Distinct Inflammatory and Oxidative Effects of Diabetes Mellitus and Hypothyroidism in the Lacrimal Functional Unit.

Diabetes mellitus (DM) and hypothyroidism (HT) are prevalent diseases associated with dry eye (DE). Their impact on the lacrimal functional unit (LFU) is poorly known. This work evaluates the changes in the LFU in DM and HT. Adult male Wistar rats had the disease induced as follows: (a) DM: streptozotocin and (b) HT: methimazole. The tear film (TF) and blood osmolarity were measured. Cytokine mRNA was compared in the lacrimal gland (LG), trigeminal ganglion (TG), and cornea (CO). Oxidative enzymes were evaluated in the LG. The DM group showed lower tear secretion (p = 0.02) and higher blood osmolarity (p < 0.001). The DM group presented lower mRNA expression of TRPV1 in the cornea (p = 0.03), higher Il1b mRNA expression (p = 0.03), and higher catalase activity in the LG (p < 0.001). The DM group presented higher Il6 mRNA expression in the TG (p = 0.02). The HT group showed higher TF osmolarity (p < 0.001), lower expression of Mmp9 mRNA in the CO (p < 0.001), higher catalase activity in the LG (p = 0.002), and higher expression of Il1b mRNA in the TG (p = 0.004). The findings revealed that DM and HT induce distinct compromises to the LG and the entire LFU.

Natural history and predictors for progression in pediatric keratoconus.

We studied the demographic and clinical predictors associated with keratoconus progression in a pediatric population. Retrospective cohort study. We evaluated 305 eyes without previous surgeries from 168 patients, 9 to < 18 years old, and with a minimum 36-month follow-up in a hospital corneal ambulatory. We used Kaplan-Meyer survival curves; the dependent variable (main outcome measure) was the interval time (months) until the event, defined as an increase of 1.5 D in the maximum keratometry (Kmax), obtained with Pentacam. We evaluated the predictors: age (< or ≥ 14 years), sex, keratoconus familial history, allergy medical history, and the baseline tomographic parameters: mean keratometry (Km), Kmax (< or ≥ 55 D); and thinnest pachymetry (TP). We used log-rank tests and compared median survival times for right (RE)/left eyes (LE) and better (BE)/worse eyes (WE). A p value < 0.05 was considered significant. The patients' mean ± SD age was 15.1 ± 2.3 years old; 67% were boys, 30% were < 14 years, 15% had keratoconus familial history, and 70% were allergic. The general Kaplan-Meyer curves showed no differences between RE/LE or BE/WE. RE with allergy and LE with Kmax ≥ 55 D had smaller survival times ((95%CI 9.67-32.1), p 0.031 and (95%CI 10.1-44.1), p 0.042, respectively). For BE and WE, Kmax ≥ 55 D had smaller survival times ((95% CI 6.42- ), p 0.031 and (95%CI 8.75-31.8), p 0.043, respectively). Keratoconus progression was similar between RE/LE and BE/WE. Steepest corneas are predictors of faster progression. Allergy is also a predictor of keratoconus progression in RE.

Multicenter prospective validation study for international chronic ocular graft-versus-host disease consensus diagnostic criteria.

