Effects of topical and subconjunctival use of bevacizumab on corneal neovascularization in rabbits' eyes / Efeitos do uso tópico e subconjuntival do bevacizumabe na neovascularização corneana de olhos de coelhos

ABSTRACT Purpose: To evaluate and compare the effects of topical application and subconjunctival injection of bevacizumab on corneal neovascularization (CNV) in rabbits' eyes after chemical burning of the cornea. Methods: The animals were randomly distributed into four groups of five animals. In one group, the drug was instilled, while in another, it was administered by subconjunctival injection. The two procedures using bevacizumab were compared with instillation and subconjunctival injection of saline solution (S). Neovascularization was evaluated according to the size of the invasion area of new blood vessels and through computerized analysis of this area. The data were analyzed using the Kruskal-Wallis test followed by Dunn's test for two-by-two comparison of the groups, to assess the external examination of CNV. Analysis of variance was used to assess the area of CNV. P<0.05 was considered statistically significant. Results: Assessing both the external examination and the invasion area of neovessels on the 5th and 10th days, there was a clear difference between the groups. The group to which saline solution had been applied showed higher scores for CNV, as well as increases in the invasion area of neovessels. Two-by-two comparison of groups revealed no significant differences. However, an analysis of the factors involved (injection vs. instillation and bevacizumab vs. saline solution) showed that injection did not differ from instillation, but that bevacizumab differed from saline solution. Conclusion: Bevacizumab showed an inhibitory effect on CNV in rabbits' eyes after chemical burning of the cornea. There was no difference between the topical or subconjunctival administration of bevacizumab in the inhibition of CNV.

RESUMO Objetivos: Avaliar e comparar o efeito do uso tópico e da injeção subconjuntival do bevacizumabe na neovascularização corneana de olhos de coelhos após queimadura química. Métodos: Os animais foram distribuídos de forma aleatória em quatro grupos de cinco animais. Em um grupo de coelhos a droga foi instilada, enquanto em outro foi aplicada injeção subconjuntival, sendo os dois procedimentos comparados com a instilação e injeção subconjuntival de soro fisiológico 0,9% (SF) e entre si. A neovascularização foi avaliada conforme o tamanho da área de invasão dos neovasos e com análise computadorizada da mesma. Na análise de dados aplicou-se o teste de Kruskal-Wallis seguido do teste de Dunn com p<0,05 para comparação dos grupos dois a dois na análise do exame externo da neovascularização corneana. Na análise da área de neovascularização corneana, aplicou-se o teste F de análise de variância. A significância estatística foi definida como valor de p<0.05. Resultados: A avaliação do exame externo e da área de invasão de neovasos, no 5º e 10º dia, mostrou nítida diferença entre os grupos. Com o uso de soro fisiológico houve maior graduação na escala de neovascularização corneana e também na área de invasão dos nevasos, o que demonstrou o efeito inibitório do bevacizumabe. Nas comparações dos grupos dois a dois não foram detectadas diferenças significativas, porém, ao analisar os fatores envolvidos (procedimentos: injeção ou instilação, e as drogas: bevacizumabe ou soro fisiológico), verificou-se que a injeção não diferiu da instilação, mas o bevacizumabe diferiu do soro fisiológico. Conclusão: O bevacizumabe apresentou efeito inibitório na neovascularização corneana de olhos de coelhos após queimadura química, tanto por via tópica como por via subconjuntival e não houve diferença entre a via tópica e a via subconjuntival de administração do bevacizumabe na inibição da neovascularização corneana.

Degeneración nodular de Salzmann / Salzmann's nodular degeneration

La degeneración nodular de Von Salzmann constituye una entidad clínico-patológica en la que aparecen nódulos lisos y opacos, blanco grisáceos o algo azulados en las capas anteriores de la córnea. La afectación suele ser bilateral y los nódulos tienden a disponerse en anillo sobre la media periferia corneal. Pueden estar relacionados o no con un proceso inflamatorio previo y su tratamiento puede ser sintomático o quirúrgico si compromete el área pupilar y con ella la visión del paciente.Se presenta un paciente masculino, de 57 años de edad, coloración de la piel blanca, quien acudió a la consulta y refirió enrojecimiento de ambos ojos (predominio del ojo derecho), con picor, escozor y lagrimeo, de más de 6 meses de evolución. Asociado a esto notó una zona de color blanquecina en ambos ojos. En la exploración oftalmológica se observaron lesiones blanquecinas sobrelevadas de aspecto hialino, en el tercio inferior de la córnea en el ojo derecho y en el ojo izquierdo hacia la región nasal. Se concluye que el caso presenta una degeneración nodular de Salzmann(AU)

