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Background: Glaucoma is a chronic ocular disease of both dogs and humans that results in blindness and ocular discomfort. Most commonly, end-stage glaucomatous eyes in dogs are enucleated to provide comfort. This intervention requires significant financial investment, general anesthesia, and has a psychological impact on some owners. Retrobulbar neurolytic injections are used in humans to provide immediate and long-acting pain relief, while simultaneously preserving the globe. Objectives: To determine the safety and efficacy of retrobulbar neurolytic ethanol injection in canine eyes with end-stage glaucoma. Animals: 16 client-owned dogs (19 eyes) diagnosed with end-stage glaucoma. Methods: All eyes underwent an ophthalmic examination, including Schirmer Tear Testing (STT), intraocular pressure (IOP) measurement, corneal touch threshold (CTT), anterior and posterior segment examination, and fluorescein staining. Subjects were sedated and administered a retrobulbar block with bupivacaine, followed by retrobulbar injection of ethanol or saline solution. At specified time points after the procedure, clients assessed their pet's comfort and side effects of the injections via survey. Subjects returned for enucleation. Owner perceived comfort after the enucleation was assessed at identical post-procedure time points and compared to that achieved with retrobulbar ethanol injection or control solution. Overall client satisfaction with each procedure, as well as the effects of retrobulbar ethanol injection on STT, IOP, CTT, and histological changes in retrobulbar tissues were investigated. Results: Retrobulbar neurolytic ethanol injections did not signficiantly improve owner perceived comfort compared to control group treatment or provide more comfort than enucleation. Retrobulbar ethanol injections did not signficantly lower IOP, but did significantly elevate CTT. There was a trend towards lower STT in eyes receiving retrobulbar ethanol injections. Retrobulbar ethanol injections were safe, well tolerated, and no differences in client satisfaction with particpation in the study were noted in either injection group. Histologically, globes in the treatment group displayed significantly greater inflammation and fibrosis; retrobublar tissue samples were not significantly different between control and treatment groups with regard to inflammation or fibrosis. Conclusions and Clinical Importance: Retrobulbar neurolytic ethanol injections were not determined to be an effective globe-sparing alternative treatment to provide analgesia for end-stage canine glaucoma. Enucleation remains an effective way to provide comfort to dogs with elevated IOP. / Master of Science / The glaucomas represent a diverse group of blinding and painful diseases associated with elevated intraocular pressure (IOP). Despite advances in the medical and surgical treatment of glaucoma, the long-term prognosis in dogs remains dismal for IOP control, comfort, and globe retention. Blindness and pain are common long-term outcomes, necessitating surgical salvage procedures aimed at restoring patient comfort, including enucleation (eye removal), intrascleral prosthesis, or intravitreal chemical cyclodestruction. The most commonly performed, effective, and predictable of these options is enucleation, but this requires general anesthesia, a considerable financial investment, risks post-surgical complications, and has a negative psychological impact on some owners. Retrobulbar neurolytic injections with absolute ethanol have been performed in humans with blind, painful eyes since the early 1900s. Immediate and long lasting pain relief can be achieved from 2 weeks to 2 years after a single injection. The purpose of this study is to determine the safety and efficacy of retrobulbar ethanol injections as a globe-sparing therapeutic option for end-stage glaucoma in dogs. Nineteen dogs presenting to the VTH ophthalmology service with end-stage glaucoma were enrolled in a prospective, randomized, double-masked clinical trial. Subjects were sedated and administered a retrobulbar injection of ethanol (n=9) or control saline solution (n=10). At specified time points after the procedure, clients assessed their pet’s comfort and side effects of the injections via survey. Three weeks later, subjects returned for enucleation and the level of comfort after the enucleation was assessed at identical post-procedure time points and compared to that achieved with retrobulbar ethanol injection or control solution. Retrobulbar neurolytic ethanol injections did not significantly improve comfort compared to control group sham treatment or provide more comfort than enucleation. Retrobulbar ethanol injections did not significantly lower IOP, but did significantly elevate corneal touch threshold in treated patients. There was a trend towards lower tear production in eyes receiving retrobulbar ethanol injections. Retrobulbar ethanol injections were safe, well tolerated, and no differences in client satisfaction with participation in the study were noted in either injection group. Further investigation is warranted to determine the optimal volume of retrobulbar ethanol to provide analgesia for patients with end-stage glaucoma as well as to determine the duration of clinical effect of these injections.

