Evaluation of porcine corneal endothelium after preservation in Eusol-C medium

Abstract

This study was conducted to evaluate the viability of porcine corneal endothelium after conditioning in the preservation medium Eusol-C. Twelve eyes from six 6-month-old male Large White pigs (Sus scrofa domesticus) were analyzed. Endothelial cell morphology and cell count of all samples were assessed prior storage using specular microscopy to ensure that only healthy corneas were included in the study. The samples were randomly divided into a control group (G1) and a test group (G2). Corneas from G1 were immediately submerged in bottles containing 2.5% glutaraldehyde, whereas corneas from G2 were stored in Eusol-C at 4°C for 14 days. The percentage of endothelial cell loss was evaluated using scanning electron microscopy (SEM). Comparisons between regions were performed using repeated measures Analysis of Variance (ANOVA). Differences were considered statistically significant at P < 0.05. Corneal samples from G1 showed no areas of endothelial cell loss. In samples from G2, the average cell loss was 3.01%. In conclusion, the hypothermic medium Eusol-C satisfactorily preserved porcine corneas for up to 14 days.

Key-words:
endothelial cells; transplantation; ophthalmology; porcine

Resumo

O objetivo deste estudo foi avaliar a viabilidade do endotélio corneano suíno após acondicionamento em meio de preservação Eusol-C. Foram incluídos 12 olhos de seis suínos da raça Large White (Sus scrofa domesticus), machos, com seis meses de idade. A morfologia e a contagem de células endoteliais de todas as amostras foram avaliadas antes do armazenamento por microscopia especular de contato, para assegurar que somente córneas hígidas seriam incluídas. As amostras foram aleatoriamente divididas em grupo controle (G1) e em grupo teste (G2). As córneas do G1 foram imediatamente submersas em frascos contendo glutaraldeído a 2,5%. As córneas do G2 foram armazenadas em Eusol-C a 4 °C por 14 dias. A porcentagem de perda de células endoteliais foi avaliada com microscopia eletrônica de varredura (MEV). As comparações entre regiões foram realizadas por meio de análise de variância de medidas repetidas (ANOVA). As diferenças foram consideradas estatisticamente significativas em P < 0,05. O G1 não apresentou perda celular. O G2 apresentou perda celular média de 3,01%. O meio hipotérmico Eusol-C preservou satisfatoriamente as córneas suínas por um período de 14 dias.

Palavras-chave:
células endoteliais; transplante; oftalmologia; suíno

1. Introduction

The corneal endothelium is a monolayer of predominantly hexagonal polygonal cells (¹). A decrease in cell density below a critical threshold leads to corneal edema and subsequent loss of corneal transparency (¹, ²). In corneal diseases resulting in loss of transparency, corneal remains the sole therapeutic option for vision restoration (³-⁵). Corneal preservation primarily aims to extend the storage time of donor tissues while maintaining endothelial cell viability (⁶). The most commonly used corneal preservation methods are hypothermic media and organ culture (⁶). Hypothermic media preserves the cornea for 10 to 14 days. Eusol-C is a hypothermic medium developed as an effective alternative to Optisol-GS. The primary advantage of Eusol-C over Optisol-GS is its lower cost, which may enhance its applicability in veterinary medicine. Porcine corneas exhibit biological and morphological characteristics comparable to those of human corneas, which allows comparison between the results of previous studies with the human cornea (⁷, ⁸). Porcine corneas have been used as study models in hypothermic preservation media to assess antibiotic use, compare different media, and observe storage performance and safety (⁹-¹¹). However, studies evaluating the Eusol-C preservation medium in this species remain recent and limited, primarily focusing on endothelial evaluation using specular microscopy (¹⁰, ¹¹).

Scanning electron microscopy (SEM) is widely used to assess endothelial morphology, with numerous studies published on medication toxicity, evaluation post-preservation, and assessments following corneal or intraocular surgical procedures (¹²-¹⁸). To the authors' knowledge, this is the first study to evaluate, using SEM, the percentage of endothelial cell loss following the preservation of porcine corneas in Eusol-C. Porcine corneas may represent a promising source of donor tissue for both human and veterinary applications (², ³, ¹⁹). The establishment of ocular tissue banks presents significant potential, and ongoing research on preservation methods in animals is critical for enhancing the safety and success of corneal transplantation procedures (²⁰-²²). This study was conducted to evaluate the viability of porcine corneal endothelium following conditioning in the preservation medium Eusol-C.

2. Material and methods

Twelve eyes from six 6-month-old male Large White pigs (Sus scrofa domesticus) were included in the study. The eyes were donated by an abattoir (Avisui, Santa Maria, Rio Grande do Sul, Brazil). Immediately following slaughter and prior to scalding, the eyes were enucleated. They were then individually placed in a humid chamber containing gauze and saline solution within a thermal box to maintain a temperature of 2ºC to 8ºC. Within 1 h post-enucleation, all eyeballs were examined using slitlamp biomicroscopy (Portable Slit Lamp SL15; Kowa, Nagoya, Japan), fluorescein staining (1% sodium fluorescein; Allergan Pharmaceuticals, Dublin, Ireland), and a contact specular microscope (Celmax Medical Service®; São Carlos, SP, Brazil).