Von Salzmann nodular degeneration constitutes a clinical pathological condition in which flat and opaque, white-grayish or bluish nodules appear in the anterior layers of the cornea. The effect is usually bilateral and the nodules tend to spread in a ring form upon the corneal peripheral media. It can be related or not with previous inflammatory process and its treatment can be symptomatic or surgical if the papillary area and the patient's vision are compromised. Here is the case of a 57 years-old male Caucasian patient, who went to the doctor´s and complained of reddened eyes (mainly the right eye), itching and tearing for more than six months. In addition to this, he observed a whitish area in both eyes. The eye screening showed overelevated whitish lesions of hyaline aspect in the lower third of the right eye cornea and in the left eye towards the nasal region. It was concluded that this is a case of Von Salzmann nodular degeneration(AU)

A linfangiogênese em enxertos corneanos humanos que evoluem para retransplante / Lymphangiogenesis in human corneal grafts that has evolved to re-transplantation

RESUMO Objetivo: Estudar botões corneanos humanos com linfangiogênese através do exame histopatológico, juntamente com os enxertos de seus transplantes anteriores e posteriores, avaliando os intervalos de tempo para sucessivas cirurgias. Métodos: Estudo descritivo, observacional, longitudinal de botões corneanos humanos com linfangiogênese, juntamente com seus transplantes anteriores e posteriores. Os tecidos foram provenientes de ceratoplastia penetrante no período compreendido entre os anos 2006 e 2013. Após revisão de prontuários em que foram obtidas principalmente as datas das cirurgias, construímos uma tábua de sobrevivência a partir da qual os intervalos de tempo para retransplante foram calculados. Resultados: Entre 89 casos de linfangiogênese corneana, foram incluídos apenas aqueles 22 que possuíam registros no prontuário de transplantes anteriores ou posteriores. Nos casos que apresentavam como provável etiologia do retransplante a linfangiogênese, isolada ou associada à hemangiogênese (grupos pré-linfangiogênese/linfangiogênese e interlinfangiogênese), foram encontrados intervalos de tempo para retransplante menores (7 e 3 meses, respectivamente) que aquele encontrado no grupo linfangiogênese/pós-linfangiogênese que apresentava outras etiologias prováveis para os retransplantes (11,31 meses). Casos que apresentavam como etiologia provável do retransplante a linfangiogênese isolada apresentaram um intervalo para retransplante (3 meses) ainda menor que aquele encontrado nos casos em que a etiologia provável era a linfangiogênese associada à hemangiogênese (7,80 meses). Conclusão: Linfangiogênese, isolada ou associada à hemangiogênese, foi encontrada nos enxertos corneanos humanos estudados que evoluíram para retransplante em pequenos intervalos de tempo. Esse achado nos leva a sugerir um possível papel para os vasos linfáticos na redução do tempo de sobrevida dos enxertos corneanos humanos.

ABSTRACT Objective: To study human corneal buttons with lymphangiogenesis through histopathological examination, together with the grafts of their preceding and subsequent transplantations, evaluating the time intervals for successive surgeries Methods: A descriptive, observational and longitudinal study of human corneal buttons that have lymphatic vessels, together with its preceding and subsequent transplants. Tissues were obtained from penetrating keratoplasty in the period between the years 2006 and 2013. After a medical records review in which information on the dates of the surgeries were mainly obtained, we built a survival table from which the time intervals for retransplantation were calculated. Results: Among 89 cases of corneal lymphangiogenesis, we included only those 22, which had previous or subsequent transplantations records in medical records. In cases where the probable regrafting etiology were lymphangiogenesis, alone or combined with hemangiogenesis (pre-lymphangiogenesis/lymphangiogenesis and interlymphangiogenesis groups), time intervals for retransplantation were found to be minor (7 and 3 months, respectively) than that found in lymphangiogenesis/post-lymphangiogenesis group that had other probable etiologies for retransplantations (11.31 months). Cases that had isolated lymphangiogenesis as probable etiology of retransplantation showed an interval time for retransplantation (3 months) lower than that found in cases in which the probable etiology was lymphangiogenesis associated with hemangiogenesis (7.80 months). Conclusion: Lymphangiogenesis, alone or combined with hemangiogenesis, was found in human corneal grafts studied that have evolved to regraft in small time intervals. This finding leads us to suggest a possible role for the lymphatic vessels in reducing the human corneal grafts survival time.

