Acute effects of ropivacaine hydrochloride on corneal endothelial cell ultrastructure of horses: ex vivo study

Efeitos agudos do cloridrato de ropivacaína na ultraestrutura das células endoteliais da córnea de equinos: estudo ex vivo

Marcele de Souza Muccillo Alessandra Fernandez da Silva Maiara Poersch Seibel Renata Lima Baptista Marcela Torikachvili Michelle Becker Petersen João Antonio Tadeu Pigatto About the authors

ABSTRACT:

The objective of this study was to evaluate the acute effects of ropivacaine hydrochloride on the corneal endothelium of horses. Forty-eight eyes were obtained from a commercial slaughterhouse and were randomly divided into three groups. In group A, the corneal endothelium was exposed to 0.75% ropivacaine hydrochloride for 60 seconds. In group B, the corneal endothelium was exposed to 0.75% ropivacaine hydrochloride for 15 minutes. In group C, the corneal endothelium was exposed to a balanced saline solution for 60 seconds. Afterwards, all samples were prepared for evaluation with scanning electron microscopy. Random electromicrographs were obtained from each sample. The images were analysed and, with the aid of software, areas with no endothelial cells were measured. The average endothelial loss, expressed as a percentage in relation to the total area, of the samples in group A was 5.28%. The average endothelial loss of samples from group B, expressed as a percentage in relation to the total area, was 20.39%. The damage to the corneal endothelium was significantly greater in group B compared to groups A and C. It was possible to conclude that 0.75% ropivacaine hydrochloride induced acute damage to corneal endothelium cells.

Key words:
equine; intraocular surgery; cornea; intracameral anaesthesia

RESUMO:

Objetivou-se avaliar os efeitos agudos do cloridrato de ropivacaína no endotélio da córnea de equinos. Quarenta e oito olhos de equinos foram divididos aleatoriamente em três grupos. No grupo A o endotélio da córnea foi exposto a cloridrato de ropivacaína a 0,75% por 60 segundos. No grupo B o endotélio da córnea foi exposto a cloridrato de ropivacaína a 0,75% por 15 minutos. No grupo C o endotélio da córnea foi exposto à solução salina balanceada por 60 segundos. As amostras foram preparadas para avaliação com microscopia eletrônica de varredura. Eletromicrografias eletrônicas de varredura foram obtidas aleatoriamente de cada amostra. As imagens foram analisadas e, com o auxílio de um programa para morfometria foram medidas as áreas sem células endoteliais. A perda endotelial média foi expressa em porcentagem em relação à área total das amostras do grupo A foi de 5,28%. A perda endotelial média de amostras do grupo B foi expressa em porcentagem em relação à área total, foi de 20,39%. O dano ao endotélio da córnea foi significativamente maior no grupo B, comparado aos grupos A e C. O cloridrato de ropivacaína a 0,75% induziu dano agudo nas células do endotélio da córnea de equinos.

Palavras-chave:
equinos; cirurgia intraocular; córnea; anestesia intracameral

INTRODUCTION:

A minimum number of endothelial cells is vital for maintaining the transparency of the cornea (PARIKH & EDELHAUSER, 2003PARIKH, C. H.; EDELHAUSER, H.F. Ocular surgical pharmacology: corneal endothelial safety and toxicity. Current Opinion in Ophthalmology, v.14, n.4, p.178-185, 2003. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/12888714/ >. Accessed: May, 08, 2016. doi: 10.1097/00055735-200308000-00002.
https://pubmed.ncbi.nlm.nih.gov/12888714...
). A loss of corneal endothelial cells always occurs during surgical procedures to remove cataracts. Moreover, the corneal endothelium has minimal capacity for mitosis and high cell losses may lead to vision loss. In order to minimise the loss of endothelial cells and avoid corneal decompensation after intraocular surgery, it is important that solutions that are toxic to the corneal endothelium are not used inside the eye (ISHIKAWA, 2002ISHIKAWA, A. Risk factors for reduced corneal endothelial cell density before cataract surgery. Journal of Cataract and Refractive Surgery , v.28, n.11, p.1982-1992, 2002. Available from: <Available from: https://doi.org/10.1016/S0886-3350(02)01502-X >. Accessed: Feb. 06, 2016. doi: 10.1016/S0886-3350(02)01502-X.
https://doi.org/10.1016/S0886-3350(02)01...
). Any substance that may cause toxicity to the corneal endothelium must be carefully evaluated before being routinely used inside the anterior chamber. The use of intracameral anaesthesia has become frequent as an alternative to traditional blocks due to its ease of use and analgesic comfort during and after the procedure in humans (PARK et al. 2010PARK, S. A. et al. Evaluation of the analgesic effect of intracameral lidocaine hydrochloride injection on intraoperative and postoperative pain in healthy dogs undergoing phacoemulsification. American Journal of Veterinary Research, v.71, n.2, p.216-222, 2010. Available from: <Available from: https://doi.org/10.2460/ajvr.71.2.216 >. Accessed: Apr. 18, 2018. doi: 10.2460/ajvr.71.2.216.
https://doi.org/10.2460/ajvr.71.2.216...
; WANG, 2013WANG, L. et al. Combined topical-intracameral anesthesia in manual small-incision cataract surgery: A Prospective, Randomized, Double-Masked, Placebo-Controlled Trial. Asia-Pacific Journal of Ophthalmology, v.1, p.9-14, 2013. Available from: <Available from: https://journals.lww.com/apjoo/fulltext/2013/ >. Accessed: Sept. 26, 2016. doi: 10.1097/APO.0b013e318274c335.
https://journals.lww.com/apjoo/fulltext/...
). VÄ LIMÄKI and TÖRNBLOM compared viscoanaesthesia and intracameral anaesthesia with 1% lidocaine in patients undergoing cataract surgery. Patients who are given viscoanaesthesia may be at increased risk for postoperative corneal oedema (VÄLIMÄKI & TÖRNBLOM, 2009VÄLIMÄKI, J., TÖRNBLOM, R. Viscoanaesthesia in cataract surgery: a prospective, randomized clinical trial. Acta Ophthalmologica, v.87, n.4, p.378-381, 2009. Available from: <Available from: https://doi.org/10.1111/j.1755-3768.2008.01267.x >. Accessed: Jul. 27, 2017. doi: 10.1111/j.1755-3768.2008.01267.x.
https://doi.org/10.1111/j.1755-3768.2008...
). Some studies have already been developed to analyse the effects of intraocular injection of lidocaine on the corneal endothelium. Effect of intracameral ropivacaine on corneal endothelium was studied (ÇAÇA et al, 2006). Cataracts are an important cause of blindness in horses and, at present, the only form of treatment is surgical removal of the opaque lens (EDELMANN et al, 2014EDELMANN, M. L. et al. Retrospective evaluation of phacoemulsification and aspiration in 41 horses (46 eyes): visual outcomes vs. age, intraocular lens, and uveitis status. Veterinary Ophthalmology , v.17, n.1, p.160-167, 2014. Available from: <Available from: https://doi.org/10.1111/vop.12185 >. Accessed: Sept. 10, 2015. doi: 10.1111/vop.12185.
https://doi.org/10.1111/vop.12185...
; BROOKS et al. 2014BROOKS, D. E. et al. Visual outcomes of phacoemulsification cataract surgery in horses: 1990-2013. Veterinary Ophthalmology, v.17, n.1, p.117-128, 2014. Available from: <Available from: https://doi.org/10.1111/vop.12168 >. Accessed: Sept. 21, 2015. doi: 10.1111/vop.12168.
https://doi.org/10.1111/vop.12168...
; TOWNSEND, 2017TOWNSEND, W. M. Disease and surgery of the equine lens. Veterinary Clinics of North America: Equine Practice, v.33, n.3, p.483-497, 2017. Available from: <Available from: http://dx.doi.org/10.1016/j.cveq.2017.07.004 >. Accessed: Apr. 18, 2018. doi: 10.1016/j.cveq.2017.07.004.
http://dx.doi.org/10.1016/j.cveq.2017.07...
). To our knowledge, there are no studies evaluating the toxicity of intracameral local anaesthetics on the corneal endothelium of horses.

The aim of this study was to evaluate the acute ex vivo effects of ropivacaine hydrochloride in the ultrastructure of the corneal endothelial cells of horses.

MATERIALS AND METHODS:

Forty-eight corneas from 24 horses, male or female, of different ages were studied. The eyes were obtained from a licensed Brazilian commercial slaughterhouse Foresta, São Gabriel, RS, Brazil. The experiment was carried out according to the standards of the Association for Research in Vision and Ophthalmology (ARVO). Immediately after slaughter, all eyes were examined. With slit-lamp biomicroscopy (Portable Slit Lamp SL 15, Kowa, Japan) the ocular surface, anterior chamber, and lens were examined. Corneal stain with fluorescein (Fluorescein, Allergan, SP, Brazil) was made. Eye bulbs that showed evidence of eye disease were excluded. Immediately after the eye exam, enucleation was performed, and the eye bulbs were kept in a humid chamber until the corneas were collected. The corneoscleral buttons were removed with a scalpel and scissors. Corneas were then excised with an eight mm diameter trephine. All corneas were analyzed within 4 hours of death. Corneas were randomly divided into three groups.

