A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy

Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (p < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (p = 0.40, p = 0.17, and p = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (p < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.

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The Effect of Phacoemulsification on Corneal Endothelial Cells Morphology and Thickness

Pakistan Journal of Ophthalmology ◽

10.36351/pjo.v36i4.1092 ◽

2020 ◽

Vol 36 (4) ◽

Author(s):

Abd Elaziz Mohamed Elmadina ◽

Raghda Faisal Abdelfatah ◽

Saif Hassan Alrasheed ◽

Mustafa Abdu ◽

Manzoor Ahmad Qureshi

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Density ◽

Corneal Endothelium ◽

Central Corneal Thickness ◽

Corneal Thickness ◽

Endothelial Cell Density ◽

P Value ◽

Corneal Endothelial Cells ◽

Before And After

Purpose: To compare the corneal endothelial cells morphology and central corneal thickness (CCT) before and after phacoemulsification in Sudanese population. Place and Duration of Study: Al-Neelain eye hospital, Khartoum, Sudan, from January 2018 to May 2018. Study Design: Observational longitudinal study. Methods: One hundred and forty eyes of 140 patients with immature senile cataract were selected by convenient sampling. The age ranged from 40 to 85 years. The patients underwent complete ocular examination including morphology of corneal endothelial cells and CCT using computerized non-contact specular microscope. Inclusion criteria for the study was eyes with normal corneal endothelial cells and cell density more than 1000 cells/mm2. We excluded patients with ocular or systemic diseases, previous history of intraocular surgery, refractive surgery or trauma as well as contact lenses wear. The patients underwent phacoemulsification by a single surgeon. The examination parameters were repeated one month after surgery. Descriptive and comparative statistical analyses were performed using SPSS for Windows Version 21.0. Results: There was significant reduction in mean endothelial cells density after phacoemulsification compared to baseline with p < 0.001. There was also significant post-operative reduction in mean endothelial cells number as compared to baseline (P value < 0.001). Mean endothelial cells hexagonality was reduced after surgery with P value of 0.003. No significant difference was found between mean coefficient variation of endothelial cells size before and after phacoemulsification (P = 0.55). Central corneal thickness showed significant increase post-operatively, P = 0.003. Conclusion: Phacoemulsification causes significant damage to corneal endothelium cells, including decrease in corneal endothelial cell density, hexagonality and cell number. Key Words: Corneal endothelium, Endothelial cell density, Central corneal thickness, Phacoemulsification.

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Crosslinking-Induced Corneal Endothelium Dysfunction and Its Protection by Topical Ripasudil Treatment

Disease Markers ◽

10.1155/2022/5179247 ◽

2022 ◽

Vol 2022 ◽

pp. 1-12

Author(s):

Xuemei Wang ◽

Yanlin Zhong ◽

Minghui Liang ◽

Zhirong Lin ◽

Huping Wu ◽

...

Keyword(s):

Dna Damage ◽

Endothelial Cells ◽

Endothelial Cell ◽

Protective Effect ◽

Corneal Endothelium ◽

Corneal Thickness ◽

Corneal Opacity ◽

Rock Inhibitor ◽

Corneal Crosslinking ◽

And Function

Purpose. To investigate the changes of corneal endothelium under different crosslinking conditions and the protective effect of ripasudil. Methods. Corneal crosslinking groups were infiltrated with riboflavin and subsequently irradiated with 0.54 J/cm2 or 1.08 J/cm2 UVA, while noncrosslinking groups included neither UVA nor riboflavin treatment, only 1.08 J/cm2 UVA and only riboflavin treatment. Corneal opacity, variations in corneal endothelial cells, and corneal thickness of all groups were observed by slit lamp, in vivo confocal microscopy, and optical coherence tomography. Immunofluorescence staining and scanning electron microscopy were performed to evaluate changes in the structure and function of the corneal endothelium. The mice that received a corneal crosslinking dose of 1.08 J/cm2 were instilled with ripasudil to explore its protective effect on the corneal endothelium. Results. Treatment with UVA and riboflavin caused an increase in corneal opacity and corneal thickness and decreased endothelial cell density. Furthermore, treatment with UVA and riboflavin caused endothelial cell DNA damage and destroyed the tight junction and pump function of the endothelium, while riboflavin or the same dose of UVA alone did not affect the endothelium. Ripasudil reduced DNA damage in endothelial cells, increased the density of cells, and protected the endothelium’s integrity and function. Conclusion. Riboflavin combined with UVA can damage the corneal endothelium’s normal functioning. The corneal endothelium’s wound healing is dose-dependent, and the ROCK inhibitor ripasudil maintains the endothelium’s pump and barrier functions.