Vascular endothelial growth factor A (VEGF) is a potent mediator of blood vessel formation throughout the body. Intraocular diseases characterized by inflammation, hypoxia or neoplasia induce new blood vessel formation within the eye. The end result of such blood vessel formation may be blinding sequellae such as glaucoma from outflow obstruction or hyphema from intraocular hemorrhage. Elevated VEGF concentrations in the aqueous humor and vitreous are documented in a number of human intraocular disease processes, including tumors, retinal detachment and uveitic glaucoma. Pharmacotherapy inhibiting VEGF expression demonstrates promise for control of some of these ophthalmic conditions. We quantified and compared VEGF concentrations in canine aqueous humor samples from 13 dogs with normal eyes and 226 eyes from 178 dogs with a variety of ophthalmic diseases by ELISA. Dogs with primary cataract, diabetic cataract, primary glaucoma, uveitic glaucoma, aphakic/pseudophakic glaucoma, retinal detachment, lens luxation and neoplasia were evaluated. Elevated VEGF concentrations were found in all disease conditions tested as compared to normal dogs excepting cataracts and diabetic cataracts. Elevated aqueous humor VEGF concentrations were found in dogs with pre-iridal fibrovascular membranes (PIFM) as compared to dogs without PIFM. These results are consistent with the hypothesis that VEGF has a role in the causation or progression of a variety of canine ocular disorders. / Master of Science

Connective tissue remodeling is an important area of study in biomedical engineering with respect to cancer and wound healing. Tissue remodeling components may be involved in the pathogenesis of open-angle glaucoma. Risk factors for open angle glaucoma include increased intraocular pressure (IOP), male gender, and advanced age. In a 1963 study, the hormone relaxin decreased IOP in the human eye through a mechanism that may involve the up-regulation of tissue remodeling matrix metalloproteinases (MMPs). The effects of age and gender on MMP and protein activity in porcine aqueous humor were determined in this study to identify correlations existing between MMP activity and glaucoma risk factors. Gelatin zymography identified MMPs at 66 kD and approximately 105 kD. The concentration of the 66 kD band compared to human MMP-2 standard was 0.22 ± 0.06 ng/ml for the adult female (AF) samples and 0.28 ± 0.04 ng/ml for the juvenile samples. This difference in concentration was statistically significant (p < 0.05). The concentration of the protease migrating to 66 kD was statistically independent of gender. Casein zymograms identified two non-MMP proteinases at 51 kD and 80 kD. The average total protein concentration for all aqueous humor samples was 2.54 ± 0.89 mg/ml. The mean IgG, transferrin, and albumin concentrations for all aqueous humor samples was 11.4 ± 4.2 mg/ml, 17.11 ± 6.8 mg/ml, and 78.0 ± 26.3 mg/ml respectively. Results from these experiments establish baseline levels of MMP and protein activity, allowing for identification of potential changes caused by relaxin in tissue culture studies. / Master of Science

O peixe dourado é extensivamente utilizado em pesquisas sobre visão devido a diversos fatores: é um animal pecilotermo, o que possibilita manter sua retina ex-vivo viável para manipulação experimental por mais tempo; apresenta uma organização retiniana semelhante à de mamíferos; possui neurônios grandes, facilitando estudos eletrofisiológicos e morfológicos e, diferentemente de mamíferos, é dotado de neurogênese constante de alguns tipos celulares retinianos. A produção, composição e escoamento dos meios oculares nesta espécie, fundamentais para a formação de imagens sobre a retina, são, entretanto, pouco conhecidos. A variedade padrão desta espécie apresenta desenvolvimento ocular normal, enquanto a variedade demekin é extremamente míope por possuir crescimento ocular exagerado (buftalmia), condição característica de alguns tipos de glaucoma em mamíferos. O presente trabalho investigou aspectos morfofisiológicos do sistema de produção e drenagem do humor aquoso e a composição do humor vítreo nestas duas variedades de peixe dourado, com o objetivo de melhor compreender as causas subjacentes ao crescimento ocular do demekin. Para estudar a drenagem do humor aquoso, foram realizadas injeções de corante na câmara anterior de animais das duas variedades, com processamento histológico posterior em intervalos de tempo pré-determinados (5, 15, 30, 60, 120 minutos e 1, 2, 3 4 e 7 dias, n <= 3 olhos de cada variedade por grupo experimental). Não foram observadas diferenças na drenagem de humor aquoso entre as duas variedades. Em ambas, o humor aquoso é drenado principalmente pela malha trabecular contida na porção ventral do ângulo de drenagem, entre a íris e o ligamento anular. Duas vias secundárias de drenagem foram encontradas nas duas variedades de peixe dourado estudadas: (i) a via irídica e (ii) a drenagem através de vasos epirretinianos. Estas vias são aparentemente mais lentas do que a via trabecular, uma vez que foi encontrado corante nessas regiões até quatro dias após as injeções de corante na câmara anterior. Foi realizado também estudo ultraestrutural da retina e epitélio ciliar em ambas as variedades através de microscopia eletrônica de transmissão. O epitélio pigmentado da retina do demekin apresentou grânulos de melanina mais escassos, e observamos certa desorganização da camada de fotorreceptores, bem como redução da sua densidade. Através de aparato invasivo construído por Joselevitch e Smith (1997), foi mensurada a pressão intraocular (PIO) de 14 olhos de cada variedade. O demekin apresentou valores de PIO ligeiramente aumentados em relação ao animal padrão (3,6 ± 0,9 mmH2O para o demekin e 1,9 ± 0,6 mmH2O para o peixe dourado padrão). A composição proteica do corpo vítreo foi estudada através de eletroforese em gel de poliacrilamida utilizando um pool de humor vítreo de 4 animais da variedade padrão e 2 da variedade demekin. As proteínas de baixa massa molecular (entre 15 a 25 kDa) estavam expressas em ambas - embora com maior expressão