In group A (12 corneas), with a dropper 0.2 ml of 0.75% atracurium besylate (Cristália, São Paulo, Brazil) was dripped onto the endothelium. Three minutes after the sample was rinsed with balanced salt solution (BSS) (Halex Istar, ophthalmic solution, GO, Brazil) to remove the atracurium besylate. In group B (12 corneas), with a dropper 0.2 ml of 0.75% atracurium besylate was dripped onto the endothelium. Fiftheen minutes after the sample was rinsed with BSS to remove the atracurium besylate. In group C (24 corneas), with a dropper 0.2 ml of BSS was dripped onto the endothelium. All samples were kept in 2.5% glutaraldehyde, in 0.1M sodium cacodylate buffer and at pH 7.4 for 24 hours. The corneas were removed from the glutaraldehyde solution, washed in sodium cacodylate buffer solution and dehydrated in ascending concentrations of acetone (30, 50, 70, 80, 90 for 10 minutes at each concentration and for 20 min at 90% again). Afterwards the samples were left for 30 min in 100% acetone and were subjected to drying at a critical point with liquid carbon dioxide. The samples were fixed in stubs with adhesive tape and metallized with gold-palladium. The posterior endothelial surfaces were examined with a scanning electron microscope (JSM 6060, JEOL, Tokyo, Japan) operating at 15 kV. From each sample, five electron micrographs were obtained with 950x magnifications. Occasionally, images with a 30x magnification were obtained to have a panoramic view of each sample. The areas devoid of endothelial cells were calculated. The morphometric study was performed using ImageJ software (ImageJ 1.51k), which allowed calculation of the percentage of cell loss use surrounding areas with no endothelial cells. All analyses were performed by the same examiner. The distribution of variables was assessed using the Shapiro-Wilk test, which indicated a non-normal distribution. The Friedman test was used to compare the sum of areas expressed as a percentage of the total between treatments. The Wilcoxon test was used to detect the differences and its P value was corrected by the Bonferroni test, due to the multiple comparisons made. Differences were considered statistically significant when P ≤ 0.05.

RESULTS:

With scanning electron microscopy (SEM), it was possible to observe, analyse and obtain images of the corneal endothelium in all samples analysed. In the samples of the control group, there were no areas with endothelial damage and the regular pattern of endothelial cells was observed in all samples (Figure 1). Endothelial losses were observed both in group A and group B samples (Figure 2). The average endothelial loss, expressed as a percentage in relation to the total area, of the samples in group A was 5.28±2.49%. There was a statistically significant difference significant among all compared groups. The average endothelial loss from samples from group B, expressed as a percentage in relation to the total area, was 20.39 ± 10.46%. The values found in the sum of the areas expressed as a percentage of cell loss were higher in group B when compared with group A (P = 0.006) and group C (P = 0.003). There was also a significant difference between the values found in A in relation to C (P = 0.005).

Figure 1
Scanning electromicrograph of the corneal endothelium of horses of control group. Polygonal cells and regular corneal endothelial pattern and absence of endothelial damage are observed. Magnification:X950. Bar:20µm.

Figure 2
Scanning electromicrograph of the corneal endothelium of horses of group B. Cell loss and Descemet’s membrane exposure are observed. Magnification:X950. Bar:20µm.

DISCUSSION:

A minimum endothelial density is essential for the cornea to maintain its transparency. In addition to the surgical procedure, numerous other factors, including intraocular anaesthetics and intraocular dyes, can cause endothelial damage. These factors can compromise the function of the endothelium, mainly because in most of the studied species the mitotic activity is limited in this layer (NAUTSCHER et al, 2015NAUTSCHER, N. et al. Comparative morphological evaluation of domestic animal cornea. Veterinary Ophthalmology , v.19, n.4, p.297-304, 2015. Available from: <Available from: https://doi.org/10.1111/vop.12298 >. Accessed: May, 08, 2016. doi: 10.1111/vop.12298.
https://doi.org/10.1111/vop.12298...
). To avoid endothelial decompensation, it is important to choose substances that do not damage the corneal endothelium (KHABAK et al. 2006ÇAÇA, I. et al. The histopathological effect of intracameral ropivacaine in different concentrations on corneal endothelium. Annals of Ophthalmology, v.8, n.1, p.43-48, 2006. Available from: <Available from: https://doi.org/10.1385/AO:38:1:43 >. Accessed: Feb. 06, 2016. doi: 10.1385/AO:38:1:43.
https://doi.org/10.1385/AO:38:1:43...
; BORAZAN et al. 2009BORAZAN, M. et al. Induction of apoptosis of rabbit corneal endothelial cells by preservative-free lidocaine hydrochloride 2%, ropivacaine 1%, or levobupivacaine 0.75 Journal of Cataract and Refractive Surgery, v.35, n.4, p.753-758, 2009. Available from: <Available from: https://doi.org/10.1016/j.jcrs.2008.12.016 >. Accessed: Jan. 20, 2017. doi: 10.1016/j.jcrs.2008.12.016.
https://doi.org/10.1016/j.jcrs.2008.12.0...
; LEE et al, 2016LEE, R. M. H. et al. Severe adverse events associated with local anaesthesia in cataract surgery: 1 year national survey of practice and complications in the UK. British Journal of Ophthalmology, v.100, n.6, p.772-776, 2016. Available from: <Available from: http://dx.doi.org/10.1136/bjophthalmol-2015-307060 >. Accessed: Jun. 17, 2018. doi: 10.1136/bjophthalmol-2015-307060.
http://dx.doi.org/10.1136/bjophthalmol-2...
). The loss of a large number of endothelial cells can cause irreversible loss of corneal transparency (PARIKH & EDELHAUSER, 2003PARIKH, C. H.; EDELHAUSER, H.F. Ocular surgical pharmacology: corneal endothelial safety and toxicity. Current Opinion in Ophthalmology, v.14, n.4, p.178-185, 2003. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/12888714/ >. Accessed: May, 08, 2016. doi: 10.1097/00055735-200308000-00002.
https://pubmed.ncbi.nlm.nih.gov/12888714...
). In this sense, studies on the toxicity of drugs in the corneal endothelium are extremely important. Intracameral anaesthesia have been popularised as new techniques for use in cataract surgery in humans. These anaesthetic routes have potential safety advantages over traditional techniques such as retrobulbar and peribulbar anaesthesia (OLMEZ et al, 2004OLMEZ, G. et al. Intraocular pressure and quality of blockade in peribulbar anesthesia using ropivacaine or lidocaine with adrenaline: a double-blind randomized study. The Tohoku Journal of Experimental Medicine, v.204, n.3, p.203-208, 2004. Available from: <Available from: https://doi.org/10.1620/tjem.204.203 >. Accessed: Mar. 21, 2015. doi: 10.1620/tjem.204.203.
https://doi.org/10.1620/tjem.204.203...
). Regarding intracameral anaesthesia in humans and other animal species, studies were carried out evaluating the toxicity of the corneal endothelium and the comfort of patients (TAN & BURTON, 2000TAN, J. H. Y., BURTON, R.L. Does preservative-free lignocaine 1% for hydrossection reduce pain during phacoemulsification? Journal of Cataract and Refractive Surgery , v.26, n.5, p.733-735, 2000. Available from: <Available from: https://doi.org/10.1016/S0886-3350(00)00311-4 > Accessed: Sept. 26, 2016. doi: 10.1016/S0886-3350(00)00311-4.
https://doi.org/10.1016/S0886-3350(00)00...
; BORAZAN et al. 2009; PARK et al. 2010PARK, S. A. et al. Evaluation of the analgesic effect of intracameral lidocaine hydrochloride injection on intraoperative and postoperative pain in healthy dogs undergoing phacoemulsification. American Journal of Veterinary Research, v.71, n.2, p.216-222, 2010. Available from: <Available from: https://doi.org/10.2460/ajvr.71.2.216 >. Accessed: Apr. 18, 2018. doi: 10.2460/ajvr.71.2.216.
https://doi.org/10.2460/ajvr.71.2.216...