Ação do bevacizumabe subconjuntival na neovascularização e re-epitelização corneana 25 dias após queimadura química / Effect of subconjunctival bevacizumab on corneal neovascularization and reepithelization 25 days after chemical burn

OBJETIVOS: Avaliar a ação do bevacizumabe subconjuntival em modelo experimental de neovascularização em córnea de coelho. Analisar se o modelo de avaliação empregado é adequado e comparar entre os grupos a extensão dos vasos, inflamação e re-epitelização da córnea. MÉTODOS: Estudo experimental, prospectivo em 20 coelhos submetidos a trauma químico com hidróxido de sódio a 1 N divididos em dois grupos, Imediatamente após a queimadura, o grupo tratado recebeu injeção subconjuntival de 0,15 ml (3,75 mg) de bevacizumabe e o grupo controle, injeção subconjuntival de 0,15 ml de soro fisiológico a 0,9 por cento. Após 25 dias, foi realizada análise fotográfica digital para avaliar a extensão e inflamação/calibre dos vasos segundo critério pré-estabelecido e estudo histopatológico da córnea, no qual foi avaliado o estado do epitélio e o número de polimorfonucleares. RESULTADOS: A extensão dos neovasos corneanos foi menor no grupo estudo em relação ao controle (p<0,001). Não houve diferença significativa entre os grupos na inflamação/calibre dos vasos. A análise histopatológica mostrou que não ocorreu diferença entre os grupos nas variáveis estado do epitélio e número de polimorfonucleares. A análise de concordância para a variável extensão dos vasos e para a variável inflamação/calibre dos vasos teve uma estimativa do coeficiente de Kappa respectivamente de 0,705 e 0,500 indicando bom grau de concordância nas diferentes avaliações cegadas, validando o método empregado. CONCLUSÕES: O modelo de avaliação foi adequado e pode ser reproduzido para avaliar outras drogas na córnea. O bevacizumabe inibiu a neovascularização corneana, porém não foi eficaz em reduzir o processo inflamatório. A droga não atrasou a re-epitelização da córnea.

PURPOSES: To evaluate the effect of subconjunctival bevacizumab in an experimental model of neovascularization in rabbit cornea. Determine its effect on vessels extension, inflammation, epithelialization of the cornea and whether the evaluation method used is appropriate to compare neovascular models. METHODS: Experimental, prospective, randomized, blinded study in twenty rabbits subjected to chemical trauma with sodium hydroxide at 1N divided into two groups. The study group received subconjunctival injection of 0.15 ml (3.75 mg) of bevacizumab and was compared with the control group that received subconjunctival injection of 0.15 ml saline solution. After 25 days, digital photographic analysis was performed to assess the vessel's extension and inflammation/diameter according to pre-established criteria. Histopathology of the cornea, which evaluated the state of the epithelium and the number of polymorphonuclear cells was also studied. RESULTS: The length of the neovessels was greater in the control group compared to the study group (P=<0.001). There was no difference in inflammation/vessel diameter between groups. Histopathology analysis showed that there was no difference between groups for the variables state of the epithelium and number of polymorphonuclear cells. The concordance analysis for the variable extension of the vessels and the variable inflammation/vessel diameter was estimated with Kappa coefficients of 0.705 and 0.500 respectively, indicating a good level of agreement in different evaluations and validating the method. CONCLUSIONS: The experimental model is adequate and can be reproduced to evaluate other drugs in the cornea. Bevacizumab inhibit the neovessels' growth but was not effective in preventing the inflammatory response. The drug did not delay the reepithelialization of the cornea.