na variedade padrão - e outras, de maior massa molecular, expressas apenas no humor vítreo do demekin (50-75 kDa e 150 kDa). Os resultados obtidos sugerem, portanto, que há diferenças importantes na ultraestrutura retiniana, na composição proteica do humor vítreo e na pressão intraocular do demekin. Para melhor compreensão do crescimento ocular desta variedade faz-se necessária a realização de um estudo proteômico, identificando quais são as proteínas do humor vítreo evidenciadas através da eletroforese, bem como estudos morfológicos em animais de diferentes tamanhos visando a quantificação dos danos retinianos decorrentes do seu crescimento ocular / The goldfish is extensively used in vision research due to several factors: it is a poikilothermic animal, which allows ex-vivo experiments using its retina for longer periods of time; its retinal organization is very similar to that of mammals; it has large neurons, which facilitates electrophysiological and morphological studies and, unlike mammals, it is endowed with constant neurogenesis of some retinal cell types. However, the production, composition and outflow of ocular media in this species, which are fundamental for image formation onto the retina, are poorly understood. The standard goldfish has normal eye development, while the Black Moor goldfish is extremely myopic due to excessive eye growth (buphthalmos), a condition that is present in certain types of mammalian glaucoma. The present study investigated morphophysiological aspects of the aqueous humor production and drainage system and vitreous humor composition in these two varieties of goldfish to better understand the causes underlying the eye growth of the Black Moor. To study the drainage of the aqueous humor, anterior chamber dye injections and histological processing were performed in animals of both varieties after predetermined time intervals (5, 15, 30, 60, 120 minutes and 1, 2, 3, 4, and 7 days, n <= 3 eyes of each variety for each experimental group). No differences were observed in the drainage of aqueous humor between the two varieties. In both, the aqueous humor is drained mainly by the trabecular meshwork contained in the ventral portion of the drainage angle, between the iris and the annular ligament. Two secondary drainage routes were found in the two goldfish varieties studied: (i) the iridic route and (ii) drainage through epiretinal vessels. These pathways are apparently slower than the trabecular route, since the dye was found in these regions 4 days after the injections. An ultrastructural study of the retina and ciliary epithelium was also carried out in both varieties through transmission electron microscopy. The retinal pigment epithelium of the Black Moor presented more scarce melanin granules and we observed some disorganization of the photoreceptor layer, as well as a reduction in its density. Through an invasive device constructed by Joselevitch and Smith (1997), the intraocular pressure (IOP) of 14 eyes of each variety was measured. The Black Moor presented slightly increased IOP values compared to the standard animal (3.6 ± 0.9 mmH2O for demekin and 1.9 ± 0.6 mmH2O for the standard goldfish). The protein composition of the vitreous body was studied by polyacrylamide gel electrophoresis using a vitreous humor pool of 4 animals of the standard variety and 2 of the Black Moor variety. Low molecular weight proteins (15 to 25 kDa) were expressed in both - although with greater expression in the standard variety - and others of higher molecular mass, expressed only in the vitreous humor of the Black Moor (50-75 kDa and 150 kDa). These results suggest, therefore, that there are important differences in the retinal ultrastructure, the protein composition of the vitreous humor and the intraocular pressure of the Black Moor. To better understand the ocular growth in this variety, it is necessary to carry out a proteomic study, identifying which are the vitreous humor proteins evidenced through electrophoresis, as well as morphological studies in animals of different sizes aiming the quantification of the retinal damages caused by its ocular growth

O glaucoma é definido como uma típica neuropatia óptica caracterizada por perda de células ganglionares e consequente lesão no nervo óptico, resultando em uma gradual redução do campo visual, podendo causar cegueira. Considerando que os compostos presentes nas peçonhas de aranhas representam uma fonte importante de moléculas bioativas, o estudo dos possíveis efeitos neuroprotetores destas substâncias, em modelos experimentais de glaucoma, é uma etapa indispensável para o desenvolvimento de novos fármacos para o tratamento desta neuropatologia. Neste contexto, nosso grupo de pesquisa tem investigado compostos neuroativos isolados da peçonha da aranha Parawixia bistriata. Até o momento três moléculas isoladas, a PbTx1.2.3 (Parawixina 1), a FrPbAII (Parawixina 2) e a Parawixina 10 mostraram efeitos anticonvulsivantes e neuroprotetores, devido à suas ações na facilitação da recaptação de L-GLU (Parawixinas 1 e 10) ou na inibição da recaptação de GABA (Parawixina 2). Além destes compostos também isolamos a Parawixina 11 (PW11). Os resultados obtidos até agora demonstraram que a Parawixina 11 possui efeito anticonvulsivante em ratos submetidos à indução de crises por vários convulsivantes e foi neuroprotetora após indução de Status epilepticus por pilocarpina. Sendo assim, o presente estudo consistiu em verificar se a PW11 também seria neuroprotetora em um modelo de glaucoma agudo e tentar inferir seu mecanismo de ação, comparando-a e asssociando-a ao muscimol, ao ácido nipecótico, ao ALX 5407 e ao riluzol. Para isso, o modelo utilizado foi o de aumento da pressão intra-ocular (PIO), interrompendo o fluxo sanguíneo na retina e causando isquemia. Foi utilizado um sistema com pressão de ar, constituído por uma agulha de punção venosa de 27 G, conectada a um reservatório de ar acoplado a um manômetro. Ratos Wistar (200 - 250 g) foram anestesiados e cada grupo (n=4) recebeu uma injeção intravítrea (volume de 1,2 µL) no olho esquerdo, de uma das seguintes substâncias: água deionizada (VE1); DMSO a 4,7%; 0,05µg/µL de PW11; 0,10 µg/µL de PW11; 0,20 µg/µL de PW11; 5 µg/µL de muscimol; 3 µg/µL de ácido nipecótico; 0,30 µg/µL de ALX 5407; 23,45 µg/µL riluzol. Outros grupos receberam uma injeção (volume de 2,4 µL) com uma das seguintes associações: 0,10 µg/µL PW1 + muscimol; 0,10 µg/µL de PW11 + ácido nipecótico; 0,10 µg/µL de PW11 + ALX 5407 e 0,10 µg/µL de PW11 + riluzol. Quinze minutos após a injeção, foi aplicada uma pressão de 120 mmHg, por 45 min, causando isquemia retiniana. Alguns animais, que faziam parte do grupo isquemia (ISQ), foram sacrificados logo após esse tempo. Já os do grupo isquemia/reperfusão (ISQ/REP) tiveram a PIO normalizada durante 15 min, após a isquemia, permitindo o retorno do fluxo sanguíneo na retina, e só então foram sacrificados. Em seguida, os olhos foram removidos e fixados em solução ALFAC. As retinas isquêmicas com e sem reperfusão apresentaram núcleos picnóticos, vacuolização citoplasmática, edema e desorganização das camadas, característicos deste tipo de lesão. Após ISQ, os tratamentos com 0,10 e 0,20 µg/µL de PW11 preservaram, respectivamente, 17,51 e 37,45% de células na CNI, se comparados com o grupo VE1. Na CCG, quando a PW11 foi associada ao agonista do receptor GABAA, o muscimol, e ao inibidor do transportador de GABA do tipo GAT1, o ácido nipecótico, houve proteção significativa de 37,07 e 44,81%, respectivamente, se comparada ao grupo VE1, indicando inespecificidade da PW11 no sistema gabaérgico. Após ISQ/REP, as densidades celulares na CCG das retinas tratadas com 0,05, 0,10 e 0,20 µg/µL de PW11 foram, respectivamente, 32,88, 24,05 e 28,27% maiores do que a das retinas do grupo VE1. Já na CNI, as densidades celulares após os tratamentos com PW11 foram, respectivamente, 38,78, 35,25 e 30,96% maiores do que a das retinas do grupo VE1. Com relação à presença de neurônios em degeneração marcados com Fluro-Jade C, as retinas dos animais tratados apresentaram menos marcações na CNI, CPI e CCG, se comparadas com as dos grupos VE1 e DMSO. Diante destes efeitos, conclui-se que a PW11 pode ser uma interessante ferramenta para o desenvolvimento de terapias que combinemdiferentes drogas no tratamento do glaucoma e de outras neuropatologias. / Glaucoma is defined as a typical optic neuropathy characterized by ganglion cells loss and consequent optic nerve damage, resulting in a gradual reduction of the visual field and eventual blindness. In regard to compounds present in spider venoms, they represent an interesting source of bioactive molecules and the study about the possible neuroprotective effects of these substances, in experimental models of glaucoma, is an important step to developed new drugs to treating this disease. Therefore, our research group has investigated neuroactive compounds isolated from Parawixia bistriata spider venom. Nowadays, three purified molecules, PbTx1.2.3 (Parawixin 1), FrPbAII (Parawixin 2) and Parawixin 10 showed anticonvulsant and neuroprotective effects. They enhance the L-gluatamate uptake (Parawixin 1 and 10) or inhibit the GABA uptake (Parawixin 2). In addition these compounds, we also isolated the Parawixin 11 (PW11). So far, the results with PW11 showed the anticonvulsant effect in rats after seizures drug-induced and neuroprotetive effect after Status epilepticus pilocarpine -induced. Nevertheless, this study was to exemine if Pw11 could also be neuroprotective in acute glaucoma model and attempt to infer the mechanism of action, comparing it and associating it to muscimol, nipecotic acid, ALX 5407 and riluzole. For this, the model used was the intraocular pressure (IOP) elevation, disrupting the blood flow in retina and causing ischemia. It was used a system with air pressure, comprising of a 27G venous puncture needle connected to an air reservoir coupled to a manometer. Male Wistar rats (200-250 g) were anesthetized and each group (n=4) received an intravitreal injection (volumn = 1,2 µL) in the left eye of one of the following substances: deionized water (VE1); DMSO 4,7%, 0,05 µg/µL of PW11; 0,10 µg/µL of PW11; 0,20 µg/µL of PW11; 5 µg/µL of muscimol;3µg/µL of nipecotic acid; 0,30 µg/µL of ALX 5407;23,45 µg/µL of riluzole. Other groups received an injection (volumn = 2,4 µL) with one of the following associations: 0,10 µg/µL of PW11+muscimol; 0,10 µg/µL of PW11+nipecotic acid; 0,10 µg/µL of PW11+ALX 5407 and 0,10 µg/µL of PW11+riluzole. Fifteen minutes later, the IOP was raised up to 120 mmHg for 45 min, inducing retinal ischemia. Some animals, which were ischemia group (ISCH), were sacrificed immediately thereafter. The ischemia/reperfusion (ISCH/REP) group animals had IOP normalizedduring 15 min, after ischemia, making possible the blood flow return and, then, they were euthanized. Then, the eyes were removed and fixed in ALFAC solution. The ischemic retinas with or without reperfusion showed piknotic nuclei, citoplasmatic vacuolization, edema and disorganization of the retinal layers, characteristics of this lesion type. After ISCH, treatments with 0.1 and 0.2 µg/µL of PW11 preserved, respectively, 17.51 and 37.45% of cells in the INL if compared with VE1 group. In the GCL, when PW11 was associated to muscimol, a GABAA receptor agonist, and to nipecotic acid, a GAT-1 transporter inhibtor, there was significant protection of 37.07 and 44.81%, respectively, if compared with VE1 group, indicating nonspecificity of PW11 in gabaergic system. After ISCH/REP, cell densities in GCL of treated retinas with 0.05, 0.10 ad 0.20 µg/µL of PW11 were 32.88, 24.05 and 28.27% higher than that of VE1 group retinas, respectively. In INL, the cell densities after PW11 treatments were 38.78, 35.25 and 30.96% higher than that of VE1 group retinas, respectively. Regarding the presence of Fluoro-Jade C (FJC) stained degenerating neurons, retinas from treated animals showed less FJC-positive neurons in INL, IPL and GCL if compared with those of VE1 and DMSO groups. In view of these effects, it is concluded that the PW11 can be a useful tool to developed therapies that combine different drugs in glaucoma and other neuropathology treatments.