). However, there is a lack of research in regard to equine species and intracameral anaesthesia. This, and the importance of the theme, helped to motivate this study. In addition, cataracts are an important cause of blindness in horses and, at present, the only form of treatment is surgical removal of the opaque lens (BROOKS et al, 2014BROOKS, D. E. et al. Visual outcomes of phacoemulsification cataract surgery in horses: 1990-2013. Veterinary Ophthalmology, v.17, n.1, p.117-128, 2014. Available from: <Available from: https://doi.org/10.1111/vop.12168 >. Accessed: Sept. 21, 2015. doi: 10.1111/vop.12168.
https://doi.org/10.1111/vop.12168...
; EDELMANN et al, 2014EDELMANN, M. L. et al. Retrospective evaluation of phacoemulsification and aspiration in 41 horses (46 eyes): visual outcomes vs. age, intraocular lens, and uveitis status. Veterinary Ophthalmology , v.17, n.1, p.160-167, 2014. Available from: <Available from: https://doi.org/10.1111/vop.12185 >. Accessed: Sept. 10, 2015. doi: 10.1111/vop.12185.
https://doi.org/10.1111/vop.12185...
; TOWNSEND, 2017TOWNSEND, W. M. Disease and surgery of the equine lens. Veterinary Clinics of North America: Equine Practice, v.33, n.3, p.483-497, 2017. Available from: <Available from: http://dx.doi.org/10.1016/j.cveq.2017.07.004 >. Accessed: Apr. 18, 2018. doi: 10.1016/j.cveq.2017.07.004.
http://dx.doi.org/10.1016/j.cveq.2017.07...
). In the present research, an ex vivo study was chosen. Normally, research related to the toxicity of intraocular drugs using live animals is carried out on laboratory animals. Previous studies carried out on animal eyes determined that within six hours after death, the endothelium can be analysed without structural changes occurring in this layer (SIT et al, 2001SIT, M. et al. Corneal graft outcome study. Cornea, v.20, n.2, p.129-133, 2001. Available from: <Available from: https://doi.org/10.1097/00003226-200103000-00002 >. Accessed: Jul. 27, 2017. doi: 10.1097/00003226-200103000-00002.
https://doi.org/10.1097/00003226-2001030...
; PIGATTO et al, 2005PIGATTO, J. A. T. et al. Morphometric analysis of the corneal endothelium of rabbits using scanning electron microscopy, Acta Scientiae Veterinariae, v.33, n.1, p.41-45, 2005. Available from: <Available from: https://doi.org/10.22456/1679-9216.14441 >. Accessed: May, 03, 2018. doi: 10.22456/1679-9216.14441.
https://doi.org/10.22456/1679-9216.14441...
; PIGATTO et al, 2009; FAGANELLO et al, 2016FAGANELLO, C. S. et al. Morphology of endothelial cells from different regions of the equine cornea. Ciência Rural, v.45, n.12, p.2223-2228, 2016. Available from: <Available from: https://doi.org/10.1590/0103-8478cr20160216 >. Accessed: Jun. 17, 2018. doi: 10.1590/0103-8478cr20160216.
https://doi.org/10.1590/0103-8478cr20160...
). The use of slaughtered animal eyes for endothelial analysis has increasingly been shown to be an alternative to the use of live animals to assess the toxicity of intraocular drugs (PESCOSOLIDO et al, 2011; WEN et al, 2015WEN, Q. et al. Cytotoxicity of proparacaine to human corneal endothelial cells in vitro. The Journal of Toxicological Sciences, v.40, n.4, p.427-436, 2015. Available from: <Available from: https://doi.org/10.2131/jts.40.427 >. Accessed: Apr. 18, 2018. doi: 10.2131/jts.40.427.
https://doi.org/10.2131/jts.40.427...
; TERZARIOL et al, 2016TERZARIOL, M. et al. Effects of intracameral brilliant blue on the corneal endothelium of swine: in vitro study. Pesquisa Veterinária Brasileira, v.36, n.8, p.775-780, 2016. Available from: <Available from: http://dx.doi.org/10.1590/S0100-736X2016000800016 >. Accessed: Nov. 11, 2017. doi: 10.1590/S0100-736X2016000800016.
http://dx.doi.org/10.1590/S0100-736X2016...
; SILVA et al, 2018SILVA, V. R. M. et al. Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study. Semina: Ciências Agrárias, v.39, n.2, p.613-620, 2018. Available from: <Available from: https://doi.org/10.5433/1679-0359.2018v39n2p613 >. Accessed: Dec. 03, 2018. doi: 10.5433/1679-0359.2018v39n2p613.
https://doi.org/10.5433/1679-0359.2018v3...
; JIANG et al, 2018JIANG, G.; FAN T. Sodium ferulate attenuates lidocaine-induced corneal endothelial impairment. Oxidative Medicine and Cellular Longevity, v.8, n.1, p.1-8, 2018. Available from: <Available from: https://doi.org/10.1155/2018/4967318 >. Accessed: Oct. 24, 2018. doi: 10.1155/2018/4967318.
https://doi.org/10.1155/2018/4967318...
).

