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Abstract

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Melanomas are the most common cancer of the eye in canines, felines and humans. The treatment approaches vary, since no gold standard exists. Therefore, this systematic review aimed to compare the treatment modalities in ocular melanoma in rabbits. Medline/PubMed, Cochrane Library, Web of Science and Embase were searched for articles published until 21 April 2021 in English, Portuguese or Spanish, reporting animal studies evaluating photodynamic therapy (PDT), laser, radiotherapy or surgical excision. Twenty-seven articles were included for the qualitative synthesis, with publication dates from 1970 to 2018. Of the selected studies, 19 used PDT, six used radiotherapy and two used laser as treatment. No studies regarding surgical therapy that met the inclusion criteria were obtained. The tumour therapy results were evaluated in a heterogeneous manner for different periods and various methods, including microscopy, angiographic, histological examination, fundoscopy, ultrasound exam and electroretinogram. The treatment modalities analysed successfully treated the ocular melanoma, with tumour necrosis being commonly observed. Despite the therapeutic efficacy shown, side effects have been reported for all the therapies. The studies showed high heterogeneity, and therefore, in the future, new studies should be carried out to increase knowledge about ocular melanoma treatment. The analysed therapies can be used successfully in the treatment of ocular melanoma, with more conservative options such as PDT presenting great potential.

Keywords

Eye lasers melanoma microsurgery photochemotherapy radiotherapy

Introduction

Melanomas are both the most common ocular neoplasia of melanocytic origin and the most common malignant primary intraocular tumours in canine,^1,2 feline^2,3 and human species.⁴ Although the vast majority of ocular melanomas originate from the uvea, they may also occur on the eyelids, on the conjunctival surface, along the limbus or within the uveal tract.^1,5 These tumours often show unclear limits regarding surrounding tissues, with frequent destruction of the implantation site⁶ and metastatic capacity,^5,7 with a significant impact on vision, comfort and longevity of companion animals.²

Diagnosis is usually made on routine eye examination due to unspecific clinical manifestations.⁸ The treatment options for ocular melanocytic neoplasia are determined by the species affected, tumour location, rate of progression and extent, as well as by the expertise and facilities available to the veterinary clinician.^5,9,10 Treatment can be performed with surgery, lasers, photodynamic therapy (PDT), radiotherapy, alone or combined regimens. Chemotherapy can be used in the case of disseminated disease. Importantly, there is no gold-standard therapy, and ocular melanomas present a varied rate of response to treatment.⁸

Besides its importance in veterinary medicine, eye melanoma treatments in animals are often used as models for human therapies. Animal models used in ocular melanoma research include mice, rats, rabbits, chick embryos, drosophila and zebrafish.^11,12 With a key advantage over the other species employed, rabbits have larger eyes, making them favourable for the procedures and ophthalmic exams.¹¹ These models play an important role in cancer research, assisting in the understanding of neoplastic behaviour in vivo, as well as possible therapies,^11,12 able to demonstrate efficacy and safety before clinical trials.¹²

Since no gold-standard therapy exists in the treatment of eye melanoma in animals, evaluating the available therapies is of the utmost importance for veterinary guidance and to support future human research.⁹ This work aimed to perform a systematic review of the literature with a qualitative comparison of the results to answer the following problem, intervention, comparison and outcome (PICO) question: ‘Which eye melanoma therapy is most effective in rabbits?’ (Table 1).

Table 1.

Problem, intervention, comparison and outcome (PICO) question.

‘Which eye melanoma therapy is most effective in rabbits?’
Population/problem	Rabbits with eye melanoma
Intervention	Photodynamic therapy, laser therapy, radiotherapy, surgical excision
Comparison	The most effective
Outcome	Tumour regression, lifespan without tumour, incidence of ocular and nervous lesions

Methods

This systematic review report followed the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines.¹³ The PRISMA checklist is available as Supplemental Material. The review protocol is registered in Prospective International Registry of Systematic Reviews (PROSPERO) under number CRD42019146667.

Search strategy

The following databases were searched for relevant studies: MEDLINE (through PubMed), Embase, Cochrane Library and Web of Science. The initial search was conducted on 14 June 2020, and the last search was completed on 19 April 2021. The search strategy for the different databases is shown in Table 2. For all databases, the following filter was applied: articles written in English, Portuguese or Spanish. No temporal limits were applied. Two authors (T.G. and C.M.M.) independently evaluated the studies by title and abstract and selected those that met the inclusion criteria. A third reviewer (M.L.) was consulted wherever there were doubts about a study’s eligibility, and a decision was reached by consensus.

Table 2.

Search strategy in databases.