PURPOSE: To validate the international chronic ocular graft-versus-host disease (GVHD) diagnostic criteria (ICCGVHD) compared to the National Institute of Health diagnostic criteria 2014 (NIH2014) for chronic ocular GVHD. METHODS: Between 2013 and 2019, the study enrolled 233 patients with or without chronic ocular GVHD combined with the presence or absence of systemic chronic GVHD in an internationally prospective multicenter and observational cohort from 9 institutions. All patients were evaluated for four clinical parameters of ICCGVHD. RESULTS: The relation between the ICCGVHD score (0-11) and NIH2014 eye score (0-4) was relatively high (r = 0.708, 95% CI: 0.637-0.767, p < 0.001). The sensitivity and specificity of ICCGVHD for NIH 2014 for 233 patients were 94.3% (95% CI: 89.6%-98.1%) and 71.7% (95% CI: 63.0-79.5%), respectively (cutoff value of the ICCGVHD score = 6). The positive predictive value was 77.1% (95% CI: 71.1%-82.1%), and the negative predictive value was 87.0% (95% CI:81.6-92.5%). For the patients with systemic GVHD (n = 171), the sensitivity and specificity were 94.2% and 67.2%, respectively (ICCGVHD-score cutoff value = 6). By receiver operating characteristic (ROC) curve analysis, the area under the curve (AUC) was 0.903 (95% CI: 0.859-0.948). For patients without systemic GVHD (n = 62), the sensitivity and specificity were 100% and 76.7%, respectively (ICCGVHD-score cutoff value = 6). The AUC was 0.891 (95% CI 0.673-1.000). CONCLUSIONS: Good sensitivity, specificity, predictive value and correlation were found between ICCGVHD and NIH2014. ICCGVHD scores ≥6 can be useful to diagnose ocular GVHD with or without systemic GVHD for clinical research.

Clinical and laboratory evaluation of sicca complaints: distinctive aspects of primary, secondary and non-Sjogren syndrome.

BACKGROUND: Sjögren Syndrome (SS) is a systemic autoimmune disease with a wide spectrum of manifestations that can lead to misdiagnosis. This study describes and compares demographic, clinical, serological, and histopathological data from subjects with SS and non-Sjögren Syndrome (NSS). It also details specific features within the primary SS (pSS) and secondary SS (sSS) groups identifying sub-groups. METHODS: The sample included individuals referred to an academic medical center in Brazil for investigation of SS from 2012 to 2020. Patients were retrospectively classified as primary SS (pSS), secondary SS (sSS), or NSS, based on the American-European Consensus Group criteria (AECG-2002), after multi-professional clinical and laboratory evaluation. RESULTS: A total of 676 individuals were screened and 510 (75.4%) completed the assessments; 198 patients were classified as pSS, 149 as sSS, and 163 as NSS. Symptoms and glandular dysfunction tests were similar in the groups. Concerning pSS, extraglandular manifestations were present in 59% of patients; the elderly had more dry symptoms and peripheral neurological disorders; and 2.5% developed non-Hodgkin lymphoma. In sSS, each overlap promoted distinct clinical and laboratory variants. Several alternative diagnoses were identified as a cause of sicca complex in NSS group. CONCLUSIONS: The diagnosis of SS remains a challenge behind dryness. Up to 31% of the suspected cases had other conditions associated to the symptoms. Histopathological analysis of LSG and SSa determined the diagnostic. Aging in pSS and overlap disease in sSS were responsible for distinct phenotypes and characteristic sub-groups in SS.

Oxidative Stress in the Protection and Injury of the Lacrimal Gland and the Ocular Surface: are There Perspectives for Therapeutics?

Oxidative stress (OS) is a major disruption in the physiology of the lacrimal functional unit (LFU). Antioxidant enzymes have dual protective activities: antioxidant and antimicrobial activities. Peroxidases have been indistinctly used as markers of the secretory activity of the LFU and implicated in the pathophysiology, diagnosis and treatment of dry eye disease (DED), even though they comprise a large family of enzymes that includes lactoperoxidase (LPO) and glutathione peroxidase (GPO), among others. Assays to measure and correlate OS with other local LFU phenomena have methodological limitations. Studies implicate molecules and reactions involved in OS as markers of homeostasis, and other studies identify them as part of the physiopathology of diseases. Despite these conflicting concepts and observations, it is clear that OS is influential in the development of DED. Moreover, many antioxidant strategies have been proposed for its treatment, including calorie restriction to nutritional supplementation. This review offers a critical analysis of the biological mechanisms, diagnostic outcomes, drug use, dietary supplements, and life habits that implicate the influence of OS on DED.