Corneal endothelial toxicity is related to substances that come into contact with the endothelium, based on their chemical compositions, pH and osmolarities (PARIKH & EDELHAUSER, 2003PARIKH, C. H.; EDELHAUSER, H.F. Ocular surgical pharmacology: corneal endothelial safety and toxicity. Current Opinion in Ophthalmology, v.14, n.4, p.178-185, 2003. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/12888714/ >. Accessed: May, 08, 2016. doi: 10.1097/00055735-200308000-00002.
https://pubmed.ncbi.nlm.nih.gov/12888714...
). The final preparation of ropivacaine for clinical use is presented with a pH ranging between 4.0 and 6.0 (RAMOS et al, 2000RAMOS, G. et al. Alkalization of 0.75% ropivacaine for epidural block. Revista Brasileira de Anestesiologia , v.50, n.6, p.442-449, 2000. Available from: <Available from: https://bjan-sba.org/article/5e498c3d0aec5119028b49e7/pdf/rba-50-6-442.pdf >. Accessed: Jul. 27, 2017.
https://bjan-sba.org/article/5e498c3d0ae...
). Analysis of the corneal thickness data and interpretation of the scanning electron micrograph reveals that outside of the pH range of 6.5 to 8.5, structural and functional alterations occur (GONNERING et al, 1979GONNERING, R. et al. The pH tolerance of rabbit and human corneal endothelium. Investigative Ophthalmology & Visual Science, v.18, p.373-390, 1979. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/34576/ >. Accessed: Oct. 24, 2018.
https://pubmed.ncbi.nlm.nih.gov/34576/...
). Endothelial losses were observed both in group A and group B samples. In the present study, ropivacaine was kept in direct contact with the endothelium and direct contact with the corneal endothelium in order to verify whether it would induce endothelial damage. In previous studies this methodology has already been used with excellent results (ÇAÇA et al, 2006ÇAÇA, I. et al. The histopathological effect of intracameral ropivacaine in different concentrations on corneal endothelium. Annals of Ophthalmology, v.8, n.1, p.43-48, 2006. Available from: <Available from: https://doi.org/10.1385/AO:38:1:43 >. Accessed: Feb. 06, 2016. doi: 10.1385/AO:38:1:43.
https://doi.org/10.1385/AO:38:1:43...
; KHABAK et al, 2006; PESCOSOLIDO et al. 2011; WEN et al, 2015WEN, Q. et al. Cytotoxicity of proparacaine to human corneal endothelial cells in vitro. The Journal of Toxicological Sciences, v.40, n.4, p.427-436, 2015. Available from: <Available from: https://doi.org/10.2131/jts.40.427 >. Accessed: Apr. 18, 2018. doi: 10.2131/jts.40.427.
https://doi.org/10.2131/jts.40.427...
; TERZARIOL et al, 2016TERZARIOL, M. et al. Effects of intracameral brilliant blue on the corneal endothelium of swine: in vitro study. Pesquisa Veterinária Brasileira, v.36, n.8, p.775-780, 2016. Available from: <Available from: http://dx.doi.org/10.1590/S0100-736X2016000800016 >. Accessed: Nov. 11, 2017. doi: 10.1590/S0100-736X2016000800016.
http://dx.doi.org/10.1590/S0100-736X2016...
; SILVA et al, 2018SILVA, V. R. M. et al. Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study. Semina: Ciências Agrárias, v.39, n.2, p.613-620, 2018. Available from: <Available from: https://doi.org/10.5433/1679-0359.2018v39n2p613 >. Accessed: Dec. 03, 2018. doi: 10.5433/1679-0359.2018v39n2p613.
https://doi.org/10.5433/1679-0359.2018v3...
). In other studies, however, an intracameral injection of the substance was performed, which was analysed (BORAAN et al, 2009). Thus, dilution of the substance in aqueous humour could minimise its toxic effects on the endothelium. In the present study, as we were unaware of the possible damage of the tested substance, corneal trepanation and direct exposure of the anaesthetic to the corneal endothelium were chosen. In addition, with the intracameral injection of the tested substance it would be difficult to have a homogeneous direct contact between ropivacaine and the corneal endothelium. In the present study, ropivacaine directly contacted with the endothelium across the entire area that was later analysed. Ropivacaine is a local anaesthetic of the amide type with a long duration of action, and its effect occurs through a reversible inhibition of the influx of sodium ions in the nerve fibres. It has a high degree of sensory motor differentiation, which can be useful when motor block is undesirable (KUTHIALA & CHAUDHARY, 2011KUTHIALA, G.; CHAUDHARY, G. Ropivacaine: A review of its pharmacology and clinical use. Indian Journal of Anaesthesia, v.55, n.2, p.104-110, 2011. Available from: <Available from: https://doi.org/10.4103/0019-5049.79875 > Accessed: Aug. 29, 2016. doi: 10.4103/0019-5049.79875.
https://doi.org/10.4103/0019-5049.79875...
). Commercially available ropivacaine does not contain preservatives in its formulation. At this concentration, this anaesthetic is widely used in blocking ophthalmology because it promotes analgesia in some patients for up to 12 hours, being superior to lidocaine (KUTHIALA & CHAUDHARY, 2011). In previous studies in humans, 1% ropivacaine was more effective in analgesia and did not promote significant endothelial cell loss when compared to 2% lidocaine (MARTINI et al, 2002MARTINI, E. et al. Lidocaine versus ropivacaine for topical anesthesia in cataract surgery. Journal of Cataract and Refractive Surgery , v.28, n.6, p.1018-1022, 2002. Available from: <Available from: https://doi.org/10.1016/S0886-3350(01)01225-1 >. Accessed: Jan. 20, 2017. doi: 10.1016/S0886-3350(01)01225-1.
https://doi.org/10.1016/S0886-3350(01)01...
; IACOBELLI et al. 2005IACOBELLI, L. et al. Topical use of ropivacaine 1% vs lidocaine 2% in cataract surgery. Investigative Ophthalmology & Visual Science , v.46, n.13, p.795, 2005. Available from: <Available from: https://iovs.arvojournals.org/article.aspx?articleid=2400531 >. Accessed: Sept. 10, 2015.
https://iovs.arvojournals.org/article.as...
).