PubMed/MEDLINE	“Melanoma” [Mesh] AND (“Photochemotherapy” [Mesh] OR “photodynamic therapy” OR “therapy, photodynamic”)“Melanoma”[Mesh] AND (Eye[Mesh] OR Eyelids [Mesh]) AND (“Surgical Procedures, Operative”[Mesh] OR Surgery) “Melanoma”[Mesh] AND (Eye[Mesh] OR Eyelids [Mesh]) AND (“Microsurgery“[Mesh] OR Microsurgery)“Melanoma“[Mesh] AND (Eye[Mesh] OR Eyelids [Mesh]) AND (“Lasers“[Mesh] OR laser OR LASER) “Melanoma”[Mesh] AND (Eye[Mesh] OR Eyelids [Mesh]) AND (“Radiotherapy”[Mesh] OR “Brachytherapy”[Mesh])
Cochrane Library	#1 (MeSH descriptor: [Melanoma] explode al trees), #2 Melanoma, #3 (MeSH descriptor: [Eye] explode al trees), #4 Eye, #5 (MeSH descriptor: [Photochemotherapy] explode al trees), #6 Photochemotherapy, #7 (#1 OR #2) AND (#3 OR #4) AND (#5 OR #6)#1 (MeSH descriptor: [Melanoma] explode al trees), #2 Melanoma, #3 (MeSH descriptor: [Eye] explode al trees), #4 Eye, #5 (MeSH descriptor: [General Surgery] explode al trees), #6 (MeSH descriptor: [Microsurgery] explode al trees), #7 surgery, #8 (#1 OR #2) AND (#3 OR #4) AND (#5 OR #6 #7)#1 (MeSH descriptor: [Melanoma] explode al trees), #2 Melanoma, #3 (MeSH descriptor: [Eye] explode al trees), #4 Eye, #5 (MeSH descriptor: [Lasers] explode al trees), #6 Laser, #7 (#1 OR #2) AND (#3 OR #4) AND (#5 OR #6)#1 (MeSH descriptor: [Melanoma] explode al trees), #2 Melanoma, #3 (MeSH descriptor: [Eye] explode al trees), #4 Eye, #5 MeSH descriptor: [Radiotherapy] explode al trees), #6 Radiotherapy, #7 (#1 OR #2) AND (#3 OR #4) AND (#5 OR #6)
Embase	('melanoma'/exp OR 'melanoma') AND ('photochemotherapy'/exp OR 'photochemotherapy' OR 'photodynamic therapy'/exp OR 'photodynamic therapy') AND('eye'/exp OR 'eye' OR 'eyelid' OR 'eyelid'/exp OR eyelid) AND ('nonhuman'/exp OR 'nonhuman') AND ([english]/lim OR [portuguese]/lim OR [spanish]/lim) ('melanoma'/exp OR 'melanoma') AND ('radiotherapy'/exp OR 'radiotherapy') AND ('eye'/exp OR 'eye' OR 'eyelid' OR 'eyelid'/exp OR eyelid) AND ('nonhuman'/exp OR 'nonhuman') AND ([english]/lim OR [portuguese]/lim OR [spanish]/lim) ('melanoma'/exp OR 'melanoma') AND ('microsurgery'/exp OR 'microsurgery' OR 'surgery'/exp OR 'surgery') AND ('eye'/exp OR 'eye' OR 'eyelid' OR 'eyelid'/exp OR eyelid) AND ('nonhuman'/exp OR 'nonhuman') AND ([english]/lim OR [portuguese]/lim OR [spanish]/lim)('melanoma'/exp OR 'melanoma') AND ('laser'/exp OR 'laser' OR 'lasers'/exp OR 'lasers') AND ('eye'/exp OR 'eye' OR 'eyelid' OR 'eyelid'/exp OR eyelid) AND ('nonhuman'/exp OR 'nonhuman') AND ([english]/lim OR [portuguese]/lim OR [spanish]/lim)
Web of Science (all databases)	TS=(melanoma) AND TS=(radiotherapy) AND TS=(eye* OR eyelid) AND TS=(rabbit)TS=(melanoma) AND TS=(microsurgery OR surgery) AND TS=(eye* OR eyelid) AND TS=(rabbit)TS=(melanoma) AND TS=(laser) AND TS=(eye OR eyelid) AND TS=(rabbit)TS=(melanoma) AND TS=(photochemotherapy OR photodynamic therapy) AND TS=(eye* OR eyelid) AND TS=(rabbit)

Inclusion and exclusion criteria

This systematic review’s inclusion criteria were: (a) studies in which the main objective was to evaluate the treatment for ocular melanoma, (b) orthotopic implantation of tumour cells in rabbit’s eyes and (c) therapeutic modalities that included PDT, laser, radiotherapy and surgery/microsurgery. Studies that did not meet the following criteria were excluded: (a) studies in which the sample included other animals or humans; (b) studies that used other forms of therapy; (c) studies reporting combined therapies; (d) heterotopic models; (e) induction of tumours other than melanoma; (f) studies in animals without tumours; (g) reviews, book chapters and conference summaries; and (h) studies that did not evaluate the therapeutic effect on ocular melanomas.

Data extraction

References (PubMed/MEDLINE and Web of Science) were managed by EndNote Web Referral Manager software (EndNote®X7; Thomson Reuters, Philadelphia, PA), and duplicate references were removed. Duplicate references from the Embase and Cochrane Library databases were handled and removed manually. For studies that met the inclusion criteria, the following information was collected: reference (author; year), animal model (strain; tumour origin; implantation site), description of treatment (initiation of treatment; type of therapy used; the form of administrations; treatment interval), clinical evaluation and histological/microscopy.