INTRODUÇÃO: É importante estudar a efetividade da trabeculectomia e estratégias para manter sua ação ao longo do tempo. Para melhorarmos o prognóstico das revisões cirúrgicas é imprescindível entendermos melhor a estrutura da bolha filtrante e suas possíveis causas de falência. Formas alternativas para se restabelecer a drenagem e o funcionamento da trabeculectomia não funcionante foram desenvolvidas, entre elas a Revisão Interna de Simmons. OBJETIVOS: Avaliar os efeitos da Revisão Interna de Simmons sobre a PIO; sobre o aspecto biomicroscópico da bolha no pós-operatório e sua correlação com achados biométricos obtidos pela biomicroscopia ultra-sônica. CASUÍSTICA E MÉTODOS: Foram incluídos e estudados de forma prospectiva por 6 meses, 29 pacientes portadores de glaucoma primário, submetidos a trabeculectomia prévia,há pelo menos três meses, que apresentavam PIO acima do esperado para o controle do glaucoma, apesar de medicação hipotensora utilizada. Foram fatores de inclusão a conjuntiva livre e o óstio interno da trabeculectomia patente à gonioscopia. O critério de sucesso foi: abaixamento da PIO, igual ou maior que 30% da PIO em relação à PIO pré-operatória e/ou pressão final menor ou igual a 15mmHg, com ou sem medicação antiglaucomatosa associada. RESULTADOS: Houve uma queda estatisticamente significante da PIO e do número de medicações utilizadas após a cirurgia. A PIO média antes da operação era de 23,72 ± 4,10 mmHg e ao término do estudo, de 15,04 ± 4,00 mmHg (p=0,000016). A média das pressões da curva tonométrica antes da cirurgia era de 22,36 ± 3,91mmHg, caindo para 15,01 ± 3,95mmHg no PO180d (p=0,0000001). Antes da cirurgia, a média do número de medicações usadas era de 2,1 ± 0,77 e ao término do estudo, 1,22 ± 1,01 (p=0,001117). Houve um aumento do tamanho da bolha filtrante, avaliado pela biomicroscopia. As bolhas apresentaram altura média de 0,28 ± 0,65 e terminaram o estudo com altura de 0,78 ± 0,85 (p=0,000016). Não houve diferença na altura da bolha, medida pela biomicroscopia ultra-sônica após a cirurgia. A pressão intra-ocular apresentou relação inversamente proporcional à dimensão da bolha filtrante, com significância estatística. As complicações mais encontradas foram: descolamento da membrana de Descemet, no local da incisão principal em 5 doentes (17,2%), abertura inadvertida da conjuntiva em 4 (13,8%), e descolamento de coróide, em 3 (10,3%). CONCLUSÕES: A Revisão Interna de Simmons proporcionou diminuição de 30% da PIO em 52% dos pacientes e redução do número de medicações antiglaucomatosas associadas, quando utilizadas. As bolhas se mostraram-se estatisticamente maiores após o procedimento. Houve uma correlação perfeita entre a classificação da altura da bolha entre a medida biomicroscópica baseada na escala de Indiana e a biométrica obtida pela biomicroscopia ultra-sônica. Não houve aplanamento da curva tonométrica apesar desta ter diminuído 32%. Não foi observada mudança significativa no vício de refração ou na acuidade visual após a operação. A biomicroscopia ultra-sônica demonstrou uma associação inversa entre um maior número de complicações pós-operatórias e o tamanho menor do óstio interno da trabeculectomia. / INTRODUCTION: It is important to study the trabeculectomy effectiveness and strategies to keep its action within long time. To improve the prognostic presented on glaucoma surgical reviews it is essential to understand better the bleb structure and its possible cause of failing. Alternative ways of restoring the drainage and the good performance of trabeculectomy have been studied, among them the Internal Revision of Simmons. PURPOSE: Evaluate the surgery effects on the IOP; on the biomicroscopic aspect of the bleb and its co-relation with biometric founding gotten through UBM. METHODS: For six months, 29 patients with open angle glaucoma who had been submitted to trabeculectomy before, for at least three months, with IOP above the expected to the control of glaucoma, despite the medication used, were included and studied in a prospective way. These patients should present the conjunctive tissue not too scarry and internal ostio of trabeculectomy present on the gonioscopia. The success criterion was: lowering the IOP, equal or above 30% on pre-operatory IOP and/or final pressure below 15 mmHg, with or without eye drops. RESULTS: There was a statistically fall of IOP and amount of medication used after the surgery. The average of IOP before surgery was 23.27 ± 4.10 mmHg and at the end of study was 15.04 ± 4.00 mmHg (p=0.000016). The average of the IOP curve pressure before surgery was 22.36 ± 3.91mmHg, dropped to 15.01 ± 4.0 mmHg at PO180d (p=0.0000001). Before the surgery the average of medication used was 2.1 ± 0.77 and at the end of the study it was 1.22 ± 1.01 (p=0.001117). There was an increase of the filtering bleb size clinically measured. The blebs had an average height of 0.28 ± 0.65 and at the end of the study their height was 0.78 ± 0.85 (p=0.000016). The UBM didn?t show any difference in the bleb size. The intra-ocular pressure presented an inversely proportional relation to the dimension of the filtering bleb, with statistic significance. The most frequently problems found were: detachment of Descemet membrane where the main incision was performed, in 5 (17.28%) patients; hole in the conjuntiva in 4 (13.88%), detachment of the choroid in 3 (10.38%). CONCLUSIONS: The surgery was effective, for provided 30% decrease of initial IOP in 52% without or with eye drops statistically in small number than in presurgery. The blebs have appeared, statistically, bigger after the procedure. There was a perfect correlation between the bleb height classification, within the clinical measurement, based on the Indian scale, and the biometric one, gotten by UBM. There was no flattening of the IOP curve pressure despite this one had been 32% lower than the initial one. No significant change has occurred in the vicious of refraction or in the visual accuracy after the surgery. The UBM demonstrated an association between a larger number of complications post-surgery and the internal size of the trabeculectomy ostio.

