The overall goal of this procedure is to study recurrent herpes simplex virus or HSV infection. This is accomplished by first infecting mice in such a way that they will become latently infected without corneal damage. After latency is fully established, the next step is to reactivate the virus by exposure to UVB light in the final step, tear film is collected for the detection of viral shedding and the miser then observed for at least five weeks to monitor the disease.
Ultimately, the extent of the disease that occurs in the mice can be assessed by disease scoring Herpetic stromal keratitis. This is a disease affecting many people worldwide. This particular disease is one of the leading causes of infectious blindness.
We have a model that we use in mice to mimic this particular disease. There are other models that actually can mimic this disease, but we feel this is the best rep representation of this particular disease. I'd like to take a few minutes today and introduce the people who you'll be seeing in this video.
This is Chloe who's been working with me for the last several months. This is Nippo, who is a summer student who's working in the lab. Begin the experiment by growing HSV one on Vero cells for three to four days to 80%co fluency in a T one 50 flask.
The cells will have rounded up by this time and will be easily dislodged by hitting the flask. Transfer the cell suspension to sterile 50 milliliter conical tubes, and then spin down the cells for 15 minutes. Add 1, 510 times G at four degrees Celsius in a benchtop centrifuge.
Next, transfer the supernatant into 50 milliliter sterile oak ridge tubes, and then split five milliliters of media total between each of the conical tubes to Resus. Suspend the large pellets, then sonicate the resuspended pellets for one minute in two 32nd bursts. After centrifuging the supernatant in a high speed floor centrifuge, a small viral pellet is produced.
After discarding the supernatant. Combine the sots with a small viral pellets and sonicate again for 45 seconds. Now centrifuge the sonicate cell and virus solution in the benchtop centrifuge again for five minutes at 280 times G and four degrees Celsius, and then aliquot the supernatant for storage as virus stock at negative 70 degrees Celsius to titer plate.
Tenfold dilutions of virus from the virus stock supernatant from 10 to the zero out to 10 to the negative eight, two times from two separate dilution sets of Vero cells in 12. Well sterile plates begin the infection procedure by anesthetizing the mouse. The mouse should remain fully anesthetized for 20 to 30 minutes to allow the virus to be absorbed before the mouse wakes up.
Then under a dissecting microscope, use a 30 gauge needle to scratch the cornea of the right eye. Now number the animal by ear punch. Next, inject the mouse with one milliliter of pooled human serum in order to protect the corneas from damage during the primary infection.
Then use a 20 microliter pipetter to infect the scratched eye with 10 to the sixth plaque forming units and place five microliters of HBSS into the other eye to keep it moist while the animal is anesthetized to confirm infection. Hydrate a sterile cotton applicator with one milliliter of Vero media. Three days post infection, swab the infected eye with the applicator, and then transfer 100 microliters of the swabbed media to Vero cells grown in 48 well plates at least five weeks following the primary infection.
Swab the infected eye of an anesthetized mouse as just shown. Mark the swabbing as day zero on the 48 well plate lid to control for spontaneous shutters. Then place the mouse in a transluminator that can emit UVB at a peak wavelength of 302 nanometers, such that only a single eye is exposed to the UVB light and expose the mouse to 250 millijoules of UVB light per centimeter squared.
Then swab the eye daily from days one to seven. Post UVB irradiation for those swabs that have infectious virus, tighter the amount of virus by a standard plaque assay using 12 well plates to evaluate the mice for clinical eye disease. Place the mouse under a binocular dissecting microscope.
Corneal opacity is rated on a scale of zero to four, where zero indicates clear S stroma one indicates mild stromal opacification. Two indicates moderate opacity with discernible iris. Features three indicates dense opacity with the loss of defined iris detail except for the pupil margins, and four indicates total opacity with no posterior view.
Neovascularization is rated on a scale of one to eight by dividing the cornea into four equal quadrants and determining the extent of vascularization in each of these quadrants. In this first figure, latently infected mice were induced to reactivate with UVB irradiation and then the mice were monitored for corneal opacity for 35 days. These data demonstrate that male C 57 black six mice display significantly less corneal opacity than do female C 57 black six mice, as there was significantly greater virus induced disease in female C 57 black six mice on days 14 through 35.
Likewise, when corneal neovascularization was evaluated under the same experimental conditions, male mice demonstrated significantly less new vessel growth in the cornea than did female mice, and once again, there is significantly greater virus induced disease in the female C 57 black six mice on days 14 through 28. While attempting this procedure, remember to fully anesthetize the mice so that they're asleep for between 20 and 30 minutes. After watching this video, I hope you have an understanding of how to use the mouse model of recurrent stromal keratitis to study this human disease.