Quality assessment

The studies’ quality was evaluated by assessing the risk of bias using SYRCLE’s bias risk tool for animal studies by two independent authors (T.G. and K.C.).¹⁴

Results

Figure 1 shows the PRISMA flow diagram of study selection, describing the process of identification, exclusion and inclusion. A total of 3058 studies were selected for analysis, with 2074 studies obtained from PubMed, 620 studies from Embase, 156 studies from Web of Science and 208 studies from Cochrane Library. Of these, 27 articles were included for the qualitative synthesis, with publication dates from 1970 to 2018. Of the selected studies, 19 used PDT as treatment,^15–33 six used radiotherapy^34–39 and two used laser.^40,41 No studies with surgical therapy that met the inclusion criteria were obtained. The detailed information of the selected studies and results is presented in Table 3.

Figure 1.

Preferred Reporting Items for Systematic Reviews (PRISMA) flow diagram of the studies selection.

Table 3.

Included studies details and results.

Study	Animal model	Treatment description	Results
Photodynamic therapy
Ozler et al., 1992¹⁵	ST: Dutch cross rabbitsN: 19TO: Greene hamster amelanotic melanomaIS/AD: Subchoroidal space; implanted through the sclera	TO: 6–10 days; diameter 3–4 mmPS: Chloroaluminum sulfonated phthalocyanine; 5 mg/kgAD: IVIR: Argon laser (675 nm, 7 to 60 J/cm²)DLI: 24 hours	Tumour growth was arrested at total light doses of 22–60 J/cm². At a total light dose of 15–21 J/cm², tumour growth was initially arrested. However, regrowth of these tumours was apparent within 7 days. Total light doses of <15 J/cm² showed no response.
Schuitmaker et al., 1990¹⁶	ST: New Zealand white rabbitN: 10TO: Greene amelanotic hamster melanomaIS/AD: Implanted trans corneally onto the iris	TO: After 7–8 days; diameter 4–7 mmPS: Bacteriochlorin; 20 mg/kgAD: IVIR: Xenon lamp (740–770 nm, 55–125 mW/cm², 150–280 J/cm²) Filter combination C was used in the first eight experiments. To improve PDT’s spectral power distribution, it was replaced by combination B in the subsequent experiments.DLI: 24 hours	After irradiation, tumour growth stopped: fluorescein angiography showed nonperfusion of the tumour. After enucleation, histopathology showed subtotal tumour necrosis with occasionally small clusters of viable cells around a blood vessel and at the tumour periphery.
Franken et al., 1985¹⁷	ST: Dutch rabbitsN: 17TO: Greene’s amelanotic melanomaIS/AD: In the iris, implanted transcorneally	TO: 8 days, diameter 4–6 mmPS: Haematoporphyrin derivative;10 mg/kgAD: IVIR: Argon laser (630 nm, 60 mW/cm, 100 J/cm²) DLI: 24 hours	Blanching and shrinkage of tumours were the first signs of tumour destruction. Angiography after PDT found non-perfusion of blood vessels at the tumour surface. Histopathological observation of vessel wall destruction is in agreement with this finding. Subtotal tumour necrosis was demonstrated in 12 out of 13 experiments. Necrosis was complete in only one experiment. Clusters of viable tumour cells were found when shielded behind pigment, at the tumour periphery and around some blood vessels.
Young, 1996¹⁸	ST: New Zealand albino rabbitN: 18TO: Mouse melanoma cells (B16F10)IS/AD: In the iris, implanted transcorneally	TO: 8 days, diameter 4–6 mmPS: Benzoporphyrin derivative monoacid (verteporfin). 2 mg/kgAD: IVIR: Argon-pumped dye laser (692 nm, 40 and 150 J/cm²)DLI: 24 hours	Tumours regressed in all eyes treated with ≥60 J/cm². With fluence of 40 J/cm², tumour regrowth was observed in one animal. Histological examination of the eyes treated with PDT immediately after treatment showed prominent vascular occlusion throughout the tumour’s full thickness. One month after treatment, tumour necrosis and mononuclear cell infiltration and pigment-laden macrophages were the predominant findings.
Kim et al., 1996¹⁹	ST: New Zealand albino rabbitN: 25TO: Mouse melanoma cells (B16F10)IS/AD: Subchoroidal space, by scleral incision	TO: After exceeded 3 mmPS: Liposomal preparation of benzoporphyrin derivative (verteporfin); 1 mg/kgAD: IVIR: Argon-pumped dye laser (692 nm, 60–120 J/cm²)DLI: 30 minutes	All animals treated with benzoporphyrin derivative and light at fluences of ≥80 J/cm² showed complete tumour arrest. Histological examination results of tumours treated with dye plus light immediately after treatment showed prominent vascular closure. Examination results of the eyes that showed tumour regression after a 4-week follow-up period showed tumour necrosis and extensive infiltration of mononuclear cells and pigment-laden macrophages at the tumour site.