Only eyes deemed healthy were included in the study. Images were acquired during specular microscopy performed to select healthy eyes before separating the corneas into groups. Subsequently, these images were processed using imaging software (Adobe Photoshop), and the morphology of 50 endothelial cells per image was analyzed. The 12 selected eyes were decontaminated by washing with a 5% povidone-iodine solution (PVP-I; Rioquímica S/A, São José do Rio Preto, SP, Brazil). Subsequently, the corneoscleral buttons were removed and the samples were randomly divided in two groups: the control group (G1) and the test group (G2). Buttons from G1 were immediately submerged in bottles containing 2.5% glutaraldehyde. Buttons from G2 were placed in bottles containing Eusol-C and remained refrigerated for 14 days. After 14 days, corneoscleral buttons were transferred to bottles of glutaraldehyde. Following fixation in 2.5% glutaraldehyde and storage at approximately 4°C, samples were individually prepared for SEM analysis at the Microscopy and Microanalysis Center of UFRGS (CMMUFRGS, Porto Alegre, Rio Grande do Sul, Brazil).

Samples were washed in cacodylate buffer and dehydrated through a series of solutions with increasing ethanol concentrations (30%, 50%, 70%, 85%, 90%, and 100%), remaining for 15 minutes at each concentration and placed three times in the 100% concentration. They were then subjected to critical point drying with liquid carbon dioxide. Corneas were placed on 10-mm aluminium stubs with double-sided adhesive tape and coated with gold-palladium. The posterior endothelial surface of each sample was analysed and photographed using a scanning electron microscope (EVO MA10; Zeiss, Oberkochen, Germany) operating at 10 kV. Five electron micrographs were obtained from each sample (1000×) within corresponding regions: I, central; II, superior; III, inferior; IV, lateral; and V, medial. Additional micrographs at 2000× magnification were obtained to provide greater detail of the endothelium. The 1000× magnification images were individually processed with ImageJ software (National Institutes of Health, Bethesda, MD, USA), using a tool that allowed circling portions of the image where endothelial loss occurred. Measurements were obtained in μm² and then converted to mm². The percentage of cell loss was calculated by subtracting the circled areas from the total image area.

2.1 Statistical analysis

Endothelial parameters obtained via specular microscopy were analysed using repeated measures ANOVA with a significance level of 5%. For SEM analysis, regions I to V of each sample were compared internally and with the corresponding regions across different samples. The median percentage of cell loss, along with minimum and maximum loss values was calculated for each group and each region. Comparisons of cell loss between G1 and G2 were performed using the Mann-Whitney U test, while comparisons across different regions were conducted using the Friedman test. The Dunn-Bonferroni test was applied to identify statistically significant differences between regions. A significance level of 5% was adopted for all statistical analyses.

3. Results

All enucleated eyes evaluated via slit lamp and specular microscopy were included in the study. Contact specular microscopy enabled visualisation of the porcine corneal endothelium, revealing a regular pattern of juxtaposed polygonal cells. The mean endothelial cell density was 1867.8 ± 78.0 cells/ mm² in the right eye and 1862.2 ± 85.2 cells/mm² in the left eye. Considering the morphology of all analysed cells, 53.8% exhibited six sides, 21.5% exhibited five sides, and 17.7% exhibited seven sides. Additionally, 5% of the cells displayed four sides, and 2% displayed eight sides. No significant differences were observed in endothelial cells when comparing right and left eyes. Corneal samples from G1 exhibited no areas of endothelial cell loss (Figure 1). Endothelial loss was observed in samples preserved in Eusol-C preservation medium (Figure 2). In samples from G2, the average cell loss was 3.01%. A significant difference was found in the endothelial loss between G1 and G2. No significant differences were found among the studied regions within G1. However, a statistically significant difference was detected between regions I and IV within G2 (Table 1).

Table 1
Comparison of the percentage of cell loss between groups and among regions.

	G1	G2	p between groups*
	n=6	n=6
I - Central	0 (0-0.47)	1.65 (1.55-1.,94)a	0.002
II - Superior	0 (0-0.56)	2.94 (2.25-4.62)	0.002
III - Inferior	0 (0-0)	3.21 (2.25-3.92)	0.002
IV - Lateral	0 (0-0)	3.70 (2.83-4.07)b	0.002
V - Medial	0 (0-0)	3.55 (2.36-4.46)	0.002
p among regions**	0.558	0.007

• Data presents as median (minimum-maximum);
• ^*
  Mann-Whitney test,
• ^**
  Friedman test followed by Dunn-Bonferroni post hoc test; abdifferent letters denote statistically different groups.

Figure 1
Scanning electromicrograph of the porcine corneal endothelium in G1. A regular corneal endothelial pattern and absence of endothelial damage are observed. Magnification: 2000×. Bar: 10 µm.

Figure 2
Scanning electromicrograph of the porcine corneal endothelium in G2. Note the area of endothelial loss (arrows). Magnification: 2000×. Bar: 10 µm.