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Morphology of endothelial cells from different regions of the cornea of dogs

Ciência Rural ◽

10.1590/0103-8478cr20180596 ◽

2018 ◽

Vol 48 (10) ◽

Author(s):

Paula Stieven Hünning ◽

Maria Cristina Caldart de Andrade ◽

André Carissimi ◽

João Pigatto

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Morphology ◽

Corneal Endothelium ◽

Central Area ◽

Alizarin Red ◽

Central Cornea ◽

Significant Difference ◽

Peripheral Areas ◽

Healthy Eyes

ABSTRACT: The aim of this study was to evaluate the morphology of endothelial cells from different areas of the cornea of dogs. Twenty healthy eyes from 10 dogs, females or males, of different ages were studied. Corneal endothelium morphology of superior, inferior, central, nasal and temporal areas was assessed by 0.2% alizarin red staining using an optic microscope. One hundred endothelial cells from each corneal area were analyzed. In all areas of the cornea studied were found endothelial cells with four sides, five sides, six sides and seven sides. There was no significant difference regarding endothelial cell morphology in all corneal regions evaluated. Thus, the morphology of the central cornea area represents the entire endothelial mosaic and may be applied to peripheral areas. Therefore, analysis of the central area is sufficient to estimate the shape of endothelial cells of peripheral areas of healthy dog corneas.

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Sodium Ferulate Attenuates Lidocaine-Induced Corneal Endothelial Impairment

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/4967318 ◽

2018 ◽

Vol 2018 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Guojian Jiang ◽

Tingjun Fan

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Morphological Changes ◽

Corneal Thickness ◽

Cell Loss ◽

Corneal Endothelial Cell ◽

Corneal Endothelial Cells ◽

Endothelial Cell Loss ◽

Sodium Ferulate ◽

Human Corneal Endothelial Cells

The introduction of intracameral anaesthesia by injection of lidocaine has become popular in cataract surgery for its inherent potency, rapid onset, tissue penetration, and efficiency. However, intracameral lidocaine causes corneal thickening, opacification, and corneal endothelial cell loss. Herein, we investigated the effects of lidocaine combined with sodium ferulate, an antioxidant with antiapoptotic and anti-inflammatory properties, on lidocaine-induced damage of corneal endothelia with in vitro experiment of morphological changes and cell viability of cultured human corneal endothelial cells and in vivo investigation of corneal endothelial cell density and central corneal thickness of cat eyes. Our finding indicates that sodium ferulate from 25 to 200 mg/L significantly reduced 2 g/L lidocaine-induced toxicity to human corneal endothelial cells, and 50 mg/L sodium ferulate recovered the damaged human corneal endothelial cells to normal growth status. Furthermore, 100 mg/L sodium ferulate significantly inhibited lidocaine-induced corneal endothelial cell loss and corneal thickening in cat eyes. In conclusion, sodium ferulate protects human corneal endothelial cells from lidocaine-induced cytotoxicity and attenuates corneal endothelial cell loss and central corneal thickening of cat eyes after intracameral injection with lidocaine. It is likely that the antioxidant effect of sodium ferulate reduces the cytotoxic and inflammatory corneal reaction during intracameral anaesthesia.