Sery and Dougherty, 1984²⁰	ST: Rabbits chinchilla pigmentedN: 14TO: Greene–Harvey amelanotic malignant melanomaIS/AD: In the iris, implanted transcorneally	TO: 10–20 days.PS: Haematoporphyrin derivative (Photofrin); 1.25–5 mg/kgAD: IVIR: Dye laser rhodamine B (632 nm,71 mW/cm², 24 minutes, 102 J/cm²) and xenon arc lamp (1000 W)DLI: After varying intervals of up to 4 days.	A dose of 2.5 mg/kg of haematoporphyrin derivative, followed by photoirradiation (102 J/cm²), was found to be lethal for tumours 4 mm in diameter. This was best accomplished with a dye laser as the source of light. A 1000 W xenon arc lamp was also effective but not as efficient.
Panagopoulos et al., 1989²¹	ST: New Zealand albino rabbitsN: 23 eyesTO: Greene melanomaIS/AD: In iris, per incision made superiorly at the limbus	TO: 5–8 daysPS: Chloroaluminum sulfonated phthalocyanine (CASPc); 5 mg/kgAD: IVIR: Argon ion pumped dye laser (irradiance, 10–48 mW/cm²; exposure, 3–10 J/cm²); (irradiance, 15–50 mW/cm²; exposure, 10–30 J/cm²); (irradiance, 50–63 mW/cm²; exposure, 57–60 J/cm²)DLI: 24 hours	Vascular occlusion was produced in a well-circumscribed area corresponding to the boundaries of PDT. Tumours successfully treated were arrested in growth and exhibited no viable tumour cells on histological examination.
Gonzalez et al., 1995²²	ST: New Zealand albino rabbitsN: 9TO: B16F10 murine melanoma cellsIS/AD: Subchoroidal space, by sclerotomy	TO: diameter 2.3–7.0 mmPS: Chloroaluminum sulfonated phthalocyanine (CASPc); 5 mg/kgAD: IVIR: Argon-pumped dye laser (675 nm, 25–70 J/cm²)DLI: 24 hours	Histological examination of tumours treated with PDT revealed prominent vascular damage early after treatment that resulted in vascular occlusion. Tumour necrosis was evident within 24 hours of treatment.
Kines et al., 2018²³	ST: New Zealand White rabbitsN: 14TO: 92.1MEL human uveal melanoma cellsIS/AD: suprachoroidal space	TO: 13 daysPS: AU-01; 50 µg, 20 µg or 5 µgAD: Intravitreal injectionIR: Zeiss Visulas 690s (690 nm, 600 mW/cm², 83 seconds, 50 J/cm²)DLI: 6–8 hours; repeated 7 days later	Histopathological analysis revealed a dose–response effect. Two days after treatment, the 50 μg dose group showed deep tumour necrosis across the tumour tissue’s entire depth. From 8–9 days after the second treatment, they had smaller tumours with evidence of necrosis. In one animal, the tumour was small and comprised mainly of monocytic infiltrate, with evidence of few cancer cells.
Schmidt-Erfurth et al., 1996²⁶	ST: New Zealand white rabbitsN: 12TO: Greene amelanotic melanomaIS/AD: Iris surface, per limbal incision	TO: 7–10 days; 3–5 mmPS: Benzoporphyrin derivative (complexed with low-density lipoprotein); 2 mg/kgAD: IVIR: Argon laser (692 nm, 150 mW/cm², 80 J/cm²)DLI: 3 hours	All tumours treated with 80 J/cm² regressed irreversibly. The principal mechanism of tumour necrosis was thrombosis following the disruption of endothelial membranes. Ultrastructure data suggested tumour cell damage, although evidence for this being the result of direct PDT-mediated tumour cell death was less clear.
Phillips et al., 1987²⁷	ST: Dutch rabbitsN: 12TO: Greene hamster melanomaIS/AD: Subchoroidal space, implanted per scleral incision	TO: 7–14 days after transplantPS: Haematoporphyrin derivative; 5 mg/kgAD: IVIR: Argon green laser (514.5 nm, 3 mm or 5 mm, 1 W/cm² for 12 minutes or 2 W/cm² for 6 minutes, 700 J/cm²)DLI: 4 hours	By 24 hours, the irradiated melanoma and surrounding retina appeared whitened and necrotic, with complete non-perfusion of the area. For 1 week, the clinical appearance did not alter. Histopathology at 24 hours and 8 days confirmed massive central tumour necrosis, but this was subtotal in all cases. Viable cells were evident at the periphery and also along the base of the tumour.
Gomer et al., 1984²⁸	ST: Pigmented rabbitsN: 44TO: Amelanotic hamster melanomaIS/AD: In the iris, through a radial cut made in the cornea	TO: Average of 4.5 mm in diameterPS: Haematoporphyrin derivative; 1–10 mg/kgAD: IVIR: Monochromatic red light (630 nm or 635 nm was generated using a tuneable rhodamine-B or kiton-red dye laser pumped by a 5 W argon laser//kiton-red dye laser pumped by an argon laser (5 W, 635 nm, 36–90 J/cm²) for animals given injections of HpD (100–400 mW/cm²)DLI: 48 hours	Toxic effect characterised by tumour blanching, oedema and haemorrhage was observed within 24 hours of treatment. Histological examination obtained 24 hours following treatment illustrated massive tumour tissue necrosis and vascular disruption. Regarding the clinical evaluation, relevant doses are shown to be effective in selectively curing the highly malignant amelanotic iris melanoma.