4. Discussion

Few recent studies have analysed corneal endothelial preservation in Eusol-C in either humans (²³-²⁴) or in animals (¹⁰-¹², ²⁵). Endothelial cell loss was evaluated in equine corneas after 7 and 14 days of storage in Eusol-C using SEM and observed satisfactory preservation for up to 14 days (¹²). Satisfactory preservation of porcine corneal endothelium after 14 days of storage in Eusol-C medium was demonstrated (¹⁰). The same group of researchers conducted a comparative study involving human and porcine corneas. After a 14-day period, the authors concluded that corneas from both species exhibited satisfactory quality parameters following conditioning in Eusol-C (¹¹). Another study evaluating calf corneas that were stored at 2-8 °C for up to 14 days in Eusol-C suggested that bovine and human corneas share comparable preservation requirements under ex vivo conditions (²⁵).

Although a study involving rabbit and human corneas demonstrated the superiority of organ culture medium in preserving corneal endothelial cells for up to 28 days (²⁶), in another retrospective study evaluating the clinical outcomes of corneal transplantation using human donor corneas stored in Eusol-C compared with corneas stored in organ culture, comparable surgical outcomes were obtained with both storage methods when the optimal storage periods for each medium were applied (²⁷). In comparisons of hypothermic media, Eusol-C has been shown to be as effective as Optisol-GS for corneal preservation for at least five days (²⁸).

Currently, human corneas are the sole donor source for transplantation. This donor source has become increasingly scarce, and this scarcity is the main limiting factor in curing corneal diseases (³⁵, ²⁹, ³⁰). To address this issue, studies have been conducted on corneal xenotransplantation, primarily using porcine species (², ³, ¹⁹). Although porcine corneas lack Bowman’s membrane, their biological and morphological characteristics have proven to be quite similar to those of human corneas (⁷, ⁸, ¹⁵, ²⁹, ³¹, ³²). This species has been widely utilized as a study model for ocular diseases and surgical procedures (⁷, ¹⁵,³³, ³⁴).

The authors opted to use eyes obtained from slaughterhouse discard material in the present study to avoid the need for euthanasia. Previous studies conducted within 4-6 hours postmortem, endothelial integrity was maintained (³⁵-³⁸). Regarding sample size, previous studies under different experimental conditions, the expected percentage of cell mortality ranges from 7,8% (SD= 3,5%) (¹¹) to 3,07% (SD=0,93%) (³⁹). With 6 animals per group, it is possible to detect differences of 7% and 1,86, considering these variabilities, a power of 90% and a significance level of 0,05. In summary, a sample size of 6 animals allows the detection of a difference of 2 standard deviations with a power of 90%, which is considered a large effect size. This calculation was performed with the help of WINPEPI 11.65 (⁴⁰).

Corneal specular microscopy was used to assess endothelial health in this study. Specular microscopy is the standard method for endothelial evaluation in both humans and animals and has been employed to analyse corneas from various species (²⁵, ³⁵-³⁸, ⁴¹, ⁴²). The parameters analysed via specular microscopy were the endothelial cell density and hexagonality. In the present study, no significant differences in endothelial density or hexagonality were observed between the right and left eyes. The values found in the present study using specular microscopy were similar to those previously reported (³⁸). This similarity was attributed to the fact that the animals in both studies were healthy and belonged to the same breed and age group. It has been established that in healthy eyes, no differences exist in corneal endothelial parameters between the left and right eyes (³⁵-³⁸).

In previous studies utilizing Eusol-C medium, corneas were analysed over periods ranging from 7 to 29 days (¹⁰, ¹², ²³, ²⁴). In the present study, a storage period of 14 days was adopted, corresponding to the storage duration recommended by the manufacturer. A 14-day storage duration has been employed in studies demonstrating satisfactory endothelial preservation within this time frame for Optisol, Optisol-GS, and Eusol-C media (¹⁰, ¹³, ²⁴, ²⁵, ⁴¹, ⁴³-⁴⁵). Refrigeration of corneas extends endothelial cell viability over prolonged periods(6).

Endothelial viability of preserved corneas can be assessed using different methods, including vital staining, optical microscopy, and SEM, among others (⁴⁶, ⁴⁷). Among the available methods, optical microscopy represents a rapid and cost-effective method for evaluating the corneal endothelium (6). In the present study, SEM was chosen to assess the percentage of endothelial loss following the preservation period. SEM is the most widely used tool for analysing endothelial morphology, with numerous studies published in humans and animals regarding drug toxicity, evaluation after preservation, and assessments following corneal or intraocular surgical procedures (¹², ¹³, ¹⁶, ¹⁸, ⁴¹). In veterinary medicine, remains in its early stages of development. Grafts typically provide tectonic support, with achievement of an optical purpose still a distant goal (²⁰-²²). However, the lower cost and minimal equipment requirements associated with the Eusol-C hypothermic medium render it a promising option for the establishment of animal eye banks, potentially transforming this reality.

