In-vivo imaging for assessing tumor growth in mouse models of ocular melanoma.

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Enzymatic Digestion of Porcine Corneas Cross-linked by Hypo- and Hyperosmolar Formulations of Riboflavin/ultraviolet A or WST11/Near-Infrared Light.

Purpose: To assess enzymatic digestion rate after Riboflavin (RF) and Water-Soluble-Taurine (WST11) based corneal cross-linking (CXL), with or without the addition of high molecular weight dextran (RF-D and WST-D). Methods: Eighty-eight paired porcine corneas were cross-linked by either RF (n = 11) or RF-D (n = 11) and ultraviolet light (UVA), or WST11 (n = 11) or WST-D (n = 11) and near-infrared (NIR) light, or used as paired control (n = 44). Corneal buttons of treated and paired control eyes were placed in a 0.3% collagenase solution. Time to full digestion and remaining dry sample weight after six hours were compared. Results: A strong treatment effect was seen with all four formulations, as all controls had been fully digested whilst all treated samples were still visible at the experiment's endpoint. After irradiation, central corneal thickness was significantly higher in samples treated with hypo-osmolar formulations, compared to dextran enriched formulations (P < 0.001). Dry sample weight after digestion was nonsignificantly different between corneas treated by the four different formulations (P = 0.102). Average dry sample weight was 1.68 ± 0.6 (n = 10), 2.19 ± 0.50 (n = 8), 1.48 ± 0.76 (n = 11), and 1.54 ± 0.60 (n = 9) mg, for RF, RF-D, WST11, and WST-D treated samples, respectively. Enzymatic resistance was similar for RF and WST based CXL (P = 0.61) and was not affected by the addition of dextran (P = 0.221). Conclusions: Both RF and WST11 based CXL significantly increases resistance to enzymatic digestion, with similar effect for hypo-osmolar and hyperosmolar (dextran enriched) formulations. Translational Relevance: Our findings indicate these formulations are interchangeable, paving the way for the development of novel PACK-CXL protocols for thin corneas and deep-seated infections.