Schuitmaker et al., 1991²⁹	ST: New Zealand white rabbitsN: 6TO: Greene amelanotic hamster melanomaIS/AD: In the iris, implanted transcorneally	TO: 7–8 days, diameter 4–7 mmPS: Bacteriochlorophyll, 20 mg/kgAD: IVIR: Xenon lamp (740–770 nm, 1250 W/m², 2–2.7 kJ/m²)DLI: 24 hours	Immediately after treatment, intracellular spaces were enlarged, and blood vessels were clotted with swollen erythrocytes. Some mitochondria had fused inner and outer membranes in the electron microscopy exam, and the mitochondrial cristae were affected. Over time, the severity of the tissue and cell damage increased, leading to almost complete tumour necrosis after 24 hours.
Hill et al., 1995³⁰	ST: Pigmented rabbitsN: 14TO: Greene hamster melanomaIS/AD: Subchoroidal space, implantation parallel to the limbus	TO: NRPS: Bis (tri-n-hexylsiloxy) silicon 2,3-naphthalocyanine; 0.5 mg/kgAD: IVIR: Argon ion pumped Ti:sapphire laser (770 nm; 40 mW/cm²; 20 J/cm²)DLI: 1 and 24 hours	Tumour destruction was evident histologically in the treated areas at a depth of at least 3.5 mm. Tumour cells within the area treated with necrosis exhibited pyknosis as manifestations of irreversible cell death.
Chong et al., 1993³¹	ST: Dutch cross rabbitN: 14 eyesTO: Greene Amelanotic melanomaIS/AD: Implanted onto the iris	TO: 6–14 daysPS: Indocyanine green, 25 mgAD: IVIR: 790 nm and 810 nm chromophore semiconductor diode laserDLI: Immediately after an intravenous injection	Tumours treated with PDT showed no growth after treatment, as judged by clinical examination and photography. Histologically, 4 of the 14 tumours treated showed total necrosis, whereas the remaining 10 tumours treated similarly demonstrated only rare viable cells around blood vessels or at the tumour periphery.
Winward et al., 1990³²	ST: New Zealand white rabbitsN: 31TO: Amelanotic Greene melanoma cellsIS/AD: Suprachoroidal space, by a transvitreal approach	TO: 4, 7 and 14 daysPS: Rose Bengal, 20 mg/kgAD: IVIR: Argon green laser (514.5 nm, 500 nm, 40–60 mW, 7.2–30.6 J)DLI: Immediately after intravenous injection of Rose Bengal	Immediately following Rose Bengal injection, 3 minutes of continuous irradiation at 20.4 W/cm² applied in three to four circumferential revolutions around the base of tumour nodules, without direct tumour irradiation, produced peripheral vascular occlusion and consequent tumour inhibition. Similar therapy at higher laser intensity (30.6 W/cm²) and multiple retreatment sessions (28.0–30.6 W/cm²) resulted in an increased tumour-inhibiting effect.
Schmidt-Erfurth et al., 1994³³	ST: New Zealand white rabbitsN: 8TO: Amelanotic Greene hamster melanomaIS/AD: Subcoronoidal space, implanted by sclerotomy	TO: 14–20 days, diameter 3–4 mmPS: Benzoporphyrin derivative monoacid; 2 mg/kgAD: IVIR: Argon-pumped dye laser (692 nm,100 J/cm²)DLI: 3 hours	Exam findings showed immediate photothrombosis after the disintegration of endothelial membranes. After complete necrosis of tumour cells within 24 hours, a small fibrotic scar slowly developed. No tumour regrowth was noted for up to 6 weeks.
Franken et al., 1988²⁴	ST: Dutch rabbitsN: 13TO: Amelanotic Greene melanomaIS/AD: Transcorneally onto the iris	TO: Diameter 3–4 mmPS: Haematoporphyrin derivative (HPD); 10 mg/kgAD: IVIR: Argon laser (1000 kJ/m²)DLI: 24 hours	Immediately after PDT, blood circulation stopped as shown by fluorescence angiography, light and electron microscopy. Direct interference in the mitochondrial structure was observed, resulting in tumour necrosis.
Gomer et al., 1985²⁵	ST: Pigmented rabbitsN: 12TO: Amelanotic Hamster MelanomaIS/AD: Heterotransplanted onto the iris	TO: Diameter 5–8 mmPS: Haematoporphyrin derivative (HPD); 2.5–20 mg/kgAD: IVIR: Argon ion laser (630 nm, 36–180 J/cm²)DLI: 24 or 48 hours	Permanent damage but not progressive in the form of a chorioretinal scar was induced. Lens opacities were not observed, and the cornea, aqueous and vitreous remained clear on all test eyes. The results showed a selective and safe approach to the treatment of ocular tumours. Tumour phase-contrast micrograph depicted an irregular area of fragmented tumour cells, suggestive of necrosis.
