward so that the vaginal opening becomes exposed. Insert a pair of curved forceps into the vaginal lumen and locate the cervix while maintaining a firm grip with the forceps.
Extract the cervix through the vaginal cavity, excise the vagina at the base of the vaginal opening, and then remove the cervix from the vagina with surgical scissors. Because the vaginal tissue is involuted, invert it to maintain the original orientation or open it into a sheet by making a lateral incision for lumbar node excision with the animal on its back. Saturate the abdomen with 70%ethanol.
Make a lateral incision starting from the lower abdomen to the chest and expose the internal organs using a pair of forceps in both hands. Move the intestines upward so that the central blood vessels become visible. Locate the inferior vena cva and the abdominal aorta.
A pair of lumbar lymph nodes can be identified adjacent to the abdominal aorta located about halfway between the origin of the renal and common iliac arteries. These lymph nodes can be visually distinguished from fat tissue by their elastic texture and are lighter and more opaque in color compared to fat tissue. They're also noticeably larger in infected animals compared to un inoculated.
Animals excised the lymph nodes by placing micro forceps under the node and then gently pull up to separate them from the surrounding tissue. After excising the tissue transfer the lymph nodes onto a sterile wire mesh screen placed inside a sterile glass Petri dish containing about 10 milliliters of Hank's balanced salt solution. To isolate lymphoid cells, refer to the written protocol.
The cellular fractions of vaginal lavage fluid from mice inoculated at least four days prior typically consist of candida epithelial cells and cellular infiltrates as shown here by wet mount microscopy. Candida can be identified by the presence of PHA as well as yeast. Here smear preparations of vaginal lavage fluid are stained.
Using the pap smear technique to examine epithelial cells and infiltrating leukocytes of which the principle cells are neutrophils, as can be identified by the traclear lobes. Very few neutrophils, if any, are detected in un inoculated mice. This figure shows an example of vaginal fungal burden.
Vaginal lavage fluid is collected at specific time points and cultured for CFU enumeration. In this example, the fluid was collected at day five post inoculation, serially, diluted plated, and incubated for 48 hours. Colonies are then counted and vaginal fungal burden calculated vaginal colonization or infection with candida persists for weeks in estrogen treated inoculated mice.
While candida fails to establish vaginal colonization in non estrogen treated inoculated mice, estrogen treated un inoculated mice remain negative for candida throughout the time. In addition, vaginal lavages can be performed either one time on separate mice at each time point or longitudinally in the same mice under anesthesia. While attempting this procedure, it's important to remember to restrain the animals securely to avoid trauma to the vagina, and please take the time necessary to lavage thoroughly to obtain accurate vaginal fungal burden when extracting vaginal tissues.
Use still PBS to keep them from dehydrating, and please be careful not to damage major veins and arteries when removing lymph nodes and while processing lymph node cells, keep them on ice or at four degrees to maintain cell viability. After watching this video, you should have a good understanding of how to employ the experimental mouse model of candida vaginitis to reproducibly quantify vaginal fungal burden and excise vaginal or lymphoid tissues for specific immunological or histological assays.
