Establishing tumors as the posterior part of the eye is challenging. This method enables the induction of reproducible choroidal melanoma tumors in mice, and live imaging of tumor growth for evaluation. This protocol utilizes OCT live imaging to assess the location of the tumors already at the time of tumor cell inoculation, and to follow tumor growth in live animals.
Uveal melanoma is a devastating disease with close to a 50%rate of metastasis. Developing experimental models is key to advancing research aimed at new therapeutic possibilities. An advantage of this method is that the immediate assessment of the success of injection allows to reach study groups with homogenous tumors and continuously follow them by live imaging.
To examine tumor progression. Injecting into the small mouse's eyes requires practice. The ability to visualize the injected cells allows you to assess your success immediately and repeat with more mice as necessary.
Demonstrating the procedure will be Dr.Dimitri Gaber from the Ophthalmology Research Laboratory at the Kaplan Medical Center. To begin, apply 0.4%topical ophthalmic anesthetic, Oxybuprocaine to both eyes of the anesthetized mouse. Then topically apply 0.5%Tropicamide to both eyes for dilating the pupils, followed by 1.4%hydroxyethyl cellulose as a lubricant to avoid drying of the eyes.
Observe one eye of the mouse under an operating microscope. Hold the eyelids open with sterile intraocular forceps. Then hold the superior temporal limbo-conjunctiva and pull toward an infra nasal position.
Secure this position by holding the limbo-conjunctiva throughout the procedure. Using a 30 gauge needle tip make a small conjunctival peritomy in the dorsal temporal area approximately one to two millimeters posterior to the limbus. Remove the excess tendons capsule from the opening of the peritomy.
Insert the needle tip to penetrate the sclera creating a track into the subchoroidal space until the brown color of the choroid appears through the exposed white matter of the sclera. Load the cells into a sterile 10 microliter glass Hamilton syringe mounted with a 32 gauge blunt needle twisted at a 45 degree angle. Insert the needle of the loaded syringe approximately two millimeters into the created tract, and inject two microliters of the cell suspension.
Hold the needle in place after injection for two to three seconds until all fluid has cleared. Remove the needle by pulling it out gently to avoid leakage from the tract. Immediately after the melanoma cell inoculation, observe the injected eye of the animal by OCT scans.
Based on the scans, classify the retinal detachment or RD patterns according to three categories local RD, Leakage Into The Vitreous, or Extended RD.Open an OCT segmentation analysis software to predict the tumor size based on RD height. Measure the height of Local RD in the OCT scans, and classify them into three groups. In the postoperative phase, apply 0.3%of Ofloxacin topically.
The OCT-based prediction of tumor growth following subchoroidal cell injection is depicted in this figure. The mice exhibited three patterns of RD, Focal, Leakage To The Vitreous, and Extended RD.There was an association between the pattern of RD immediately after injection, and the localization of the tumors five days after injection. Focal RD was associated with the tumor cell growth that was limited to the choroid.
However, observation of cells in the vitreous after injection in animals displaying Focal RD indicated tumor growth in the vitreal cavity and the choroid. when Extended RD was observed after injection, tumors were dispersed throughout the choroid and vitreous after five days. The range of tumor sizes induced in this model can be divided into small, medium, and large.
Shown here are representative images of the OCT measurement of RD height after injection, and the corresponding fundus image, a horizontal OCT scan of the tumor height and width on day five after injection, and its corresponding fundus image, and the corresponding H&E stained eye section demonstrating comparable tumor measurements. Tumor volume measurements of mice that presented small, medium or large RD immediately after cell injection are presented here. Adjusting the force of injection can affect tumor size and location.
It is important to calibrate this and to evaluate the pattern of induced retinal detachment by OCT after injection. Live imaging of choroidal tumors is an important experimental tool that allows to examine the effect of different factors or potential therapies on tumor progression.