Radiotherapy
Yang et al., 1993³⁴	ST: New Zealand white rabbitN: 18TO: Greene melanomaIS/AD: Suprachoroidal	TO: 1 weekRT: ¹²⁵I irradiation (10,000 cGy, 3341.5 cGy/h (1 cGy=1 rad) and 239.9 cGy/h	For tumours >10 mm tall, two out of two eyes in the low-activity plaque group and one of four eyes in the high-activity plaque group failed to show complete tumour regression.
Wollensak et al., 1990³⁵	ST: New Zealand white rabbitsN: 55TO: Greene melanomaIS/AD: Anterior chamber	TO: 12th day (45% 75% of the anterior chamber was filled with the tumour)RT: Protons generated by the 72 MeV 50 Gy/min	In animals with positive tumour growth after irradiation, growth retardation, and even regression occurred 2 weeks after treatment. The tumour temporarily changed its colour from pink to yellow, that is, it became partially necrotic. In animals with negative tumour growth after irradiation, regression began 1 week after treatment with maximum regression, typically reaching 4–6 weeks after irradiation. At the time of irradiation, the tumours showed relative necrosis in about 2–4% of their total area. Viable tumour cells showed an elevated nucleus/plasma ratio and presented spindle or epithelioid appearance. Microscopically, relative necrosis could be found in up to 80% of the total area of irradiated tumours showing positive growth; in irradiated lesions exhibiting negative tumour growth, there was regression or relative necrosis in >80% of the tumour area.
Rand et al., 1987³⁶	ST: Both albino and pigmented rabbitsN: 9TO: Amelanomic epithelioid melanoma cellsIS/AD: Anterior chamber	TO: 7 days, 3–4 mmRT: Leksell stereotactic radiosurgical unit or ‘gamma knife’ (6000 to 9000 rads)	A single treatment at a dose of 6,000 to 9,000 rads was able to destroy melanoma and preserve vision. In six of nine animals, no clinical or pathological signs of recurrence were seen. In these animals a small scarred region and necrosis was frequently present. In the other three animals, treatment was not successful due to experimental constraints.
Krohn et al., 1970³⁷	ST: New Zealand White rabbitsN: 25TO: Amelanotic variant of a spontaneous hamster melanoma (Greene tumour)IS/AD: Anterior chamber	TO: NRRT: thermal neutron (200 kV and exposures of ≤4.5 minutes), associated gamma dose (21–163 r)	In some eyes, neither tumour growth nor radiation effect occurred. Other eyes were tumour free after two months but showed several degrees of radiation effects. Others survived intact for ≥24 days after irradiation and finally developing erosion tumours. Other eyes were intact for <24 days after radiation.
Kurhanewicz et al., 1994³⁸	ST: New Zealand albino rabbitsN: 18TO: Greene amelanotic malignant melanomaIS/AD: Suprachoroidal space	TO: Approximately 5 mm RT: ¹²⁵I irradiation (10–30 Gy)	Animals that received only 10 Gy of brachytherapy showed rapid tumour growth over a 1- to 2-week period. Those who received 25–30 Gy had tumour regression in 1–2 weeks. In those who received 15–20 Gy, 50% had slow regression over 4–6 weeks, while the other 50% demonstrated delayed tumour growth over that same period. The changes in ³¹P metabolite ratios after radiation were not predictive of therapeutic outcome. There was also a significant alkalosis.
Steeves et al., 1995³⁹	ST: Pigmented rabbitsN: 9TO: Greene melanomasIS/AD: Subretinal space, using a transvitreal approach	TO: Diameter 3–7 mmRT: ¹²⁵I irradiation (35 and 45 Gy)	Radiation dose required to achieve 50% tumour control with ¹²⁵I was 43 Gy.
Laser
Damico et al., 2014⁴⁰	ST: New Zealand albino rabbitsN: 52TO: B16F10 melanomasIS/AD: Subcoronoidal space, by sclerotomy	TO: 2–4 weeks, ≥2.1 mmLT: Nd:YLF laserIR: At 1047 nm, 125 J/cm²	Immediately after treatment, angiography showed diffuse hypofluorescence in 71%. Light microscopy showed vascular occlusion extending at least two thirds of the tumour thickness from the base. Seven of the 14 tumours followed for 15±8 days were eradicated. There was no correlation between tumour height and eradication.
Krause et al., 2003⁴¹	ST: New Zealand albino rabbitsN: 64TO: B16F10 melanomasIS/AD: Subcoronoidal space, by sclerotomy	TO: 2–4 weeks, ≥2.1 mmLT: Diode-pumped Nd:YLF laser (1047 nm, 13–125 J/cm²)IR: At 1047 nm, 125 J/cm²	The complete tumour eradication rate was 91% (10 of 11 animals) at a dosage of 125 J/cm² and 75% (9/12) at 63–87 J/cm². The eradication rate dropped to 25% (1 of 4) at ≤38 J/cm². Continuous tumour growth was observed in all animals treated with collimated radiation.