Comparison of the effects of corneal and lacrimal gland denervation on the lacrimal functional unit of rats / Comparação dos efeitos da denervação da córnea e da glândula lacrimal na unidade funcional lacrimal de ratos

ABSTRACT Purpose: This study aimed to compare the changes in the lacrimal functional unit in the following two models of neurogenic dry eye syndrome: sensory denervation of the cornea versus autonomic denervation of the lacrimal gland. Methods: The neural network supports the lacrimal functional unit. It can be divided into afferent (sensory) and efferent (autonomic) pathways and is affected by severe diseases that compromise the lacrimal functional unit. Male Wistar, 8-week-old rats were divided into the following three groups: 1) control naïve (n=16 animals); 2) autonomic denervation: where rats were subjected to right lacrimal gland nerve ablation and evaluated after 1 and 2 months (1M and 2M) after the procedure (n=7 animals per subgroup, autonomic denervation 1M and autonomic denervation 2M, respectively); 3) sensory denervation induced by 0.2% benzalkonium chloride eye drops, twice a day for 7 days in the right eye (n=10 animals). The corneal sensitivity was measured using the eye wipe test with capsaicin (10 µM). The quantitative real-time PCR was performed to compare the mRNA expressions of proinflammatory cytokines, such as Il-1β, Il-6, Tnf, Mmp9, in the cornea, trigeminal ganglion, and lacrimal gland. In addition, the mRNA of the promitotic factors in the lacrimal gland, such as Bmp7, Runx1, Runx3, Fgf10, and Smad1, was compared. Results: Sensory denervation induced corneal hyperalgesia (p=0.001). Sensory denervation and autonomic denervation increased the mRNA of proinflammatory cytokines in the cornea and lacrimal gland (p<0.05), but only sensory denervation increased the mRNA levels of Il-1β and Tnf in the trigeminal ganglion (p<0.05) compared with the control naïve. Conclusions: Autonomic denervation and sensory denervation models can have common features, such as inflammation of different parts of the lacrimal functional unit. However, hyperesthesia and inflammatory markers in the trigeminal ganglion because of sensory denervation and the expression of regenerative mediators in the lacrimal gland owing to autonomic denervation are the distinguishing features of these diseases that can be explored in future studies assessing dry eye syndrome secondary to neural damage of the lacrimal functional unit.

RESUMO Objetivo: O nosso objetivo neste estudo foi comparar as alterações na unidade funcional lacrimal em dois modelos de síndrome do olho seco neurogênica: desnervação sensorial da córnea versus desnervação autonômica da glândula lacrimal. Métodos: A rede neural é um importante suporte para a unidade funcional lacrimal. Pode ser dividido em vias aferentes (sensoriais) e eferentes (autonômicas), sujeitas a doenças graves que comprometem a unidade funcional lacrimal. Ratos Wistar machos, com 8 semanas de idade, foram divididos em três grupos: 1) Controle naïve (n=16 animais); 2) Desnervação autonômica: onde os ratos foram submetidos à ablação do nervo da glândula lacrimal direita e avaliados após um e dois meses (1 M a 2 M) do procedimento (n=7 animais por subgrupo, desnervação autonômica 1M e desnervação autonômica 2M, respectivamente); 3) Desnervação sensorial induzida por colírio a 0,2% de cloreto de benzalcônio, duas vezes ao dia por 7 dias no olho direito (n=10 animais). A sensibilidade da córnea foi medida pelo teste de movimento pata-olho com capsaicina (10 µM). A PCR quantitativa em tempo real foi aplicada para comparar a expressão relativa de mRNA de citocinas pró-inflamatórias: Il1b, Il6, Tnf, Mmp9, na córnea, gânglio trigêmio e glândula lacrimal. O mRNA dos agentes pró-mitóticos Bmp7, Runx1, Runx3, Fgf10 e Smad1 foram comparados na glândula lacrimal. Resultados: A desnervação sensorial induziu hiperalgesia da córnea (p=0,001). Desnervação sensorial e desnervação autonômica aumentaram o mRNA de citocinas pró-inflamatórias no córnea e glândula lacrimal (p<0,05), mas apenas desnervação sensorial aumentou o mRNA de Il1b e Tnf no gânglio trigêmio (p<0,05) quando comparado ao controle naïve. Conclusões: Os modelos de desnervação autonômica e desnervação sensorial podem ter características comuns, como inflamação de diferentes partes da unidade funcional lacrimal. No entanto, a hiperestesia e os marcadores inflamatórios no gânglio trigêmio de desnervação sensorial e a expressão de mediadores regenerativos na glândula lacrimal na desnervação autonômica são características que distinguem essas doenças, podendo ser investigadas em estudos futuros que abordam o olho seco secundário ao dano neural da unidade funcional lacrimal.