In a recently published study, an ex vivo porcine cornea model has been suggested as an alternative to the human as an alternative to the human cornea for analysing endothelial preservation after storage under hypothermic conditions or organ culture (¹⁰). Images were obtained with a high-resolution camera from light microscopy with trypan blue, and the cell mortality rate and other parameters were assessed with the aid of software. Porcine and human corneas exhibited comparable trends under the investigated storage conditions for up to 14 days. Preservation of human corneas was studied in a new hypothermic preservation medium called Kerasave with Optisol-GS (³⁹). For this comparison, SEM was not employed, instead trypan blue staining and specular microscopy were used to analyze cell density, mortality, and morphology. On day 14, the mean mortality rate in the central region of the cornea was 0.54% ± 0.40% for corneas in Kerasave and 0.14% ± 0.14% for those in Optisol-GS. In the peripheral regions of the cornea, the mortality rate was 3.07% ± 0.93% for corneas in Kerasave and 3.38% ± 0.78% for those in Optisol-GS. These mortality rates were considered consistent with satisfactory preservation over the analysed period.

In the only published study analyzing porcine corneas preserved in hypothermic medium via SEM (⁹), porcine corneas were stored in 2.5% chondroitin sulphate for up to 10 days. Cell mortality rates of 12% on day 5 and 26% on day 10 of storage were observed, and these rates were considered similar to those for human corneas in this medium. Rodella and collaborators (¹⁰) compared the effects of preservation in Eusol-C and organ culture medium by evaluating cell density and mortality using trypan blue as a cell marker after a storage period of up to 14 days. Mortality rate was estimated using ImageJ software. At the end of the storage period, porcine corneas stored in organ culture medium and Eusol-C showed mortality rates of <10% and <20%, respectively. In a similar study, Giurgola et al. (¹¹) investigated the density, morphology, and cell mortality of human and porcine corneas after preservation in Eusol-C for 14 days, washing with cleaning solution, and employing trypan blue and specular microscopy. At the end of storage period, the endothelial mortality rate increased by 3.1% ± 3.3% in human corneas and by 7.8% ± 3.5% in porcine corneas. Based on these results, the authors concluded that the use of preservation medium and cleaning solution was safe and effective, emphasizing the similar behavior between corneas from both species.

In the present study, the percentage of endothelial loss was 3.01% after 14 days of storage, considering the average losses in the evaluated regions. Compared to previous studies on porcine corneas, the percentage of cell loss obtained in this study was lower than that found using the same storage period (¹¹, ¹⁰). However, the analysis methods employed in these previous studies did not involve SEM. In previous studies of human corneas in which endothelial mortality was established using vital dyes and visualisation under a microscope, cell loss ranged from 4% to 16% after 14 days of storage in preservation media (⁴⁸-⁴⁹). These rates were also slightly higher than those found in our research, although still comparable despite differences in the analysis methods. Although the difference in endothelial loss between G1 and G2 was statistically significant in this study, it was not considered clinically relevant, as a certain degree of loss is expected. This finding is consistent with previously published data in the literature, indicating satisfactory preservation during the observed period. In the current study, the percentage of cell loss was evaluated as a parameter of satisfactory preservation based on the abovementioned previous studies because the cell density can be significantly different depending on the fixation method used in the sample for SEM. Furthermore, these values may also vary depending on the analysis method utilized (³⁹).

This study evaluated the percentage of cell loss following preservation in Eusol-C medium. Cell density and cell morphology were not evaluated after the preservation period. In a previous study, endothelial density of porcine corneas evaluated by SEM decreased after 10 days of storage in preservation medium (⁹). This likely occurred because of cell shrinkage, and it did not follow the same pattern as cell mortality, which increased during both periods. Rodella et al. (¹⁰) did not observe significant differences in the endothelial density of porcine and human corneas stored in Eusol-C for 14 days. However, endothelial mortality increased significantly over time. These results reinforce that cell density is not a reliable parameter for this type of evaluation. Cellular morphology was assessed only to ensure the inclusion of healthy corneas in the study. No significant changes in cellular morphology are expected in an acute study.

Study limitations include the reduced sample size, the single preservation time, the absence of functional evaluation and the absence of studies designed with SEM, Eusol-C and percentage of cell loss analysis in pigs to compare results more accurately.

This is the first study to associate the evaluation of porcine corneal endothelium preserved in hypothermic Eusol-C medium with SEM, enabling the calculation of the percentage of endothelial cell loss following storage. The results demonstrated satisfactory preservation over the analysed period, with cell loss rates comparable to those reported in previous studies, despite differences in analysis methods. The authors suggest further studies with larger samples and morphological evaluation of preserved corneas after transplantation.

5. Conclusion

The results of this study demonstrated that the hypothermic preservation medium Eusol-C was effective in preserving the corneal endothelium of pigs for up to 14 days.

Data availability statement

The data will be provided upon request.

Acknowledgments

The authors thank Avisui and the Microscopy and Microanalysis Center of UFRGS (CMM-UFRGS) for their contributions to this study and to thank Coordination for the Improvement of Higher Education Personnel (CAPES), which provided a fellowship for two of the authors (M.P.S. and N.P.M).