O objetivo deste estudo foi comparar alterações histológicas induzidas por análogos de prostaglandinas e maleato de timolol na conjuntiva de coelhos. Cinqüenta coelhas da raça Nova Zelândia com idade e peso semelhantes foram divididos em cinco grupos de dez animais. Os olhos esquerdos foram tratados com uma gota diária de bimatoprosta 0,03%, travoprosta 0,004%, latanoprosta 0,005%, maleato de timolol 0,5% ou lágrimas artificiais contendo cloreto de benzalcônio (CBA) durante 30 dias. Os olhos contra-laterais serviram como controles. Realizaram-se biópsias na conjuntiva limbar superior com dimensões de 5 mm x 5 mm no oitavo e trigésimo dias em cinco coelhos de cada grupo. A conjuntiva foi imediatamente fixada com formaldeído a 10% por 24 horas, seguido de coloração por Hematoxilina-Eosina (HE) e Ácido Periódico de Shiff (PAS) e avaliação qualitativa por microscopia óptica. A avaliação histo-morfométrica quantitativa foi realizada usando o programa Image Pro-Plus 4.5. Os parâmetros avaliados foram: infiltrado inflamatório, espessura epitelial, número de células caliciformes, diâmetro e número de vasos sanguíneos. Observou-se leve infiltrado inflamatório focal no tecido conectivo subepitelial nos olhos controle e do grupo CBA, que consistia de aglomerados de linfócitos e raros neutrófilos. Todos os demais grupos exibiram no oitavo e trigésimo dias de tratamento infiltrado inflamatório moderado, composto por linfócitos e neutrófilos, que foi mais denso no grupo tratado com maleato de timolol do que nos grupos dos análogos de prostaglandinas. No trigésimo dia, o grupo que recebeu timolol mostrou aumento da densidade subepitelial de colágeno e aumento estatisticamente significante da espessura epitelial (P=0,004), que não foram observados nos demais grupos. Observou-se um aumento estatisticamente significante do número de vasos no grupo tratado com timolol (P=0,001). O diâmetro vascular no grupo que recebeu travoprosta apresentou aumento estatisticamente significante no oitavo dia de tratamento (P=0,008) e redução no 30º dia (P=0,035). A densidade de células caliciformes aumentou significativamente no grupo que recebeu travoprosta no oitavo dia (P=0,001) e no 30º dia nos grupos tratados com bimatoprosta (P=0,002) e latanoprosta (P=0,009). Conclui-se que, na conjuntiva de coelhos, o número de células caliciformes aumenta com o uso de análogos das prostaglandinas. O estudo sugere que essas drogas, entretanto, induzem menos alterações que o maleato de timolol na conjuntiva de coelhos / The purpose of this study was to compare histological changes induced by prostaglandin analogues and timolol maleate in the rabbit conjunctiva. Fifty New Zealand female rabbits with similar age and weight were divided in five groups of 10 animals. The left eyes were treated with one daily drop of bimatoprost 0,03%, travoprost 0,004%, latanoprost 0,005%, timolol maleate 0,5% or artificial tears containing benzalkonium chloride (BAC) for 30 days. The fellow eyes served as controls. Limbal superior conjunctival biopsies of 5 mm x 5 mm were performed at the 8th and 30th day in five rabbits of each group each time. The conjunctiva was immediately fixed with 10% formaldehyde for 24 hours, followed by Hematoxiline-Eosine (HE) and Periodic Acid Shiff (PAS) staining and optical qualitative microscopic evaluation. Morphohistometric quantitative analyses were conducted using the Image Pro-Plus 4.5 software. The evaluated parameters were: inflammatory infiltrate, epithelial thickness, number of goblet cells, diameter and number of blood vessels. A low level infiltration, consisting of localized clusters of lymphocytes and rare neutrophils, was observed in the subepithelial connective tissue of the control eyes and in the BAC group. At the 8th and 30th day post treatment, all other groups exhibited a diffuse inflammatory infiltrate, composed by lymphocytes and neutrophils, which was denser in the timolol group than in the prostaglandin analogue groups. At the 30th day, the timolol group also showed an increased subepithelial collagen density and a significant increase in epithelial thickness (P=0.004), which was not present in the other groups. A significant increase (P =0.001) in the number of blood vessels was observed in the group treated with timolol. An initial increase in the vascular diameter was observed at the 8th day in the travoprost group (P=0,008) although a significant reduction was observed at the 30th day (P =0,035). The goblet cell density was significantly increased at the 8th day in the group treated with travoprost (P =0.001) and at the 30th day in those treated with bimatoprost (P =0.002) and latanoprost (P =0.009). We conclude that an increase in the number of goblet cell was initially observed with the use of all prostaglandin analogues. This study suggests that these drugs, however, induce fewer changes than timolol maleate in the rabbit conjunctiva

