Protocol for Vital Dye Staining of Corneal Endothelial Cells

Park, Sunju; Fong, A.; Cho, Hyung; Zhang, Cheng; Gritz, David C.; Mian, Gibran; Herzlich, Alexandra A.; Gore, Patrick; Morganti, Ashley; Chuck, Roy S.

doi:10.1097/ico.0b013e31824d0dda

Cited by 26 publications

(25 citation statements)

References 9 publications

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“…As previously described (Park et al, 2012), rabbit and/or human cornea button was briefly immersed and rinsed with pH 4.2 0.9% saline, then stained with pH 4.2 0.2% Alizarin Red 0.9% saline for 2 min, and rinsed with pH 7.2 0.9% saline.…”

Section: Methodsmentioning

confidence: 99%

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Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Zhang

Ogando

et al. 2017

EBioMedicine

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Corneal endothelium (CE) is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis) through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH3:H+ cotransporter Solute Carrier Family 4 Member 11 (SLC4A11). Considering SLC4A11 mutations leads to corneal endothelial dystrophy and sensorineural deafness, our results indicate that SLC4A11-associated developmental and degenerative disorders result from altered glutamine catabolism. Overall, our results describe an important metabolic mechanism that provides CE cells with the energy required to maintain high level transport activity, reveal a direct link between glutamine metabolism and developmental and degenerative neuronal diseases, and suggest an approach for protecting the CE during ophthalmic surgeries.

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Section: Methodsmentioning

confidence: 99%

“…After the experiment, Ringer solution (~ 350 μL) is collected and prepared for GC–MS analysis for residual glutamine and glucose. Then cornea button was stained with Alizarin Red for corneal endothelium visualization and cell density was counted under light microscope (Park et al, 2012). …”

Section: Methodsmentioning

confidence: 99%

Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Zhang

Ogando

et al. 2017

EBioMedicine

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“…The contrast between live and dead cells appears more prominent in a live/dead cell assay. In addition, the vital dye staining with alizarin red and trypan blue has no reproducible staining protocol, and obtaining reliable staining results by replicating the protocols previously described is difficult [27]. Therefore, the live/dead cell assay is a more sensitive tool for the detection of damaged corneal endothelial cells after laser iridotomy than combined staining with alizarin red and trypan blue.…”

Section: Discussionmentioning

confidence: 99%

Effects of Argon Laser Iridotomy on the Corneal Endothelium of Pigmented Rabbit Eyes

Youm

Heo

Kim

et al. 2014

Korean J Ophthalmol

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PurposeIn Asian countries, laser iridotomy for the treatment of angle-closure glaucoma is a common cause of bullous keratopathy, which may be associated with a shallow anterior chamber and dark iris pigmentation in Asians. Several cases of corneal decompensation after argon laser iridotomy have been reported. In the present study, we evaluated the harmful effects of argon laser iridotomy on the corneal endothelium.MethodsArgon laser iridotomy was performed on the right eyes of pigmented rabbits. Changes in corneal thickness and endothelial cell density after laser iridotomy were evaluated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed for assessment of corneal endothelial cell apoptosis. Combined staining with alizarin red and trypan blue, as well as a live/dead cell assay, were performed for evaluation of damage to the corneal endothelium induced by laser iridotomy.ResultsCorneal thickness did not change immediately after laser iridotomy; however, a significant increase was observed 24 hours after iridotomy (p = 0.001). The endothelial cell density of laser-treated eyes four days after laser iridotomy was significantly decreased compared with control eyes (p < 0.001). TUNEL staining showed many TUNEL-positive cells in the corneal endothelium and corneal stroma. No endothelial trypan blue-stained cell nuclei were observed after laser iridotomy; however, several large endothelial cells with damaged membrane integrity were observed. The live/dead cell assay clearly showed a large number of dead cells stained red in several areas throughout the entire corneal button 24 hours after iridotomy.ConclusionsArgon laser iridotomy induces corneal endothelial cell apoptosis in pigmented rabbit eyes, resulting in decreased endothelial cell density.

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“…Each tissue was stained according to the protocol described by Park et al 3 The tissue was first carefully rinsed with BSS in a syringe through a 30-gauge cannula to remove the coating of Healon. The tissue was then gently dried with a Weck-Cel optical spear (Beaver-Visitec International, Waltham, MA) and covered with a 0.4% solution of trypan blue (MP Biomedicals, Solon, OH) for 90 seconds.…”

Section: Stainingmentioning

confidence: 99%

In Vitro Evaluation of Endothelial Cell Loss Using the Neusidl Corneal Inserter

Davis-Boozer¹,

Terry²,

Greiner³

et al. 2013

Cornea

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Protocol for Vital Dye Staining of Corneal Endothelial Cells

Abstract: We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Cited by 26 publications

References 9 publications

Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium

Effects of Argon Laser Iridotomy on the Corneal Endothelium of Pigmented Rabbit Eyes

In Vitro Evaluation of Endothelial Cell Loss Using the Neusidl Corneal Inserter

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