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Evaluation of Corneal Endothelium in Adolescents with Juvenile Glaucoma

Journal of Ophthalmology ◽

10.1155/2015/895428 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Beata Urban ◽

Alina Bakunowicz-Łazarczyk ◽

Marta Michalczuk ◽

Małgorzata Krętowska

Keyword(s):

Endothelial Cell ◽

Carbonic Anhydrase ◽

Beta Blocker ◽

Corneal Endothelium ◽

Central Corneal Thickness ◽

Open Angle Glaucoma ◽

Corneal Thickness ◽

Control Group ◽

Open Angle ◽

Juvenile Glaucoma

Purpose. To evaluate the endothelial cell density (ECD) and central corneal thickness (CCT) in adolescents with juvenile open-angle glaucoma (JOAG) and ocular hypertension (OH) and to investigate the influence of topical antiglaucoma medications on ECD and CCT in adolescents with JOAG.Methods. ECD and CCT were investigated in 66 eyes of 33 adolescents with JOAG. Depending on the topical treatment the eyes were classified into 4 groups: (1) topical carbonic anhydrase inhibitor, (2) prostaglandin analogs, (3) beta-blocker, and (4) CAI-beta-blocker combination. ECD and CCT were also checked in 24 adolescents with OH and in control group (33 persons).Results. ECD was significantly lower in eyes with JOAG (2639.5 cells/mm2) compared with ECD in eyes with OH (2924.5 cells/mm2) and in control group (2955.5 cells/mm2). CCT was 0.554 mm in eyes with JOAG, 0.55 mm in eyes with OH, and 0.544 mm in control group. ECD in patients with JOAG was 2730 cells/mm2(1 group), 2773.5 cells/mm2(2 group), 2539.5 cells/mm2(3 group), and 2551 cells/mm2(4 group). CCT was 0.556 mm in 1 group, 0.558 mm in 2 group, 0.532 mm in 3 group, and 0.544 mm in 4 group.Conclusions. Our findings indicate that JOAG and OH did not affect CCT, but JOAG has influence on ECD in adolescents. There were no significant differences between ECD and CCT of eyes treated with different kinds of antiglaucoma medications.

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Monitoring Endothelial Cell Activation In Vivo during Thrombus Formation.

Blood ◽

10.1182/blood.v110.11.294.294 ◽

2007 ◽

Vol 110 (11) ◽

pp. 294-294

Author(s):

Ben T. Atkinson ◽

Prathima Nandivada ◽

Bruce Furie ◽

Barbara C. Furie

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Intracellular Calcium ◽

Cell Activation ◽

Thrombus Formation ◽

Single Cells ◽

Calcium Mobilization ◽

Endothelial Cell Activation ◽

Calcium Elevation

Abstract The endothelium serves as a metabolically active interface between the blood and underlying tissues and offers response to changes in its microenvironment. The endothelium is rapidly activated in response to stimuli associated with fluctuations in shear stress, physical trauma, oxidative stress, and thrombotic and inflammatory mediators. To study endothelial cell activation we have monitored calcium mobilization in vitro in cultured endothelial cells and also in situ in the living mouse, following vessel wall injury. The addition of exogenous ADP (10 μM) or thrombin (1 U/mL) to Human Umbilical Vein Endothelial Cells (HUVECs) loaded with the calcium sensitive dye, Fluo-4 AM, is followed by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. To further investigate the ability of the endothelium to activate in response to mediators potentially localized in the microenvironment of the endothelium during thrombus formation, we investigated the ability of IL-6 (0.1 ng/mL), IL-8 (80 ng/mL), MCP-1 (15 ng/mL), NAP-2 (10 ng/mL) and SDF-α (60 ng/mL) to mobilize calcium in HUVECs. These potential agonists were selected based on their primary roles in inflammation and thrombosis, and proposed roles in atherosclerosis and tumor angiogenesis. SDF-α and NAP-2 rapidly mobilized calcium in HUVECs, with similar maximum responses, but NAP-2 initiated a more prolonged (4-fold longer) rise in intracellular calcium. IL-6, IL-8, and MCP-1 also mobilized calcium, but the rise in intracellular calcium was almost 4-fold lower than that observed with SDF-α and NAP-2. Comparable calcium mobilization occurs in HUVECs subjected to a single pulse of a nitrogen dye-tuned laser. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation in the targeted cell and was followed by a wave of calcium elevation in surrounding cells. To determine whether this endothelial cell activation, and more specifically the calcium elevation, occurs in vivo, we conducted experiments using the laser-induced model of thrombus formation to look for calcium elevation in the arteriolar endothelium in live mice. Calcium elevation was monitored by Fluo-4AM introduced intravenously into the mouse circulation via the jugular vein. Fluo-4AM is non-specific in its uptake among cell types, and endothelial cell uptake of the dye in the cremaster muscle microcirculation was observed. Upon laser-induced injury, calcium elevation in the endothelium could be monitored by intravital microscopy. The observed calcium elevation was rapid (within 30 s) and preceded detection of platelets in the developing thrombus. These results add to the evidence of a dynamic endothelium and demonstrate that the endothelium activates rapidly prior to thrombus formation in the laser induced thrombosis model.