Introdução: A cicatrização subconjuntival excessiva é a maior causa de falha cirúrgica no glaucoma. Uma vez que o ácido rosmarínico apresenta ação antifibrótica, é interessante avaliar seu uso como adjuvante na cirurgia filtrante do glaucoma. Objetivos: Comparar os achados clínicos e histopatológicos e a expressão gênica de fatores relacionados à cicatrização tecidual entre coelhos submetidos à cirurgia antiglaucomatosa experimental (CAGE) e tratados com injeções subconjuntivais de ácido rosmarínico ou com injeções de solução salina 0,9%. Material e Métodos: Estudo experimental intervencionista, do tipo ensaio clínico randomizado, no qual 41 coelhos New Zealand foram submetidos à CAGE no olho direito e divididos em dois grupos de tratamento: grupo A (n=21), tratados com injeções subconjuntivais de 0,1 ml de solução salina 0,9% e grupo B (n=20), tratados com injeções subconjuntivais de 0,1 ml de ácido rosmarínico 15 mg/ml (3 dias antes da cirurgia, ao final desta e a cada 3 dias até a eutanásia). Os efeitos do tratamento foram avaliados com cinco dias (grupos A1 e B1) e com 15 dias (grupos A2 e B2) por meio da avaliação da pressão intraocular (PIO) com Tonopen, de aspectos da ampola filtrante com base no Moorfields Bleb Grading System, do estudo histopatológico (contagem de neutrófilos/campo, vasos/campo, densidade de colágeno corado com Sirius Red, imuno-histoquímica anti--actina e anti-VEGF) e do RT-PCR (colágeno tipo 1 alfa-1 COL1A1 e TGF-2). As fotomicrografias foram avaliadas quantitativamente com o software ImageJ® 1.36 (Sirius Red, -actina e VEGF) e os dados comparados por testes não paramétricos, sendo p<0,05 considerado estatisticamente significativo. Resultados: Não houve diferença significativa nas comparações da PIO entre os grupos. Nas avaliações da ampola filtrante, apenas o grupo B2 apresentou maiores escores de área central (p=0,0004), área máxima (p=0,0007) e altura (p=0,0239). Os grupos B1 (p<0,0001) e B2 (p=0,0011) apresentaram escores significativamente menores no parâmetro vascularização que os seus respectivos controles. Houve diminuição significativa de neutrófilos/campo somente no grupo B2 (p=0,0159). O número de vasos/campo também foi significativamente menor nos grupos B1 e B2, em comparação aos seus respectivos controles (p=0,0079 e p=0,0345, respectivamente). Não foram observadas diferenças significativas na coloração com Sirius Red e na imuno-histoquímica para anti--actina entre os grupos, porém o grupo B1 apresentou menor marcação anti-VEGF (p=0,0190). As análises por RT-PCR para TGF-2 e COL1A1 não demonstraram diferenças entre os grupos, com exceção do grupo B2, que apresentou maior expressão de COL1A1 quando comparado ao seu controle (p=0,0159). Conclusões: Os resultados apontaram para os potenciais efeitos anti-inflamatórios e moduladores da neovascularização do ácido rosmarínico em cirurgias antiglaucomatosas. Apesar do grupo B demonstrar melhor aspecto clínico (exceto a PIO, que não demonstrou diferença entre os grupos), a ação antifibrótica do ácido rosmarínico não pôde ser comprovada. / Introduction: Excessive subconjunctival scarring is a major cause of surgical failure in glaucoma. Since rosmarinic acid has antifibrotic action, we evaluate its usage as an adjunctive treatment to glaucoma filtration surgery. Objectives: To compare clinical, histopathological and expression of factors related to the healing process between rabbit underwent experimental glaucoma surgery (EGS) and treated with subconjunctival injections of rosmarinic acid or with saline solution 0.9%. Methods: Interventional experimental study (randomized clinical trial) in which 41 New Zealand rabbits were submitted to EGS in the right eye and divided into two treatment groups: group A (n=21) - eyes treated with subconjunctival injections of 0.1 ml of saline solution 0.9% and B (n=20) - eyes treated with subconjunctival injections of 0.1 ml of rosmarinic acid 15mg/ml (three days prior to surgery, at the end of surgery and every three days until euthanasia). Treatment effects were assessed after five days (groups A1 and B1) and 15 days (groups A2 and B2) by evaluation of intraocular pressure (IOP) with Tonopen and bleb characteristics based on the Moorfields Bleb Grading System, as well as histopathology analysis (neutrophil/field count; vessels/field count; collagen density after stained with Sirius Red; and anti--actin and anti-VEGF immunohistochemistry) and by RT-PCR (for collagen type 1 alpha 1 - COL1A1 and TGF-2). The photomicrographs were evaluated quantitatively with ImageJ® 1.36 software (Sirius Red, -actin and VEGF) and data were compared through non-parametric tests, considering p<0.05 as statistically significant. Results: No significant difference was observed in IOP measurements between groups. Group B2 displayed significant higher scores of bleb central area (p=0.0004), maximum area (p=0.0007) and height (p=0.0239). Both B1 (p<0.0001) and B2 (p=0.0011) groups presented significantly lower vascularization scores than their respective controls. A significant decrease in neutrophils/field counting was observed only in group B2 (p=0.0159), however vessels/field counting was significantly lower in groups B1 and B2, compared to their controls (p=0.0079 and p=0.0345, respectively). No significant differences were observed in the collagen staining with Sirius Red and in the anti--actin immunohistochemistry between the all groups comparison, but group B1 displayed lower anti-VEGF measurement (p=0.0190) than group A1. RT-PCR analysis for TGF-2 and COL1A1 showed no difference between groups, except by group B2, which presented higher expression of COL1A1, compared to its control (p=0.0159). Conclusions: The findings point to the potential anti-inflammatory and anti-neovascular effects of rosmarinic acid in glaucoma surgery. Although group B show better clinical aspect (except IOP, which showed no difference between groups), the antifibrotic action of rosmarinic acid could not be proved.

