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* These authors contributed equally
The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.
Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.
Uveal melanoma (UM) is the most frequent intraocular primary malignancy in adults. Approximately 90% of ocular melanomas originate from melanocytes in the choroid region of the uveal tract1. UM is a major cause of morbidity and mortality, as it is estimated that close to 50% of patients develop metastatic disease, with the liver being the major site of metastasis2. Early treatment of primary lesions may reduce the chance of metastases, yet no effective treatment prevents metastases formation3.
The standard treatment of uveal melanoma includes irradiation therapy, which ....
The experiments in the protocol were approved by the Israeli National Council on Animal Experimentation and comply with the ARVO Statement for using Animals in Ophthalmic and Vision Research. Female C57BL/6 mice, aged 8-10 weeks, were used for the present study and were exposed to 12/12 h light-dark cycles. The animals were obtained from a commercial source (see Table of Materials).
1. Cell culture
Eyes were examined via OCT immediately after injection of the B16LS9 cells. Local retinal detachment was observed after injection. The mice exhibited three patterns of RD: focal (Figure 2, upper panel), leakage to the vitreous (Figure 2, middle panel), and extended RD (Figure 2, bottom panel). Extended RD is likely caused by damage from the injection. There was an association between the pattern of RD immediately after inje.......
Uveal melanoma is a devastating disease for which novel therapeutic approaches are greatly needed. However, research on uveal melanoma and potential treatments is limited by the technical challenges of uveal melanoma animal models1,25. Ocular tumors, which are induced by intraocular injection of cancer cells, are highly variable in both localization and size, likely due to the small dimensions of the mouse eye. Such variability is an obstacle to the comprehensive.......
This study was supported in part by grant 1304/20 from the Israel Science Foundation (ISF), Israel, for Arie Marcovich. We thank Shahar Ish-Shalom and Ady Yosipovich, from the Department of Pathology, Kaplan Medical Center, Rehovot, Israel, for histology analysis.
....Name | Company | Catalog Number | Comments |
10 μL glass syringe (Hamilton Co., Bonaduz, Switzerland) | Hamilton | 721711 | |
30 G needles | BD Microbalance | 2025-01 | |
Atipamezole hydrochloride | Orion Phrma | ||
B16LS9 cells | from Hans Grossniklaus USA | ||
Buprenorphine | richter pharma | 102047 | |
C57BL/6 female mice | Envigo | ||
Essential vitamin mixture | satorius | 01-025-1A | |
Fetal bovine serum | rhenium | 10270106 | |
HEPES | satorius | 03-025-1B | |
Hydroxyethylcellulose 1.4% eye drops | Fisher Pharmaceutical | 390862 | |
InSight OCT segmentation software | Phoenix Micron, Inc | ||
Ketamine | bremer pharma GMBH (medimarket) | 17889 | |
L-glutamine | satorius | 03-020-1B | |
Medetomidine | zoetis (vetmarket) | 102532 | |
Ofloxacin 0.3% eye drops | allergan | E92170 | |
Optical coherence tomography | Phoenix Micron, Inc | ||
Oxybuprocaine 0.4% | Fisher Pharmaceutical | 393050 | |
Penicillin-streptomycin-amphoteracin | satorius | 03-033-1B | |
Phosphate buffered saline (PBS)Â | satorius | 02-023-1a | |
RPMI cell media | satorius | 01-104-1A | |
Sodium pyruvate | satorius | 03-042-1B | |
Surgical microscope | Zeiss | OPMI-6 CFC | |
Tropicamide 0.5% | Fisher Pharmaceutical | 390723 |
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