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Endothelial Progenitor Cells (EPCs) Isolated from the Rhesus Macaque but Not the Mouse Are Phenotypically and Functionally Similar to Human EPCs.

Blood ◽

10.1182/blood.v104.11.3910.3910 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3910-3910

Author(s):

David A. Ingram ◽

Laura E. Mead ◽

Daniel B. Moore ◽

Theresa Krier ◽

Ann Farese ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Rhesus Macaque ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Peripheral Blood ◽

Single Cells ◽

Preclinical Model ◽

Proliferative Potential ◽

Endothelial Colony Forming Cells

Abstract We recently identified a novel hierarchy of human endothelial progenitor cells (EPCs), which are functionally defined by their proliferative and clonogenic potential (Blood, 2004). Emerging evidence suggests that EPCs may be used as angiogenic therapies, or as biomarkers to assess cardiovascular disease risk. Thus, identification of animal models, which phenocopy the human EPC hierarchy, is an important priority for preclinical testing of experimental therapeutics. Given the importance of the Rhesus Macaque as a preclinical model, we tested whether EPCs could be isolated from the peripheral blood of the Rhesus Macaque and compared to EPCs isolated from human adult peripheral blood. Mononuclear cells were isolated from 20 ml of Rhesus peripheral blood and cultured in EGM-2 medium, which promotes the formation of EPC colonies. After 7 days in culture, we identified approximately 20 endothelial cell colonies (n=9), which appeared identical to human EPC colonies. We subcultured the endothelial cell colonies into monolayers for immunophenotyping and functional analysis. Endothelial cells (ECs) derived from the Rhesus EPC colonies formed vessels in matrigel, and demonstrated uptake of acetylated LDL, which are characteristics of ECs. Similar to ECs derived from human EPCs, Rhesus ECs expressed the endothelial cell antigens, CD31, CD144, CD105, CD146, and Flk1. Importantly, Rhesus ECs did not express the hematopoietic cell specific antigens, CD45 and CD14. Similar to ECs derived from human peripheral blood EPC colonies, Rhesus ECs could be serially passaged for at least 40 population doublings without signs of cellular senescence. A hallmark of stem and progenitor cells is their ability to proliferate and give rise to functional progeny. Analogous to a paradigm established in the hematopoietic cell system, we recently developed a single cell deposition assay to reproducibly identify the following human EPCs: (1) high proliferative potential - endothelial colony forming cells (HPP-ECFC), which form macroscopic colonies that form secondary and tertiary colonies upon replating, (2) low proliferative potential - endothelial colony forming cells (LPP-ECFC), which form colonies greater than 50 cells, but do not form secondary colonies upon replating, (3) endothelial cell clusters (EC-clusters) that contain less than 50 cells, and (4) mature terminally differentiated endothelial cells (EC), which do not divide (Blood, 2004). To determine whether these different populations of EPCs could be identified in the ECs derived from Rhesus EPCs, we performed single cells deposition assays on 1,000 cells. All types of EPCs could be identified in the Rhesus ECs (Table I). Further, ECs derived from the Rhesus EPCs rapidly form chimeric vessels with human ECs derived from adult blood, implying that the molecular mechanisms critical for vessel formation are conserved between the two species. Finally, while the murine model is an animal model widely used for studying EPCs, a similar hierarchy of EPCs could not be established from the peripheral blood of mice. Thus, given the diversity of therapeutic applications of EPCs for treating a variety of human diseases, these studies establish the Rhesus Macaque as an important preclinical model. Percent of 1,000 Single Cells Plated Mature EC EC-Cluster LPP-ECFC HPP-ECFC Rhesus ECs 85.8±2.1 4.2±1.1 7.8±0.5 1.3±0.5 Human ECs 80.8±9.6 8.6±1.4 12.4±8.1 0.2±0.2