The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model

PURPOSE: To evaluate the use of subconjunctival bevacizumab on corneal neovascularization in an experimental rabbit model for its effect on vessel extension, inflammation, and corneal epithelialization. METHODS: In this prospective, randomized, blinded, experimental study, 20 rabbits were submitted to a chemical trauma with sodium hydroxide and subsequently divided into two groups. The experimental group received a subconjunctival injection of bevacizumab (0.15 m; 3.75 mg), and the control group received an injection of 0.15 ml saline solution. After 14 days, two blinded digital photograph analyses were conducted to evaluate the inflammation/diameter of the vessels according to pre-established criteria. A histopathological analysis of the cornea evaluated the state of the epithelium and the number of polymorphonuclear cells. RESULTS: A concordance analysis using Kappa's statistic showed a satisfactory level of agreement between the two blinded digital photography analyses. The neovascular vessel length was greater in the control group (p<0.01) than in the study group. However, the histopathological examination revealed no statistically significant differences between the groups in terms of the state of the epithelium and the number of polymorphonuclear cells. CONCLUSIONS: Subconjunctival bevacizumab inhibited neovascularization in the rabbit cornea. However, this drug was not effective at reducing inflammation. The drug did not induce persistent corneal epithelial defects.

[Corneal neovascularization: review article]

Corneal neovascularization occurs through inordinate wound healing after infection, injury or surgery. Neovascularization is formation of new vascular structures in the locations which had not already vessels. The two overlapping mechanisms including vasculogenesis and angiogenesis are probably involved in neovascularization process, and the last mechanism is more involved in tumor growth and corneal and retinal disorders. In fact, corneal neovascularization is a visual threatening status that usually occurs along with inflammatory or infectious disorders of the eye surface. The studies of angiogenesis-related cancer showed that there is a balance between angiogenic factors [such as VEGF and FGF] and antiangiogenic molecules [such as angiostatin, endostatin and pigment epithelium-derived factor; EPDF] in cornea. Problems such as inflammation, infection, injury and lesions result in corneal neovascularization, which are due to stimulation of angiogenesis in this tissue. Corneal neovascularization may be influenced by matrix inetalloproteinase [MMPs] and other proteolytic enzymes. The application of new medical and surgical therapies such as angiostatic steroids, non-steroidal anti inflammatory drugs, argon laser photocoagulation and photodynamic therapy [PDT] in animal models had been efficient to some extent for inhibition of corneal neovascularization. In this study we reviewed neovascularization-dependent corneal disorders and molecular processes involved in this disorder, and also their potential therapies

The effects of the subconjunctival injection of bevacizumab (Avastin®) on angiogenesis in the rat cornea

The purpose of this study was to evaluate the effects of the use of the subconjunctival injection of bevacizumab (Avastin®) on angiogenesis in the rat cornea. Corneas of 20 Wistar male rats were cauterized with silver nitrate crystal. Animals were divided in four groups: control group (GC) that received subconjunctivally 0.02 ml of 0.9 percent saline solution on the day of the lesion; group GO that received subconjunctivally 0.02 ml of bevacizumab just after the lesion; group G3 that received bevacizumab on day 3 and group G5 that received bevacizumab on day 5 after lesion. Animals were euthanized on day 7. The newly formed vessels were quantified after China Ink perfusion and photographs were obtained and analyzed in a computerized system (Image Pro-Plus®). In the control group, neovascularization covered 53.56 percent ± 15.11 (mean ± SD) of the corneal surface, compared with 35.57 percent ± 18.80 (mean ± SD) in the G0 group, 30.60 percent±11.82 (mean±SD) in the G3 and 35.86 percent±0.07 (mean±SD) in the G5. The results showed an inhibition of angiogenesis when the control group was compared with all treated groups. These results suggest that subconjunctival injection of bevacizumab is able to inhibit corneal angiogenesis independently of the day of treatment.

O objetivo deste estudo foi avaliar os efeitos da aplicação subconjuntival de bevacizumab (Avastin®) na angiogênese corneal em ratos. Vinte ratos Wistar, machos, foram submetidos a cauterização química com cristal de nitrato de prata. Os animais foram divididos em 4 grupos: O grupo controle (GC), recebeu injeção de 0,02 ml de solução fisiológica pela via subconjuntival no momento da lesão. O grupo G0 recebeu 0,02 ml de bevacizumab (Avastin®) imediatamente depois da lesão. O grupo G3 recebeu 0,02 ml de bevacizumab no terceiro dia após a lesão.O grupo G5 recebeu 0,02 ml de bevacizumab no quinto dia após a lesão. Os animais foram eutanasiados 7 dias após a cauterização. Os vasos neoformados foram quantificados após preenchimento do leito vascular com Tinta da China e imagens foram obtidas e analisadas em sistema computadorizado (Image Pro-Plus®). No grupo controle a neovascularização ocupou 53,56 por cento ± 15,11 (média ± DP) da superfície corneal comparando a 35,57 por cento ± 18,80 no grupo G0, 30,60 por cento ± 11,82 (média ± DP) no G3 e 35,86 por cento ± 0,07 (média ± DP) no G5. Os resultados mostram uma inibição da angiogênese quando se compara GC com os grupos tratados. Os resultados sugerem que a injeção subconjuntival de Bevacizumab é capaz de inibir a angiogênese corneal independentemente do dia de aplicação.