Referências

• ¹ Joyce NC. 2012. Capacidade proliferativa das células endoteliais corneanas. Exp Eye Res 95: 16-23. Available from: https://doi.org/10.1016/j.exer.2011.08.014
  » https://doi.org/10.1016/j.exer.2011.08.014
• ² Lee SE, Mehra R, Fujita M, Roh DS, Long C, Lee W, Funderburgh JL, Ayares DL, Cooper DKC, Hara H. 2013. Caracterização do endotélio corneano de porco para xenotransplante. Semin Ophthalmol 29: 127-135. Available from: https://doi.org/10.3109/08820538.2013.787104
  » https://doi.org/10.3109/08820538.2013.787104
• ³ Yoeruek E, Bayyoud T, Maurus C, Hofmann J, Spitzer MS, Schmidt KUB, Szurman P. 2012. Reconstrução do estroma corneano com xenógenos de córnea porcina decelularizada em um modelo de coelho. Acta Ophthalmol 90: 206-210. Available from: https://doi.org/10.1111/j.1755-3768.2011.02300.x
  » https://doi.org/10.1111/j.1755-3768.2011.02300.x
• ⁴ Isidan A, Liu S, Li P, Lashmet M, Smith LJ, Hara H. 2019. Métodos de decelularização para o desenvolvimento de xenotransplantes de córnea porcina e perspectivas futuras. Xenotransplantation 26: e12564. Available from: https://doi.org/10.1111/xen.12564
  » https://doi.org/10.1111/xen.12564
• ⁵ Yoon CH, Choi HJ, Kim MK. 2021. Xenotransplante de córnea: Qual é o nosso estágio atual? Prog Ret Eye Res 80: 1-40. Available from: https://doi.org/10.1016/j.preteyeres.2020.100876
  » https://doi.org/10.1016/j.preteyeres.2020.100876
• ⁶ Saad HA, Terry MA, Shamie N, Chen ES, Friend DF, Holiman JD, Stoeger C. 2008. Um método simples e barato para análise quantitativa de dano endotelial utilizando coloração vital e software Adobe Photoshop. Cornea 27: 818-824. Available from: https://doi.org/10.1097/ICO.0b013e3181705ca2
  » https://doi.org/10.1097/ICO.0b013e3181705ca2
• ⁷ Menduni F, Davies LN, Madrid-Costa D, Fratini A, Wolffsohn JS. 2018. Caracterização do globo ocular suíno como modelo in vitro para síndrome do olho seco. Cont Lens Anterior Eye 41: 13-17. Available from: https://doi.org/10.1016/j.clae.2017.09.003
  » https://doi.org/10.1016/j.clae.2017.09.003
• ⁸ Crespo-Moral M, García-Posadas L, López-Garcia A, Diebold Y. 2020. Caracterização histológica e imuno-histoquímica da superfície ocular de suínos. PLoS One 15: 1-17. Available from: https://doi.org/10.1371/journal.pone.0227732
  » https://doi.org/10.1371/journal.pone.0227732
• ⁹ Ellis MF, McGhee CNJ, Lee WR. 1992. Estudo do endotélio corneano suíno armazenado em sulfato de condroitina por microscopia eletrônica de varredura. Cornea 11: 127-132. Available from: https://doi.org/10.1097/00003226-199203000-00006
  » https://doi.org/10.1097/00003226-199203000-00006
• ¹⁰ Rodella U, Bosio L, Ferrari S, Gatto C, Giurgola L, Rossi O, Ciciliot S, Ragazzi E, Ponzin D, Tothova JD. 2023. Modelo de armazenamento ex vivo de córnea suína como alternativa ao tecido de doadores humanos para investigações sobre preservação da camada endotelial. Transl Vis Sci Technol 12: 1-13. Available from: https://doi.org/10.1167/tvst.12.4.24
  » https://doi.org/10.1167/tvst.12.4.24
• ¹¹ Giurgola L, Rodella U, Gatto C, Rossi O, Honisch C, Ragazzi E, Ruzza P, Ferrari S, Tothova JD. 2024. Avaliação de desempenho e segurança do armazenamento hipotérmico em câmara corneana e lavagem corneana com PSS-L em córneas humanas e suínas. BMJ Open Ophthalmol 9: e001453. Available from: https://doi.org/10.1136/bmjophth-2023-001453
  » https://doi.org/10.1136/bmjophth-2023-001453
• ¹² Albuquerque L, Pigatto AM, Pigatto JAT. 2020. Avaliação do endotélio corneano equino (Equus caballus) armazenado no meio de preservação Eusol-C®. Semina: Cienc Agrar 41: 3155-3164. Available from: https://doi.org/10.5433/16790359.2020v41n6Supl2p3155
  » https://doi.org/10.5433/16790359.2020v41n6Supl2p3155
• ¹³ Arndt C, Reese S, Köstlin R. 2001. Preservação de tecido corneoescleral de cães e gatos em Optisol® GS. Vet Ophthalmol 4: 175-182. Available from: https://doi.org/10.1046/j.1463-5216.2001.00146.x