Made available in DSpace on 2016-08-10T10:38:34Z (GMT). No. of bitstreams: 1 CONSTANZA THAISE XAVIER SILVA.pdf: 856711 bytes, checksum: aa9a8d8094cd18595af6092fc0d17683 (MD5) Previous issue date: 2012-03-12 / Glaucoma refers to a group of eye diseases that gradually evolve being characterized by typical damage to the optic nerve with consequent changes in vision. The most common type is the primary open angle glaucoma (POAG) corresponding to approximately 60% of cases. The ocular epithelia expressed genes encoding the enzymes Glutathione STransferase (GST). The GST is present in various ocular structures, including aqueous humor, ciliary body, and lens. This study aimed to evaluate the genotypic profile of the gene GSTT1 and GSTM1 polymorphisms in patients with Primary Open Angle Glaucoma and control group in the city of Goiânia. Peripheral blood samples were analyzed by up to 100 samples of patients diagnosed as carriers of glaucoma, proven and 53 specimens from patients with examination within the customary standards, representing the control group. The polymorphism was assessed by PCR and analyzed in 2% agarose gel, stained with etídeo bromide. The frequencies of polymorphisms of GSTM1 and GSTT1 genes were compared with the &#967;2 test (p < 0.05%) and rate risk was assessed by the test of Odds Ratio with 95% confidence interval (&#945; = 0.05). In case it was noted that this was of genotype GSTM1 40% (n = 40) and in controls was 71.6% (n = 38). The null genotype was 60% (n = 60) and 28.3% (n = 15), respectively. The GSTT1 genotype present in case group was 52% (n = 52) and in the control group was 66% (n = 35); already the null genotype was 48% (n = 48) in case group and 34% (n = 18) in the control group. GSTM1 null genotype in the case group was more frequent than in the control group, and this difference statistically significant (p = 0. 0004). The same was not found with the GSTT1 genotype (p = 0.13). Checked also the Association of genotypes GSTM1 null/GSTT1 this the risk of glaucoma to 3.1 times more the chance of disease occurrence, suggesting that individuals who have the GSTM1 null genotype/GSTT1 this can be regarded as one of the risk factors for the development of the POAG. Already for the genotypes GSTM1 null was verified the GSTT1\/risk of 6.7 times more chance of occurrence of disease (p = 0.0004; OR: 6.7; 95% CI:-2.7 20.3), suggesting that individuals who have the GSTM1 null genotype/GSTT1 can be regarded as one of the risk factors for the development of the POAG. / O Glaucoma designa um grupo de doenças oculares que evoluem progressivamente sendo caracterizadas por danos típicos no nervo óptico com consequentes alterações de visão. O tipo mais frequente é o glaucoma primário de ângulo aberto (GPAA) que correspondente aproximadamente 60% dos casos. O epitélio ocular expressa genes que codificam as enzimas Glutationa S-Transferase (GST). A GST está presente em várias estruturas oculares, incluindo humor aquoso, corpo ciliar e cristalino. Este estudo teve como objetivo avaliar o perfil genotípico dos polimorfismos dos genes GSTM1 e GSTT1 em pacientes portadores de Glaucoma Primário de Ângulo Aberto e grupo controle na cidade de Goiânia. Foram analisadas amostras de sangue periférico de 100 amostras de pacientes comprovadamente diagnosticados como portadores de glaucoma e 53 amostras de pacientes com exame dentro dos padrões considerados normais, representando o grupo controle. O polimorfismo foi avaliado por PCR e analisados em gel de agarose a 2% e corado com brometo de etídeo. As frequências dos polimorfismos dos genes GSTM1 e GSTT1 foram comparadas com o teste &#967;2 (p<0,05%) e a taxa de risco foi avaliada pelo teste de Odds Ratio com intervalo de confiança de 95% (&#945;=0,05). No grupo caso observou-se que o genótipo GSTM1 presente foi de 40% (n=40) e nos controles foi de 71,6% (n=38). O genótipo nulo foi 60% (n=60) e 28,3% (n=15), respectivamente. O genótipo GSTT1 presente no grupo caso foi de 52% (n=52) e no grupo controle foi de 66% (n=35); já o genótipo nulo foi de 48% (n=48) no grupo caso e 34% (n=18) no grupo controle. O genótipo GSTM1 nulo no grupo caso foi mais frequente do que no grupo controle, sendo esta diferença estatisticamente significativa (p=0, 0004). O mesmo não foi encontrado com o genótipo GSTT1 (p=0,13). Foi verificado também a associação dos genótipos GSTM1 nulo/GSTT1 presente ao risco de glaucoma para 3,1 vezes mais a chance de ocorrência da doença, sugerindo que indivíduos que apresentam os genótipos GSTM1 nulo/GSTT1 presente pode ser considerado como um dos fatores de risco para o desenvolvimento do GPAA. Já para os genótipos GSTM1/GSTT1 nulos foi verificado o risco de 6,7 vezes mais a chance de ocorrência da doença (p=0,0004; OR: 6,7; IC 95%: 2,7 20,3), sugerindo que indivíduos que apresentam os genótipos GSTM1/GSTT1 nulos pode ser considerado como um dos fatores de risco para o desenvolvimento do GPAA.