Focal lymphocytic sialadenitis and ectopic germinal centers in oral reactive lesions and primary Sjögren's syndrome: a comparative study.

Focal lymphocytic sialadenitis (FLS), an important diagnostic criterion for Sjögren's syndrome (SS) diagnosis, can also be observed when assessing minor salivary gland (mSG) biopsies from healthy asymptomatic individuals (non-SS patients). Fifty cases of primary SS (pSS group) and 31 cases of oral reactive lesions (non-SS non-sicca group) containing also typical FLS features, were assessed by morphological and immunohistochemical (CD10, CD23 and Bcl-6) analysis, aiming at the detection of GCs. All pSS cases showed FLS with focus score (FS) ≥ 1. In the non-SS non-sicca group, 12, 10 and 9 cases showed FLS with FS ≥ 1, FLS with FS < 1 and FLS associated with chronic sclerosing sialadenitis with FS < 1, respectively. The morphological analysis revealed similar frequency of GCs in pSS (20%) and non-SS non-sicca group (19%). The area (p = 0.052) and largest diameter (p = 0.245) of GCs were higher in pSS than non-SS non-sicca group. The FS and number of foci were significantly higher in pSS than non-SS non-sicca group with FS < 1. Immunohistochemistry confirmed all morphological findings (GCs showing CD23 and Bcl-6 positivity, with variable CD10 expression) and additionally in 3 and 1 cases of the pSS and non-SS non-sicca group, respectively. Moreover, another 6 and 2 cases of the pSS and non-SS non-sicca group with FS ≥ 1, respectively, showed positivity only for CD23. FLS can also be observed when assessing oral reactive lesions, which showed similar frequency of GCs with those found in pSS patients. Further studies, including functional analysis of lymphocytic populations and GCs in FLS, are encouraged.

Comparison of the effects of corneal and lacrimal gland denervation on the lacrimal functional unit of rats.

PURPOSE: This study aimed to compare the changes in the lacrimal functional unit in the following two models of neurogenic dry eye syndrome: sensory denervation of the cornea versus autonomic denervation of the lacrimal gland. METHODS: The neural network supports the lacrimal functional unit. It can be divided into afferent (sensory) and efferent (autonomic) pathways and is affected by severe diseases that compromise the lacrimal functional unit. Male Wistar, 8-week-old rats were divided into the following three groups: 1) control naïve (n=16 animals); 2) autonomic denervation: where rats were subjected to right lacrimal gland nerve ablation and evaluated after 1 and 2 months (1M and 2M) after the procedure (n=7 animals per subgroup, autonomic denervation 1M and autonomic denervation 2M, respectively); 3) sensory denervation induced by 0.2% benzalkonium chloride eye drops, twice a day for 7 days in the right eye (n=10 animals). The corneal sensitivity was measured using the eye wipe test with capsaicin (10 µM). The quantitative real-time PCR was performed to compare the mRNA expressions of proinflammatory cytokines, such as Il-1ß, Il-6, Tnf, Mmp9, in the cornea, trigeminal ganglion, and lacrimal gland. In addition, the mRNA of the promitotic factors in the lacrimal gland, such as Bmp7, Runx1, Runx3, Fgf10, and Smad1, was compared. RESULTS: Sensory denervation induced corneal hyperalgesia (p=0.001). Sensory denervation and autonomic denervation increased the mRNA of proinflammatory cytokines in the cornea and lacrimal gland (p<0.05), but only sensory denervation increased the mRNA levels of Il-1ß and Tnf in the trigeminal ganglion (p<0.05) compared with the control naïve. CONCLUSIONS: Autonomic denervation and sensory denervation models can have common features, such as inflammation of different parts of the lacrimal functional unit. However, hyperesthesia and inflammatory markers in the trigeminal ganglion because of sensory denervation and the expression of regenerative mediators in the lacrimal gland owing to autonomic denervation are the distinguishing features of these diseases that can be explored in future studies assessing dry eye syndrome secondary to neural damage of the lacrimal functional unit.