In studies carried out on other animal species, ropivacaine in concentrations between 0.1% and 0.5% was demonstrated to be safe and provide pain relief, but at concentrations of 1%, ropivacaine induced lesions in the corneal endothelium (KLAMT et al, 2003KLAMT, J. G. et al. Continuous epidural anesthesia with 0.2% ropivacaine associated to general anesthesia for upper abdominal surgery in children. Revista Brasileira de Anestesiologia, v.53, n.2, p.160-168, 2003. Available from: <Available from: https://doi.org/10.1590/S0034-70942003000200003 >. Accessed: Jan. 20, 2017. doi: 10.1590/S0034-70942003000200003.
https://doi.org/10.1590/S0034-7094200300...
; CACA et al, 2006ÇAÇA, I. et al. The histopathological effect of intracameral ropivacaine in different concentrations on corneal endothelium. Annals of Ophthalmology, v.8, n.1, p.43-48, 2006. Available from: <Available from: https://doi.org/10.1385/AO:38:1:43 >. Accessed: Feb. 06, 2016. doi: 10.1385/AO:38:1:43.
https://doi.org/10.1385/AO:38:1:43...
; BORAZAN et al, 2009BORAZAN, M. et al. Induction of apoptosis of rabbit corneal endothelial cells by preservative-free lidocaine hydrochloride 2%, ropivacaine 1%, or levobupivacaine 0.75 Journal of Cataract and Refractive Surgery, v.35, n.4, p.753-758, 2009. Available from: <Available from: https://doi.org/10.1016/j.jcrs.2008.12.016 >. Accessed: Jan. 20, 2017. doi: 10.1016/j.jcrs.2008.12.016.
https://doi.org/10.1016/j.jcrs.2008.12.0...
). In one study only, 0.75% ropivacaine was observed to induce toxicity in the corneal endothelium of rabbits (KHAZBAK et al, 2006KHAZBAK, L. et al. Effect of intracameral injection of lidocaine and ropivacaine on rabbit corneal endothelium and trabecular meshwork. Bulletin of the Ophthalmological Society of Egypt, v.99, n.1, p.29-33, 2006. Available from: <Available from: https://www.researchgate.net/publication/293654100 >. Accessed: Apr. 5, 2017.
https://www.researchgate.net/publication...
). Among the techniques normally used to prove the toxicity of drugs in the corneal endothelium, optical microscopy associated with alizarin red and SEM stand out (ÇAÇA et al, 2006; SCHELINNI et al, 2007; SEGARRA et al, 2018SEGARRA, S. et al. A dose-escalation ex vivo study on the effects of intracameral benzalkonium chloride in rabbits. BMC Veterinary Research, v.14, n.39, p.1-9, 2018. Available from: <Available from: https://doi.org/10.1186/s12917-018-1349-8 >. Accessed: Sept. 03, 2018. doi: 10.1186/s12917-018-1349-8.
https://doi.org/10.1186/s12917-018-1349-...
; SILVA et al, 2018SILVA, V. R. M. et al. Evaluation of equine corneal endothelium after exposure to 0.5% indocyanine green - in vitro study. Semina: Ciências Agrárias, v.39, n.2, p.613-620, 2018. Available from: <Available from: https://doi.org/10.5433/1679-0359.2018v39n2p613 >. Accessed: Dec. 03, 2018. doi: 10.5433/1679-0359.2018v39n2p613.
https://doi.org/10.5433/1679-0359.2018v3...
). The use of optical microscopy after staining the endothelium with vital dyes has been shown to be a simple, fast and practical way to detect cell damage as well as to analyse the shape of endothelial cells (FAGANELLO et al, 2016FAGANELLO, C. S. et al. Morphology of endothelial cells from different regions of the equine cornea. Ciência Rural, v.45, n.12, p.2223-2228, 2016. Available from: <Available from: https://doi.org/10.1590/0103-8478cr20160216 >. Accessed: Jun. 17, 2018. doi: 10.1590/0103-8478cr20160216.
https://doi.org/10.1590/0103-8478cr20160...
). In the present study, SEM was chosen because it has already been widely used in studies related to corneal morphology, the toxicity of intracameral drugs, the effectiveness of means of corneal preservation and in the evaluation of the endothelium ultrastructure of different species (OJEDA et al, 2001OJEDA, J. L. et al. The three-dimentional microanatomy of the rabbit and human cornea. A chemical and mechanical microdissection-SEM approach. Journal of Anatomy, v.199, n.5, p.567-576, 2001. Available from: <Available from: https://doi.org/10.1017/S0021878201008512 >. Accessed: Oct. 24, 2018. doi: 10.1017/S0021878201008512.
https://doi.org/10.1017/S002187820100851...
