Corneal Endothelial Dysfunction Model in Monkeys

The monkey corneal endothelial dysfunction model was created by scraping the corneal endothelium completely from the Descemet’s membrane with a 20-gauge silicone needle (Soft Tapered Needle; Inami & Co., Ltd., Tokyo, Japan) while the animal was under general anesthesia, as described previously8 (link). The MCEC injection experiments were conducted on the following 3 groups: 1) MCECs (5.0 × 10⁵ cells) were suspended in 200 μl of DMEM supplemented with 100 μM of Y-27632 (ROCK inhibitor; Wako Pure Chemical Industries, Ltd.) and injected into the anterior chamber (n = 6), (2) MCECs (5.0 × 10⁵ cells) were suspended in 200 μl of DMEM and injected into the anterior chamber (n = 2), and (3) no MCECs were injected (n = 2). The HCEC injection experiments were conducted on the following 4 groups:1) HCECs (5.0 × 10⁵ cells) were suspended in 200 μl of OptiMEM-I supplemented with 100 μM of Y-27632 (Wako Pure Chemical Industries, Ltd.) and injected into the anterior chamber (n = 8), (2) HCECs (5.0 × 10⁵ cells) were suspended in 200 μl of OptiMEM-I and injected into the anterior chamber (n = 2), (3) human pre-cut donor corneas were transplant using a DSAEK procedure (n = 2), and (4) no HCECs were injected(n = 2). The eyes were kept in the face-down position for 3 hours under general anesthesia, except for the eyes of monkeys that underwent DSAEK. The corneal transparency and thickness of the anterior segments were evaluated by slitlamp microscopy. A Pentacam^® (OCULUS Optikgeräte GmbH, Wetzlar, Germany) instrument was used to visualize the corneal shape. Corneal thickness was determined with an ultrasound pachymeter (SP-2000; Tomey, Nagoya, Japan), and the mean of 10 measured values was calculated (up to a maximum thickness of 1200 μm, the instrument’s maximum reading). Intraocular pressure was determined by a Tonovet^® (icare Finland, Vantaa, Finland) instrument. The corneal endothelium was evaluated by non-contact specular microscopy (FA-3809, Konan Medical, Nishinomiya, Japan). Eyes that exhibited clinical features such as the presence of keratic precipitates, progression of corneal edema, and conjunctival injection were diagnosed as having undergone graft rejection.

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Okumura N., Sakamoto Y., Fujii K., Kitano J., Nakano S., Tsujimoto Y., Nakamura S.I., Ueno M., Hagiya M., Hamuro J., Matsuyama A., Suzuki S., Shiina T., Kinoshita S, & Koizumi N. (2016). Rho kinase inhibitor enables cell-based therapy for corneal endothelial dysfunction. Scientific Reports, 6, 26113.

Publication 2016

Animal Anterior chamber Cells Clinical Conjunctival Corneal edema Corneal endothelium Descemet membrane Donor Eyes Face General anesthesia Graft rejection Hcecs Human Intraocular pressure Microscopy Monkeys Needle Oculus Progression Silicone Slitlamp microscopy Transplant Ultrasound Y 27632

Corresponding Organization :

Other organizations : Doshisha University, Senju Pharmaceutical (Japan), Shiga University of Medical Science, Kyoto Prefectural University of Medicine, National Institute of Biomedical Innovation, Health and Nutrition, Tokai University

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Variable analysis

independent variables

Injection of MCECs (5.0 × 10^5 cells) suspended in 200 μl of DMEM supplemented with 100 μM of Y-27632
Injection of MCECs (5.0 × 10^5 cells) suspended in 200 μl of DMEM
Injection of HCECs (5.0 × 10^5 cells) suspended in 200 μl of OptiMEM-I supplemented with 100 μM of Y-27632
Injection of HCECs (5.0 × 10^5 cells) suspended in 200 μl of OptiMEM-I
DSAEK procedure using human pre-cut donor corneas

dependent variables

Corneal transparency
Corneal thickness
Corneal shape
Intraocular pressure
Corneal endothelium evaluation
Presence of clinical features (keratic precipitates, corneal edema, conjunctival injection)

control variables

General anesthesia during the procedure
Keeping the eyes in the face-down position for 3 hours after the injection, except for the DSAEK group

positive controls

DSAEK procedure using human pre-cut donor corneas

negative controls

No MCEC injection
No HCEC injection

Annotations

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