CCM analysis was performed with the Heidelberg Retinal Tomograph III Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany). The cornea was locally anesthetized by instilling 1 drop of 0.4% benoxinate hydrochloride (Chauvin Pharmaceuticals, Chefaro, United Kingdom) and Viscotears gel (Carbomer 980, 0.2%, Novartis, United Kingdom) was used as the coupling agent between the cornea and the TomoCap as well as between the TomoCap and the objective lens. Subjects were instructed to fixate on a target with the eye not being examined. Several scans of the sub-basal nerve plexus in the central cornea were captured per eye for ∼2 min. The field of view of each image is 400 × 400 μm. At a separate time, three high clarity images per eye were selected by one researcher blind to the patient diagnosis using established criteria based on depth, focus position and contrast (Kalteniece et al., 2017 (link)). Corneal nerve fiber density (CNFD) (fibers/mm2), branch density (CNBD) (branches/mm2) and fiber length (CNFL) (total fiber length mm/mm2) were quantified manually using CCMetrics, a validated image analysis software (Dabbah et al., 2011 (link)).
Viscotears gel
Manufactured by Novartis
Sourced in United States, Australia
Viscotears gel is a sterile, clear, and colorless gel-like ophthalmic lubricant. It is designed to provide temporary relief of dry eye symptoms by maintaining moisture in the eyes.
Automatically generated - may contain errors
Lab products found in correlation
D luciferin potassium salt, by GoldBio (2 mentions)
Xenogen ivis lumina, by PerkinElmer (2 mentions)
Isoflurane, by Abbott (2 mentions)
Atropine 1, by Alcon (2 mentions)
Proparacaine hydrochloride alcaine, by Alcon (2 mentions)
Heidelberg retinal tomograph 3 rostock cornea module, by Heidelberg Engineering (1 mentions)
Fluorescite, by Alcon (1 mentions)
Rlt lysis buffer, by Qiagen (1 mentions)
Micron 3, by Phoenix Pharmaceuticals (1 mentions)
5 protocols using viscotears gel
1
Quantifying Corneal Nerve Parameters
2
In Vivo Bioluminescence Imaging of Murine Eyes
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All mice used for live imaging were aged between 12 and 25 weeks. For imaging, mice were anesthetized using 1.5 to 2% isoflurane (Abbott Laboratories Ltd., Berkshire, UK) in approximately 1.5 L/min flow of oxygen. A mix of luciferin substrate (30 mg/mL D-luciferin potassium salt; Gold Biotechnology, St. Louis, MO, USA) mixed 1:1 w/v with Viscotears gel (Novartis, Camberley, UK) was dropped onto the eye of heterozygous Krt12+/luc2 transgenic mice immediately prior to imaging. A Xenogen IVIS Lumina (Perkin Elmer, Cambridge, UK) was used to quantify luminescence. A region of interest encircling the mouse eye was selected for quantification whose size and shape was kept constant throughout, using protocols as previously described.9 (link)
3
In Vivo Corneal Luminescence Imaging
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All mice used for live imaging were aged between 12 and 25 weeks old. For imaging, mice were anaesthetised using 1.5–2% isoflurane (Abbott Laboratories Ltd., Berkshire, UK) in ~1.5 l/min flow of oxygen. A mix of luciferin substrate (30 mg/ml D-luciferin potassium salt; Gold Biotechnology, St. Louis, USA) mixed 1:1 w/v with Viscotears gel (Novartis, Camberley, UK) was dropped onto the eye of heterozygous Krt12 +/luc2 transgenic mice immediately prior to imaging. A Xenogen IVIS Lumina (Perkin Elmer, Cambridge, UK) was used to quantify luminescence. Live images of mice (n = 4) were taken every 24 hours for 7 days, then once every week thereafter for six weeks (42 days) in total. Quantification of luciferase inhibition was determined by calculating the right/left ratio, with values normalised to those at day 0 (as 100%).
4
Quantifying Laser-Induced Choroidal Neovascularization
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Seven days and 30 days following laser induced CNV, fluorescein angiography was performed to quantify the number and size of laser-induced lesions. Animals were anesthetized (ketamine 67 mg/kg and xylazine 13 mg/kg) and corneas were anaesthetized with topical proparacaine hydrochloride (Alcaine; 0.5%, Alcon) and dilated with topical atropine 1% (Alcon). Corneas were lubricated with viscotears gel (Novartis, Macquarie Park, NSW, Australia) to prevent corneal drying. Animals were injected subcutaneously with 100 μL of 1% sodium fluorescein (Fluorescite, Alcon). Brightfield and fluorescent fundus images were taken with the Micron III fundus camera 5 min following injection. Following fluorescein angiography, and whilst animals remained under anaesthetic, they were killed by cervical dislocation, their eyes removed and either placed in a fixative containing 60% Ethanol, 5% acetic acid, 4% paraformaldehyde, 3% sucrose in distilled water for histological analysis, or dissected and the RPE/choroid placed in a buffer containing RLT lysis buffer (Qiagen, Valencia, CA, USA) and 1% 2-mercaptoethanol and frozen at − 80 °C until required.
5
Laser-Induced Optic Nerve Lesions in Mice
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Mice were anaesthetized with a combination of ketamine 67 mg/kg and xylazine 13 mg/kg via intraperitoneal injection. Corneas were anaesthetized with topical proparacaine hydrochloride (Alcaine; 0.5%, Alcon, Macquarie Park, NSW, Australia) and dilated with topical atropine 1% (Alcon). Corneas were lubricated with viscotears gel (Novartis, Macquarie Park, NSW, Australia) to prevent corneal drying. Four laser induced lesions were placed around the optic nerve (at the 3, 6, 9 and 12 o’clock positions) using an image guide laser photocoagulation system (Micron III, Phoenix Research Laboratories, Pleasanton, CA, USA; 532 nm continuous wave laser, 350 mW, 70 ms duration) as previously described33 (link). Animals were intravitreally injected with a 0.5 μL aliquot of 400 μM cinnamoyl anthranilate derivative (final concentration 50 μM) (OCX063; OccuRx Pty Ltd, Melbourne, VIC, Australia) or sterile PBS using a 30-gauge needle either immediately following laser (day 7 endpoint) and 7 days following laser photocoagulation (day 30 endpoint).
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