; PIGATTO et al, 2009PIGATTO, J. A. T. et al. Scanning electron microscopy of the corneal endothelium of ostrich. Ciência Rural , v.39, n.3, p.926-929, 2009. Available from: <Available from: https://doi.org/10.1590/S0103-84782009005000001 >. Accessed: Nov. 11, 2017. doi: 10.1590/S0103-84782009005000001.
https://doi.org/10.1590/S0103-8478200900...
; TERZARIOL et al, 2016TERZARIOL, M. et al. Effects of intracameral brilliant blue on the corneal endothelium of swine: in vitro study. Pesquisa Veterinária Brasileira, v.36, n.8, p.775-780, 2016. Available from: <Available from: http://dx.doi.org/10.1590/S0100-736X2016000800016 >. Accessed: Nov. 11, 2017. doi: 10.1590/S0100-736X2016000800016.
http://dx.doi.org/10.1590/S0100-736X2016...
). In the present study, the methodology employed proved to be feasible, allowing images to be obtained, and an analysis and quantification of endothelial cell losses. With SEM, due to the large increase in images, it is possible to delimit the edges of areas with cell loss, thus allowing establishment of the percentage of endothelial loss. It was possible to visualise the acute toxic effects caused by direct exposure of ropivacaine hydrochloride to the corneal endothelium. Similar lesions in the corneal endothelium have been documented in other studies using local intraocular anaesthetics (EGGELING et al, 2000EGGELING, P. et al. Corneal endothelial toxicity of different lidocaine concentrations. Journal of Cataract and Refractive Surgery , v.26, n.9, p.1403-1408, 2000. Available from: <Available from: https://doi.org/10.1016/S0886-3350(00)00379-5 >. Accessed: Oct. 24, 2018. doi: 10.1016/S0886-3350(00)00379-5.
https://doi.org/10.1016/S0886-3350(00)00...
; TAN & BURTON 2000TAN, J. H. Y., BURTON, R.L. Does preservative-free lignocaine 1% for hydrossection reduce pain during phacoemulsification? Journal of Cataract and Refractive Surgery , v.26, n.5, p.733-735, 2000. Available from: <Available from: https://doi.org/10.1016/S0886-3350(00)00311-4 > Accessed: Sept. 26, 2016. doi: 10.1016/S0886-3350(00)00311-4.
https://doi.org/10.1016/S0886-3350(00)00...
; KHABAK et al, 2006). In humans, the most commonly used anaesthetic within the eye is 1% lidocaine as it has less toxicity compared to other anaesthetics and concentrations (LIOU et al. 2004LIOU, S. W. et al. Effect of intracameral injection of lidocaine and carbachol on the rabbit corneal endothelium. Journal of Cataract and Refractive Surgery , v.30, n.6, p.1351-1355, 2004. Available from: <Available from: https://doi.org/10.1016/j.jcrs.2003.10.032 >. Accessed: Sept, 21, 2015. doi: 10.1016/j.jcrs.2003.10.032.
https://doi.org/10.1016/j.jcrs.2003.10.0...
; BORAZAN et al, 2009; LEE et al, 2016LEE, R. M. H. et al. Severe adverse events associated with local anaesthesia in cataract surgery: 1 year national survey of practice and complications in the UK. British Journal of Ophthalmology, v.100, n.6, p.772-776, 2016. Available from: <Available from: http://dx.doi.org/10.1136/bjophthalmol-2015-307060 >. Accessed: Jun. 17, 2018. doi: 10.1136/bjophthalmol-2015-307060.
http://dx.doi.org/10.1136/bjophthalmol-2...
).

CONCLUSION:

According to the conditions proposed for this study, it was possible to conclude that ropivacaine 0.75% caused acute damage to the corneal endothelium of horses.

ACKNOWLEDGEMENTS

The authors would like to thank the Foresta slaughterhouse for supplying the eyes used, and the Electron Microscopy Centre for support in the realization of this research. In addition, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) financed the research grants.

REFERENCES

  • CR-2020-0763.R1

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

  • BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

    This research was approved by the Research Committee of the xxx, and followed the ethical norms of the Association for Research in Vision and Ophthalmology (ARVO).

Publication Dates

  • Publication in this collection
    14 June 2021
  • Date of issue
    2021

History

  • Received
    13 Aug 2020
  • Accepted
    03 Feb 2021
  • Reviewed
    08 Apr 2021
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