Avaliações clínica e morfométrica da capacidade angiogênica da membrana de quitosana em córneas de coelhos / Clinical and morphometric evaluations of the angiogenic capacity of chitosan membrane in rabbit corneas

OBJETIVO: Estudar a vascularização corneal (VC) induzida pela membrana de quitosana (MQ) implantada por enxertia interlamelar na córnea de coelhos. MÉTODOS: Foram utilizados 16 coelhos. No olho esquerdo procedeu-se enxertia interlamelar de um fragmento de 0,25 x 0,25 cm de MQ (olho tratado). No olho direito realizou-se apenas a microbolsa estromal (olho controle). Avaliaram-se clinicamente os animais aos 1, 3, 7, 15 e 30 dias de pós-operatório. Aos 30 dias mensurou-se a VC pelo Sistema Analisador de Imagens LEICA QWIN-550®. RESULTADOS: Aos sete dias observou-se VC a 1,5±0,93 mm do limbo em direção ao eixo visual. Aos 15 dias houve aumento da VC (4,75±3,20 mm), que se manteve aos 30 dias (4,25±4,10 mm). Os olhos controles não apresentaram quaisquer alterações oculares. Houve diferença estatística (p<0,05) entre as áreas corneais vascularizadas dos olhos tratados e controles aos 15 e 30 dias de pós-operatório. CONCLUSÕES: A MQ induziu angiogênese corneal quando aplicada à córnea de coelhos por enxertia interlamelar, a qual persistiu de forma leve até 30 dias de pós-operatório. Embora estudos adicionais sejam necessários a MQ poderá ser mais uma opção de membrana para enxertos em ceratoplastias.

PURPOSE: To evaluate corneal vascularization (CV) induced by interlamellar graft chitosan membrane (CM) in rabbit cornea. METHODS: An interlamellar graft with a 0.25 x 0.25 cm CM fragment was performed in the left eye (treated eye). In the right eye, an estromal tunnel was done (control eye). The clinical evaluation was done at 1, 3, 7, 15 and 30 days postoperatively. CV analysis was after 30 days by the Images Analizator System LEICA QWIN-550®. RESULTS: After 7 days, CV at 1.5±0.92 mm from the limbus in direction of the cornea axial area was observed. After 15 days CV increased (4.75±3.19 mm), remaining until day 30 (4.25±4.06 mm). The control eyes did not present any changes. There was a statistical differences of the vascularizated corneal areas between control and treated eyes from the 15th to the 30th postoperative day. CONCLUSION: The chitosan membrane induced corneal angiogenesis when applied to rabbit cornea through an interlamellar graft, which was maintained at low levels until 30 days postoperatively. Although further studies are necessary, the results found here demonstrated the usefulness of chitosan membrane in keratoplasties.

The effects on inhibition of corneal neovascularization after human amniotic membrane transplantation in severely damaged rabbit corneas

Human amniotic membrane isolated from the placenta contained basement membrane components such as type IV collagen, laminin, and 6 and 4 integrins, all of which remained detectable while preserved in glycerin for one week. One month after the n-heptanol removal of the total corneal epithelium and the limbal lamellar keratectomy, all rabbit eyes carried features of limbal deficiency, including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Ten control eyes then received a total keratectomy, and 13 experimental eyes received an additional amniotic membrane transplantation. Three-month follow-ups revealed that all control corneas were revascularized to the center with granuloma and retained a conjunctival phenotype. In contrast, in the experimental groups, 5 corneas became clear with either minimal or no vascularization; the rest had either mild peripheral (5) or total (3) vascularization and more cloudy stroma. Using monoclonal antibodies for epithelial markers and matrix components, we concluded that the success correlated with the return of a cornea-like epithelial phenotype and the preservation of the amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. Measures taken to facilitate the former might prove this procedure clinically useful for ocular surface reconstruction.