  » https://doi.org/10.1046/j.1463-5216.2001.00146.x
• ¹⁴ Kim SY, Park YH, Lee YC. 2008. Comparação do efeito da moxifloxacina, levofloxacina e cefazolina intracamerais sobre células endoteliais da córnea de coelhos. Clin Exp Ophthalmol 36: 367-370. Available from: https://doi.org/10.1111/j.14429071.2008.01771.x
  » https://doi.org/10.1111/j.14429071.2008.01771.x
• ¹⁵ Kunzmann BC, Hellwinkell OJC, Klameth C, Wenzel D, Bartz-Schmidt KU, Spitzer MS, Schultheiss M. 2017. Estabelecimento de um modelo de cultura organotípica do endotélio corneano suíno para fins de pesquisa. Cell Tissue Banking 19: 269-276. Available from: https://doi.org/10.1007/s10561-017-9669-7
  » https://doi.org/10.1007/s10561-017-9669-7
• ¹⁶ Liou SW, Chiu CJ, Wang IJ. 2004. Efeito da injeção intracameral de lidocaína e carbacol sobre o endotélio corneano de coelhos. J Cataract Refract Surg 30: 1351-1355. Available from: https://doi.org/10.1016/j.jcrs.2003.10.032
  » https://doi.org/10.1016/j.jcrs.2003.10.032
• ¹⁷ Pigatto JAT, Abib FC, Pizzeti JC, Laus JL, Santos JM, Barros PSM. 2005. Análise morfométrica do endotélio corneano de coelhos por microscopia eletrônica de varredura. Acta Sci Vet 33: 41-45. Available from: http://hdl.handle.net/10183/20027
  » http://hdl.handle.net/10183/20027
• ¹⁸ Tamayo-Aranjo LJ, Baraldi-Artoni SN, Laus JL, Vicenti FAM, Pigatto JAT, Abib FC. 2009. Morfologia ultraestrutural e morfometria do endotélio corneano normal de suínos mestiços adultos. Cienc Rural 39: 117-122. Available from: https://doi.org/10.1590/S0103-84782009000100018
  » https://doi.org/10.1590/S0103-84782009000100018
• ¹⁹ Fujita M, Mehra M, Lee SE, Roh DS, Long C, Fundenburgh JL, Ayares DL, Cooper DKC, Hara H. 2013. Comparação da capacidade proliferativa de células endoteliais corneanas geneticamente modificadas de suínos e humanos. Ophthalmic Res 49: 127-138. Available from: https://doi.org/10.1159/000342978
  » https://doi.org/10.1159/000342978
• ²⁰ Brooks DE, Plummer CE, Kallberg ME, Barrie KP, Ollivier FJ, Hendrix DVH, Baker A, Scotty NC, Utter ME, Blackwood SE, Nunnery CM, Ben-Sholmo G, Gelatt KN. 2008. Transplante de córnea para ceratopatias inflamatórias em equinos: Resultado visual em 206 casos (1993-2007). Vet Ophthalmol 11: 123-133. Available from: https://doi.org/10.1111/j.1463-5224.2008.00611.x
  » https://doi.org/10.1111/j.1463-5224.2008.00611.x
• ²¹ Henriksen ML, Plummer CE, Mangan B, Ben-Sholmo G, Tsujita H, Greenberg S, Toft N, Brooks DE. 2012. Resultado visual após transplante de córnea por perfuração corneana e prolapso de íris em 37 cavalos: 1998-2010. Equine Vet J 44: 115-119. Available from: https://doi.org/10.1111/j.2042-3306.2012.00657.x
  » https://doi.org/10.1111/j.2042-3306.2012.00657.x
• ²² Usha B, Kontsevaya S, Lutsay V, Nityaga IM, Lukashina UE. 2019. Ceratoplastia penetrante com córnea de doador preservada na oftalmologia veterinária. J Mol Biol Res 9: 71. Available from: http://dx.doi.org/10.5539/jmbr.v9n1p71
  » http://dx.doi.org/10.5539/jmbr.v9n1p71
• ²³ Kanavi MR, Javadi MA, Chamani T, Fahim P, Javadi F. 2014. Comparação dos índices quantitativos e qualitativos de córneas doadas mantidas em Optisol-GS com aquelas mantidas em Eusol-C. Cell Tissue Bank 16: 243-247. Available from: https://doi.org/10.1007/s10561-014-9466-5
  » https://doi.org/10.1007/s10561-014-9466-5
• ²⁴ Yuksel MB, Uzunel UD, Küsbeci T. 2016. Viabilidade de células endoteliais de córneas doadoras preservadas no meio de armazenamento corneano Eusol-C. Exp Clin Transplant 14: 441-444. Available from: https://doi.org/10.6002/ect.2014.0295
  » https://doi.org/10.6002/ect.2014.0295
• ²⁵ Rodella U, Piaia M, Giurgola L, Gatto C, Tóthová JD. 2025. Modelo ex vivo de córnea e globo ocular bovino para simular condições de armazenamento de córnea humana e cirurgias oculares. Cell Tissue Bank 26: 1-13. Available from: https://doi.org/10.1007/s10561-025-10162-7