PDT: photodynamic therapy; PS: photosensitizer; AD: administration protocol; IV: intravenously; IR: irradiation; DLI: drug light interval; ST: strain; TO: tumour origin; IS: implantation site; TO: treatment onset; LT: laser type; RT: radiotherapy; NR: not reported; N: number.

Studies using PDT as a treatment have used Dutch Cross rabbits, Dutch rabbits, New Zealand albino rabbits, New Zealand white rabbits, Pigmented rabbits and chinchilla pigmented rabbits. New Zealand white rabbits, both albino and pigmented, were employed in studies with radiotherapy treatment, and New Zealand albino rabbits were used in laser studies. With regard to the origin of the tumours, murine melanoma (B16F10), Greene amelanotic hamster melanoma and human uveal melanoma (92.1MEL) were reported. Tumours were implanted in the anterior chamber, iris, subretinal space or subchoroidal space. Treatment initiation was performed within a range of 5–28 days after tumour implantation, with the tumours reaching a diameter range of 2.1–7 mm. The photosensitisers used in PDT, concentration, route of administration and activating light are shown in Table 3. The irradiation agents in radiotherapy studies and the lasers employed in laser therapy are also shown in Table 3.

Outcome of tumour therapy were evaluated by various methods, including biomicroscopy exams,^{17,20,21,24,26,27,30,31,33,40,41} angiographic examination^{15–19,21–24,26–28,31,33,40,41} histological examination,^{15–23,26–28,30,31,33,34,36,37,40,41} fundoscopy, ^{18,19,22,23,27,28,32–34,38–41} magnetic spectroscopy,³⁸ electron microscopy,^24,29,35 ultrasound exam^{18,19,32,34,38,39} and electroretinogram.^32,36 All treatments led to tumour inhibition.^18,19,23,32 However, complete tumour destruction was not always achieved,^15,17,22 with incomplete tumour necrosis²⁹ and tumour development in the periphery of the treated area.^16,27 It was demonstrated absence of tumour growth after PDT,³¹ occurring irreversible regression,²⁶ and tumour destruction.^20,30 Until the time animals were killed to collect tissues, no tumour growth was observed.³³ No tumour cells were detected in the histological examination ²¹ and necrosis was present.^{15–24,26–33} Radiotherapy is effective, destroying melanoma.³⁶ Since the height of the tumour may influence its complete regression,³⁴ partial^38,39 to total responses were observed at varying periods of days after treatment,³⁷ with necrosis areas of variable dimensions.³⁵ The energy used in laser therapy was related to therapeutic responses.^40,41 Complete regression of tumours was achieved.⁴¹ According to the laser dose, tumour necrosis was present,⁴¹ and intact tumour cells were also found.^40,41 Despite the therapeutic efficacy shown by the treatments, several side effects have been reported in different eye structures, depending on the therapy used, as described in Table 4. In terms of quality assessment, most of the studies included presented an unclear bias. The risk assessment for individual study bias is represented in Figure 2 and detailed in Supplemental Table S1.

Table 4.

Side effects observed after treatments.

Reported changes
PDT	Vascularised cornea,¹⁷ inflammation,¹⁷ epithelial injury,¹⁷ oedema^15,17,20,21 and haemorrhagic keratitis.¹⁶ Conjunctiva with hyperaemia,^15,26 inflammation,²⁰ subconjunctival chemosis¹⁹ and haemorrhagic conjunctivitis.²⁰ Cataract development in the lens.^16,17,31 Interruption of surrounding choroidal vasculature²² inflammatory process,²⁰ hyperaemia^16,29 and choroid atrophy.^27,33 Retinal oedema,^{16,20,22,28,32,33} haemorrhage,^{18,22,27,32,33} detachment^{18,19,22,25,27,28,32,33} and necrosis.^25,28 Scars^18,²⁵ and chorioretinal atrophy.^18,19,32 Iris with inflammation²⁰ and haemorrhage.^20,21 Hyphaemia and fibrinous veil in the aqueous humour of the anterior chamber²¹ and eyeball atrophy (phthisis bulbi).²⁰
Radiotherapy	Several changes were observed, including corneal opacification, iris atrophy, cataract formation,³⁷ hair loss, epiphora, symblepharon, blepharitis,³⁵ haemorrhagic choroidal detachments,³⁸ retinal detachment and vitreous haemorrhage.³⁴
Laser therapy	Serous retinal detachment and occasional areas of haemorrhage,^40,41 and fibrotic pigmented chorioretinal scars surrounded by choroidal atrophy.⁴¹

Figure 2.

Summary of the quality assessment of the studies included in the systematic review.

Discussion

The present study’s objective was to evaluate the effectiveness of ocular melanoma treatment options in rabbits. The obtained results allow us to conclude that PDT, radiotherapy and lasers may be effective in eye melanoma. Nevertheless, complete remission was sometimes not achieved. For all treatments, the outcomes are dependent on the protocol intensity. Further studies should compare the best protocols for each treatment modality in a randomised controlled trial. A study of this nature will allow conclusions to be made over which option is the most effective.

Currently, the evidence available does not support one treatment as a gold standard for eye melanoma. Since this systematic review supports the efficacy of PDT, radiotherapy and lasers, the decision over which one to use should consider factors such as clinician preference and proficiency, financial constraints and patient health.^42–44 Several animal models of ocular melanoma exist, and the decision as to which is the most appropriate is determined by factors such as cost, scientific principles, available data, the technique to be used and suitability of the model for the project.⁴⁵ For ophthalmic research, rabbits have the main advantage of larger eye size that allows for easier examination, retinal and choroidal assessment, background photography and surgical procedures,¹¹ as well as an accurate injection of cells in the posterior compartment and easy use of diagnostic techniques for follow-up.⁴⁶ Other advantages in choosing this species are the easy handling; short gestation, lactation and puberty cycles; the standardisation of the environment; the genetic standardisation; and the easy transplantation of tumours. Among the disadvantages are the need to live in an artificial environment, the need for a standardised diet and the execution of most experiments in animals with artificially induced diseases.⁴⁵

Rabbits are widely used in research, not only in the field of ophthalmic diseases but also in several other pathologies.^47,48 The New Zealand white rabbit (albino) and American, Dutch and Californian rabbits are the most used breeds for research, representing both pigmented and albino animals.⁴⁹ Amelanotic Greene hamster melanoma cells were used in almost all revised studies, being used in 16 of the 19 PDT studies^{15–18,20,21,24–33} and in five of the six radiotherapy ones.^34,35,37 Mus musculus melanoma cells (B16F10) were used in two PDT studies^19,22 and in two laser studies.^40,41 Human uveal melanoma (92.1MEL cells) were used in a PDT study.²³ One of the articles with radiotherapy classifies the cancer cells used as amelanotic epithelioid melanoma.³⁶ The preference for amelanotic Greene hamster melanoma cells is probably due to the success of establishing tumours in the eye, brain, testis, muscle and subcutaneous space.⁵⁰ They also grow faster after inoculation, and no immunosuppression is required in rabbits for the cells to grow.¹¹ Nevertheless, immunosuppression treatment was used in one study.¹⁸