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Specular microscopic observations of the corneal endothelium in the normal rabbit

Laboratory Animals ◽

10.1258/002367781780952924 ◽

1981 ◽

Vol 15 (4) ◽

pp. 393-395 ◽

Cited By ~ 11

Author(s):

Denise Montanaro Sailstad ◽

R. L. Peiffer

Keyword(s):

Endothelial Cells ◽

New Zealand ◽

Corneal Endothelium ◽

Corneal Thickness ◽

Specular Microscopy ◽

Clinically Normal ◽

New Zealand White Rabbits ◽

Mean Diameter ◽

The Mean ◽

Number Of Cells

Endothelial specular microscopy and pachometry were performed on both eyes of 14 young adult New Zealand white rabbits with clinically normal eyes. Endothelial cells of the central corneas formed a mosaic-like pattern of homogenous hexagonal cells with a mean diameter of 20·6 ± 1·0, μm sd. The mean number of cells per mm was 2998 ± 326 sd and the mean corneal thickness was 0·38 ± 0·02 mm sd.

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A new storage medium containing amphotericin B versus Optisol-GS for preservation of human donor corneas

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2020-317136 ◽

2020 ◽

pp. bjophthalmol-2020-317136

Author(s):

Raffaela Mistò ◽

Laura Giurgola ◽

Francesca Pateri ◽

Anna Limongelli ◽

Eugenio Ragazzi ◽

...

Keyword(s):

Endothelial Cell ◽

Amphotericin B ◽

Corneal Thickness ◽

Storage Period ◽

Blue Staining ◽

Specular Microscopy ◽

Storage Medium ◽

Human Donor ◽

Corneal Transparency ◽

Donor Corneas

Background/AimWe compared the quality of human donor corneas stored in a cold storage medium containing 2.5 μg/ml of amphotericin B (Kerasave, AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy) and Optisol-GS (Bausch & Lomb Inc., Bridgewater, NJ, USA) for 14 days.MethodsSixteen pairs of human donor corneas were collected in Eusol-C (AL.CHI.MI.A. S.R.L., Ponte San Nicolò, Italy). Next, all tissues underwent the first evaluation that included the assessments of central corneal thickness (CCT), endothelial cell density (ECD) measured using both trypan blue staining and specular microscopy, endothelial cell (EC) mortality and morphology, and corneal transparency within 24 hours from recovery (Day 1). Afterwards, one cornea of each pair was transferred into Kerasave or Optisol-GS. ECD and CCT were also assessed at Day 7, and all the metrics were evaluated again at the end of the storage period (Day 14).ResultsAt all tested time points, no differences were found in the qualitative (corneal transparency, EC morphology) and quantitative metrics (ECD, CCT, EC mortality) between the Kerasave and the Optisol-GS storage groups. At Day 14, the corneas stored in Kerasave and Optisol-GS showed ECD of 2312±98 and 2335±128 cells/mm2 (p=0.886), CCT of 717±17 and 697±19 μm (p=0.454) and central EC mortality of 0.54%±0.40% and 0.14%±0.14% (p=0.719), respectively.ConclusionsThe new amphotericin B−containing medium Kerasave was comparable to Optisol-GS in terms of preservation of corneal characteristics at 2–8°C for 14 days.

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