  » https://doi.org/10.1007/s10561-025-10162-7
• ²⁶ Wang Q, Liu Y, Gao Y, Zhang M, Yang L, Hu J, Ren Y, Zhou Q, Zhu M. 2024. Comparação da integridade estrutural e qualidade do endotélio corneano armazenado em meio de cultura de órgão versus Eusol-C. Cell Tissue Bank 25: 839-850. Available from: https://doi.org/10.1007/s10561-024-10138-z
  » https://doi.org/10.1007/s10561-024-10138-z
• ²⁷ Gisoldi RAMC, Pocobelli G, Rodella U, Giurgola L, Gatto C, Lodato G, Buffon G, Nucci C, Tóthová JD, Pocobelli A. 2024. Resultados clínicos retrospectivos de ceratoplastia utilizando córneas humanas doadoras preservadas em meio de armazenamento hipotérmico Eusol-C. J Clin Med 13: 1-13. Available from: https://doi.org/10.3390/jcm13247606
  » https://doi.org/10.3390/jcm13247606
• ²⁸ Mena-Linares EE, Vásquez-Maya L, García-Carmona K, Martínez-Mijared MA, Baldivieso-Hurtado R. 2023. Viabilidade de células endoteliais comparando Eusol-C versus Optisol-GS como meio de preservação corneana. Rev Med Hosp Gen Mex 86: 129-134. Available from: http://dx.doi.org/10.24875/HGMX.22000083
  » http://dx.doi.org/10.24875/HGMX.22000083
• ²⁹ Kim DH, Kim J, Jeong HJ, Lee HJ, Kim MK, Wee WR. 2016. Compatibilidade biofísico-funcional da córnea de mini suíno da Universidade Nacional de Seul (SNU) como enxerto xenocorneano para uso em ensaio clínico humano. Xenotransplantation 23: 202-210. Available from: https://doi.org/10.1111/xen.12234
  » https://doi.org/10.1111/xen.12234
• ³⁰ Shi Y, Bikkuzin T, Song Z, Jin X, Jin H, Li X, Zhang H. 2017. Avaliação abrangente da córnea suína descelularizada após transplante clínico. Xenotransplantation 24: 1-7. Available from: https://doi.org/10.1111/xen.12338
  » https://doi.org/10.1111/xen.12338
• ³¹ Heichel J, Wilhelm F, Kunert KS, Hammer T. 2016. Achados topográficos da córnea suína. Med Hypothesis Discov Innov Ophthalmol 5: 125-131. Available from: https://mehdijournal.com/index.php/mehdiophthalmol/article/view/308
  » https://mehdijournal.com/index.php/mehdiophthalmol/article/view/308
• ³² Sanchez I, Martin R, Ussa F, Fernandez-Bueno I. 2011. Os parâmetros do globo ocular suíno. Graefes Arch Clin Exp Ophthalmol 249: 475-482. Available from: https://doi.org/10.1007/s00417-011-1617-9
  » https://doi.org/10.1007/s00417-011-1617-9
• ³³ Chan KY, Cho P, Boost M. 2014. Viabilidade de células epiteliais corneanas em modelo ex vivo de olho suíno. Clin Exp Optom 97: 337-340. Available from: https://doi.org/10.1111/cxo.12128
  » https://doi.org/10.1111/cxo.12128
• ³⁴ Hwang, H, Kim M. 2009. Dano endotelial de uma córnea doadora dependendo do método de inserção do doador durante a ceratoplastia endotelial automatizada com retirada de Descemet em olhos suínos. Jpn J Ophthalmol 53: 523-530. Available from: https://doi.org/10.1007/s10384-009-0713-4
  » https://doi.org/10.1007/s10384-009-0713-4
• ³⁵ Albuquerque L, Freitas LVRP, Pigatto JAT. 2016. Análise do endotélio corneano em olhos de galinhas utilizando microscopia especular de contato. Semina: Ciênc Agrár 36: 4199-4205. Available from: https://doi.org/10.5433/16790359.2015v36n6Supl2p4199
  » https://doi.org/10.5433/16790359.2015v36n6Supl2p4199
• ³⁶ Azevedo MG, Méndez NP, Cargnin LS, Rocha RS, Seibel MP, Silva AF, Pigatto JAT. 2023. Microscopia especular das células endoteliais da córnea de bovinos: um estudo ex vivo. Open Vet J 13: 1554-1561. Available from: https://doi.org/10.5455/ovj.2023.v13.i12.5
  » https://doi.org/10.5455/ovj.2023.v13.i12.5
• ³⁷ Bercht BS, Albuquerque L, Araújo ACP, Pigatto JAT. 2015. Microscopia especular para determinação da morfologia e morfometria das células endoteliais da córnea em chinchilas (Chinchilla lanigera) in vivo. Vet Ophthalmol 48: 137-142. Available from: https://doi.org/10.1111/vop.12236
  » https://doi.org/10.1111/vop.12236
