Our protocol allows for the reproducible introduction of microorganism and therapeutic agents into the mouse eye to study the pathogenesis of intraocular infection and novel treatment effectiveness. Our technique allows intraocular infections to be established in a mouse model and facilitates the quantitation of microorganisms, host immune responses, infection related host and pathogen gene expression, and novel treatment effectiveness. Visualization of the injection site, needle angle, and delivery are critical for successful execution of this technique and a proper eye harvest is essential for quantification of the infection parameters.
In a biosafety level two facility, add five milliliters of BHI broth to a 10 milliliter snap cap tube and use a sterile loop to transfer a single B.cereus colony from an auger plate culture into five milliliters of broth. After brief vortexing, incubate the sample in a 37 degrees Celsius rotating incubator at 200 revolutions per minute for 18 hours. At the end of the incubation, dilute the culture to a 200 colony forming units per microliter concentration in 10 milliliters of fresh BHI, and after vortexing, add one milliliter of the freshly diluted culture to a 1.5 milliliter microcentrifuge tube on ice.
For intravitreal injection, turn on the microinjector and open the gas valve on the compressed air tank attached to the microinjector. Press Mode on the microinjector until the screen shows Balance. Press Balance and place the computer mouse on the operating table.
Connect the stainless steel pipette holder to the tubing attached to the fill output on the injector and tightly screw the pipette holder connector around a beveled glass capillary pipette tip needle to allow insertion of the capillary needle into the other end of the pipette holder. Turn on the ophthalmic microscope and set the light intensity to 50%Position the microscope over the procedure site and adjust the microscope to the desired focus. After confirming a lack of response to pedal reflex, place the anesthetized mouse on its left side on the medical underpads with the nose pointed to the right.
Locate the right eye through the ophthalmic microscope objective and place the tongs of reverse action forceps on either side of the eye to expose the injection site. Left click the mouse connected to the microinjector to fill the prepared capillary needle with the diluted bacteria solution, and securing the head with the left hand, place the tip of the needle, bevel side up, at the limbus of the eye. With the needle at a 45 degree angle, puncture the eye, taking care that only 0.5 millimeters of the sharp tip of the needle is inserted.
Once the needle tip has been inserted, use the left hand to right click on the mousepad to inject 0.5 microliters of the B.cereus solution. To prevent leakage, leave the needle tip inside the mouse eye for two to three seconds before removing, then release the forceps and the eye will go back into the socket by itself. Transfer the mouse to a recovery cage on a warming pad with monitoring until full recumbency.
At the appropriate experimental endpoint, add 400 microliters of PBS supplemented with protease inhibitor in one labeled, autoclaved harvest tube per eye on ice and place open fine tip forceps on either side of the infected eye. Push the tips down towards the head to proptose the eye. Once the tongs are behind the eye globe, squeeze the tongs together and pull the forceps away from the head to detach the eyeball.
Then immediately place the eyeball into the appropriately labeled harvesting tube. Within 60 minutes of sample collection, place the closed harvesting tubes into a tissue homogenizer and homogenize the samples for two, one minute homogenization periods with a 30 second period of rest in between. After homogenization, place the samples on ice and use 20 microliter aliquots of homogenate to serially dilute the samples in 180 microliters of PBS per dilution, until a factor of one times 10 to the negative eight is reached.
Next, label each row of a square pre-warmed BHI plate with the appropriate dilution concentration, and with the plate tilted at a 45 degree angle, add 10 microliters of each dilution into individual rows at the top of the plate. Let each sample run until it almost reaches the bottom of the plate before laying the plate flat. When the samples have been absorbed into the agar, transfer the plate to a 37 degrees Celsius incubator.
Colonies should begin to be visible after eight hours of incubation. For an accurate representation of the concentration in the sample, count the row that has between 10 to 100 colonies. The construction of a beveled glass needle tip is critical for delivering the bacteria into the mid-vitreous of the mouse eye.
In these images, Bacillus cereus growing on a blood agar plate and in broth culture tubes are shown. As illustrated in this graph of interocular bacterial counts from five different mouse eyes at 10 hours post-infection between one times 10 to the fifth and one times 10 to the seventh colony forming units of bacteria can typically be recovered from a single infected eye. The most important thing to remember when attempting this procedure is to inject that appropriate volume of bacteria into the mouse eye.
Following this procedure, we can study inflammation by histology, inflammatory mediators by ELISA, and gene expression by high throughput RNA sequencing to facilitate a better understanding of the pathogenesis of this disease.