Limitations and advances in new treatments and future perspectives of corneal blindness / Limitações e avanços em novos tratamentos e perspectivas futuras na cegueira corneal

ABSTRACT This review is intended to describe the therapeutic approaches for corneal blindness, detailing the steps and elements involved in corneal wound healing. It also presents the limitations of the actual surgical and pharmacological strategies used to restore and maintain corneal transparency in terms of long-term survival and geographic coverage. In addition, we critically review the perspectives of anabolic agents, including vitamin A, hormones, growth factors, and novel promitotic and anti-inflammatory modulators, to assist corneal wound healing. We discuss the studies involving nanotechnology, gene therapy, and tissue reengineering as potential future strategies to work solely or in combination with corneal surgery to prevent or revert corneal blindness.(AU)

RESUMO O presente trabalho traz uma revisão das abordagens terapêuticas para a cegueira da córnea. O estudo detalha as etapas e os elementos envolvidos na cicatrização da córnea. Ele mostra as limitações das estratégias cirúrgicas e farmacológicas usadas para restaurar e manter a transparência da córnea em termos de sobrevida a longo prazo e alcance geográfico. As perspectivas dos agentes anabólicos, incluindo vitamina A, hormônios, fatores de crescimento e novos moduladores pró-mitóticos e anti-inflamatórios para auxiliar a cicatrização da ferida na córnea, são revisadas criticamente. Aqui, apresentamos estudos envolvendo nanotecnologia, terapia gênica e reengenharia de tecidos como possíveis estratégias futuras para atuar de maneira isolada ou combinada com a cirurgia da córnea para prevenir ou reverter a cegueira corneana.(AU)

Ocular findings among patients surviving COVID-19.

To describe the medium-term ophthalmological findings in patients recovering from COVID-19. Patients recovered from the acute phase of COVID-19 underwent a complete ophthalmological evaluation, including presenting and best-corrected visual acuity (BCVA), refractometry, biomicroscopy, tonometry, break-up time and Schirmer tests, indirect ophthalmoscopy, color fundus picture, and retinal architecture evaluation using optical coherence tomography. Socio-demographic data and personal medical history were also collected. According to the severity of systemic manifestations, patients were classified into mild-to-moderate, severe, and critical. Sixty-four patients (128 eyes) were evaluated 82 ± 36.4 days after the onset of COVID's symptoms. The mean ± SD duration of hospitalization was 15.0 ± 10.7 days. Seven patients (10.9%) had mild-to-moderate, 33 (51.5%) severe, and 24 (37.5%) critical disease. Median [interquartile ranges (IQR)] presenting visual acuity was 0.1 (0-0.2) and BCVA 0 (0-0.1). Anterior segment biomicroscopy was unremarkable, except for dry eye disease, verified in 10.9% of them. The mean ± SD intraocular pressure (IOP) in critical group (14.16 ± 1.88 mmHg) was significantly higher than in severe group (12.51 ± 2.40 mmHg), both in the right (p 0.02) and left eyes (p 0.038). Among all, 15.6% had diabetic retinopathy, and two patients presented with discrete white-yellowish dots in the posterior pole, leading to hyporreflective changes at retinal pigment epithelium level, outer segment, and ellipsoid layers. The present study identified higher IOP among critical cases, when compared to severe cases, and discrete outer retina changes 80 days after COVID-19 infection. No sign of uveitis was found.