• ³⁸ Vargas EVB, Pigatto AM, Rocha RS, Franceschini MEM, Pigatto JAT. 2023. Microscopia especular das diferentes regiões da córnea em olhos suínos enucleados - avaliação ex vivo. Ciênc Anim Bras 24: 1-6. Available from: https://doi.org/10.1590/18096891v24e-75138E
  » https://doi.org/10.1590/18096891v24e-75138E
• ³⁹ Mistò R, Giurgola L, Pateri F, Limongelli A, Ragazzi E, Tothova JD. 2020. Um novo meio de conservação contendo anfotericina B versus Optisol-GS para preservação de córneas humanas doadoras. Br J Ophthalmol 106: 1-6. Available from: https://doi.org/10.1136/bjophthalmol-2020-317136
  » https://doi.org/10.1136/bjophthalmol-2020-317136
• ⁴⁰ Abramson, JH. 2011. WINPEPI atualizado: programas de computador para epidemiologistas e seu potencial didático. Epidemiol Perspect Innov 8: 1. Available from: https://doi.org/10.1186/1742-5573-8-1
  » https://doi.org/10.1186/1742-5573-8-1
• ⁴¹ Andrew SE, Samuelson DA, Lewis PA, Kubilis PS. 1999. Comparação entre Optisol-GS e solução oftálmica de neomicinapolimixina B-gramicidina para armazenamento de córneas em cães. Vet Ophthalmol 2: 155-161. Available from: https://doi.org/10.1046/j.1463-5224.1999.00070.x
  » https://doi.org/10.1046/j.1463-5224.1999.00070.x
• ⁴² Chaurasia S, Vanathi M. 2021. Microscopia especular na prática clínica. Indian J Ophthalmol 69: 517-524. Available from: https://doi.org/10.4103/ijo.IJO_574_20
  » https://doi.org/10.4103/ijo.IJO_574_20
• ⁴³ Sharma N, Shaikh F, Nagpal R, Manumuraleekrishna, Maharana PK, Agarwal T, Sinha R, Satpathy G, Velpandian T, Titiyal JS. 2021. Avaliação de diversos meios de preservação para armazenamento de córneas doadoras. Indian J Ophthalmol 69: 24522456. Available from: https://doi.org/10.4103/ijo.IJO_258_21
  » https://doi.org/10.4103/ijo.IJO_258_21
• ⁴⁴ Gimenes I, Pintor AVB, Sardinha MS, Marañón-Vázquez GA, Gonzalez MS, Presgrave OCF, Maia LC, Alves GG. 2022. Meios de armazenamento a frio versus Optisol-GS na preservação da qualidade corneana para ceratoplastia: uma revisão sistemática. Appl Sci 12: 1-30. Available from: https://doi.org/10.3390/app12147079
  » https://doi.org/10.3390/app12147079
• ⁴⁵ Javadi MA, Arkbarzadeh AR, Chamani T, Kanavi MR. 2021. Sinasol versus Optisol-GS para preservação a frio da córnea humana: estudo prospectivo ex vivo e clínico. Cell Tissue Bank 22: 563-574. Available from: https://doi.org/10.1007/s10561-02109930-y
  » https://doi.org/10.1007/s10561-02109930-y
• ⁴⁶ Guimarães CB, Albuquerque L, Torikachvili M, Vargas EV, Dall-A’gnol CC, Silva TC, Pigatto JAT. 2019. Efeitos do besilato de atracúrio sobre o endotélio corneano de galinhas: estudo in vitro. Pesq Vet Bras 39: 70-74. Available from: https://doi.org/10.1590/1678-5150-PVB-5595
  » https://doi.org/10.1590/1678-5150-PVB-5595
• ⁴⁷ Park S, Fong A, Cho H, Zhang C, Gritz D, Mian G, Herzlich A, Gore P, Morganti A, Chuck R. 2012. Protocolo para coloração vital de células endoteliais da córnea. Cornea 31: 1476-1479. Available from: https://doi.org/10.1097/ico.0b013e31824d0dda
  » https://doi.org/10.1097/ico.0b013e31824d0dda
• ⁴⁸ Means TL, Geroski DH, Hadley A, Lynn MJ, Edelhauser HF. 1995. Viabilidade do endotélio corneano humano após armazenamento em Optisol-GS. Arch Ophthalmol 113: 805-809. Available from: https://doi.org/10.1001/archopht.1995.01100060131047
  » https://doi.org/10.1001/archopht.1995.01100060131047
• ⁴⁹ Parekh M, Salvalaio G, Ferrari S, Amoureux MC, Albrecht C, Fortier D, Ponzin D. 2014. Um método quantitativo para avaliar a qualidade do tecido corneano doador utilizado em um estudo comparativo entre dois meios de preservação por hipotermia. Cell Tissue Bank 15: 543-554. Available from: https://doi.org/10.1007/s10561-014-9424-2
  » https://doi.org/10.1007/s10561-014-9424-2

Edited by

• Editor:
  Luiz Augusto B. Brito

Publication Dates

• Publication in this collection
  15 Sept 2025
• Date of issue
  2025

History

• Received
  25 June 2024
• Accepted
  05 May 2025
• Published
  31 July 2025