As stated, melanoma can affect any anatomical region in which melanocytes are present.⁵¹ Ocular melanoma in dogs and cats often originate in the iris and the ciliary body,² and in humans, they commonly affect the choroid.⁸ For the studies in this review, tumours were implanted in the iris,^{16–18,20,21,24–26,28,29,31}anterior chamber,^35–37 subretinal space³⁹ and subchoroidal space.^{15,19,22,23,27,30,32,33,38,40,41} The studies therefore represent the most common locations for ocular melanomas, for both animals and humans, making the results more comprehensive. As a suggestion for future studies, a more detailed description of the animal model used must be performed.

PDT is based on the selective retention of photosensitisers in malignant cells and the subsequent destruction of tumours by appropriate wavelength irradiation.⁵² PDT largely depends on the properties of the photosensitising agent,^53–55 generating photophysical and photochemical reactions, and exerting its outcomes by producing reactive oxygen species (ROS).^56,57 ROS rapidly lead to significant toxicity in the target tissue microenvironment, resulting in tumour cell death by apoptosis, necrosis or autophagy.⁵⁷ Radiation exposure causes direct or indirect cellular stress due to the generation of ROS, DNA damage and damage to subcellular organelles.⁵⁸

Necrosis was the primary type of death observed after treatment.^{15,16,18,19,21–24,26,29,31,33,36,41} Death by necrosis caused by PDT can be accompanied by an inflammatory response, which can boost an anti-tumour immune response.⁵⁹ The results support that despite PDT not being the primary treatment option for ocular melanomas, it presents several advantages regarding the other therapies. It is the most conservative, since it has selectivity for tumour cells, and the light source can easily be applied to the eye. In the future, PDT should be assessed as a primary option for ocular melanoma treatment in animals and possibly in humans.

The included studies’ follow-up periods ranged from immediately^{18,19,29,40,41} to two years after treatment.³⁶ Since ocular melanoma can be recurrent,^40,41 it will be of interest to present longer follow-ups to evaluate the long-term effects. Nevertheless, the immediate results allow the rapid effect of the treatments on tumour reduction to be verified.^{18,19,28,29,40,41}

Inflammatory response and retinal detachment were the most common side effects, as shown in Table 4. Inflammatory response was common, contributing to tissue repair. Retinal detachment is expected, since most treatments are administered through the retina or in the proximity of this structure. Of importance, most of these complications were solved spontaneously, in a short period and without the need for additional treatment.^{15,18,19,26,27,32}

One of the limitations observed was the heterogeneity between the studies due to different anatomical sites of the melanoma implantation, therapy intervals, time of exposure, energies and doses used, forms of evaluation, times of evaluation and times of follow-up. Also, differences exist between the numbers of studies included for each treatment, which means that the sample included in some groups is reduced, limiting the conclusions obtained. The decision to perform a systematic review aimed to reduce the heterogeneity among studies using defined inclusion and exclusion criteria, thus allowing more evidence-based conclusions. Regarding the inclusion and exclusion criteria, we opted to include all the options currently available in veterinary clinics. Additionally, PDT was included because it is a therapy with promising results that has been underused, despite the availability of commercial photosensitisers. Experimental therapies were excluded because they are not available to be used in the clinics and their efficacy is not fully established. We also decided to exclude combination studies, since they do not allow each therapy effect to be distinguished, and that would compromise the answer to the PICO question.

The present systematic review aimed to compare the available treatment modalities for ocular melanoma in rabbits. We conclude that all of the analysed therapies can be successfully used in ocular melanoma treatment, since they proved to be effective, even more conservative approaches such as PDT. Despite the therapeutic success, all modalities presented side effects, with PDT being the safest therapeutic option. The studies presented high heterogeneity. In the future, new studies should be performed to increase our knowledge of ocular treatment options.

Supplemental Material

sj-pdf-1-lan-10.1177_00236772211039333 - Supplemental material for Evaluation of eye melanoma treatments in rabbits: A systematic review

Supplemental material, sj-pdf-1-lan-10.1177_00236772211039333 for Evaluation of eye melanoma treatments in rabbits: A systematic review by Tarcísio Guerra Guimarães, Carlos Miguel Marto, Karla Menezes Cardoso, Nuno Alexandre, Maria Filomena Botelho and Mafalda Laranjo in Laboratory Animals

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by the Foundation for Science and Technology (FCT), Portugal, through a PhD fellowship awarded to Tarcísio G. Guimarães (SFRH/BD/139319/2018). CIBB is funded by National Funds via FCT (Foundation for Science and Technology) through the Strategic Projects UID/NEU/04539/2019, UIDB/04539/2020, UIDP/04539/2020, and by COMPETE-FEDER (POCI-01-0145-FEDER-007440).

ORCID iD

Mafalda Laranjo

Supplemental material

Supplemental material for this article is available online.

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