Lacrimal Gland as a Target Organ for Adenovirus Gene Therapy Encoding Erythropoietin for Dry Eye Induced by Benzalkonium Chloride.

Purpose: The aims of this work were a) to describe the histology of the lacrimal gland (LG) and cornea induced by an adenovirus (Ad) vector encoding the human erythropoietin (Epo) gene delivered to the LG and b) to evaluate the therapeutic potential of this strategy to prevent benzalkonium chloride (BAK) corneal toxicity.Methods: Structure and function of male Wistar rats LG were compared in the groups: 1) naïve control and 2) Ad-hEpo in the right LG (RLG). The protective response against BAK eye drops was compared among the groups 1) naïve control, 2) BAK in the right eye, 3) Ad-hEpo RLG + BAK and 4) Ad-hEpo in the right salivary gland (RSG)+BAK. Ad-hEpo groups received an injection of AdLTR2EF1a-hEPO (25 ul, 1010 particles/ml) in the right LG or SG (positive control). The BAK groups received 0.2% BAK in the right cornea twice a day. The tests applied after 7 days, included tear secretion, hEPO mRNA detection by qRT-PCR, LG and cornea histology, LG ELISA for cytokines and hematocrit.Results: hEPO mRNA was present in the Ad-hEpo RLG and RSG, but not kidney or liver samples (negative controls). TNF-α and IL-1ß increased in the LG exposed to Ad-hEpo compared to naïve control (p = .0115 and p = .0397, respectively). BAK reduced tear secretion, but this reduction was prevented by Ad-hEpo RLG+BAK and Ad-hEpo RSG+BAK (p = .017). The corneal epithelia were thinner in the BAK-treated groups independent of Ad-hEpo (p = .0009). Hematocrit increased only in the Ad-hEpo RSG group (p = .01).Conclusions: Ad-hEpo infection of rat LG and SG induces local, but only the SG infection induced systemic changes in rats. Importantly, Ad-hEpo attenuated the BAK-mediated toxic reduction in tear flow. Future studies must consider viral vector tissue tropism, biodistribution and effective therapeutic gene products for ocular surface diseases.

Limitations and advances in new treatments and future perspectives of corneal blindness.

This review is intended to describe the therapeutic approaches for corneal blindness, detailing the steps and elements involved in corneal wound healing. It also presents the limitations of the actual surgical and pharmacological strategies used to restore and maintain corneal transparency in terms of long-term survival and geographic coverage. In addition, we critically review the perspectives of anabolic agents, including vitamin A, hormones, growth factors, and novel promitotic and anti-inflammatory modulators, to assist corneal wound healing. We discuss the studies involving nanotechnology, gene therapy, and tissue reengineering as potential future strategies to work solely or in combination with corneal surgery to prevent or revert corneal blindness.

Lacrimal gland atrophy and dry eye related to isotretinoin, androgen, and prolactin: differential diagnosis for Sjögren's syndrome.

This report is of three cases of sicca syndrome, initially suspected to be Sjögren's syndrome, which was ruled out by clinical and laboratory investigations. The patients were a 24-year-old woman, a 32-year-old man, and a 77-year-old woman with chronic symptoms of sicca syndrome, including dry eye syndrome. The first case was associated with the use of isotretinoin, a retinoic acid. The second was associated with the use of anabolic androgenic steroids, and the third was related to a prolactin- secreting pituitary adenoma. All cases manifested sicca, including dry eye syndrome, after those events, and the manifestations persisted. Magnetic resonance imaging revealed bilateral atrophy of the lacrimal gland. The medical history, ocular examinations, laboratory exams, and magnetic resonance images confirmed dry eye syndrome; however, the exams were all negative for Sjögren's syndrome. The lacrimal gland was absent on magnetic resonance imaging in all three cases. The clinical history revealed that the signs and symptoms appeared after chronic exposure to retinoic acid, anabolic androgenic steroids, and a prolactin-secreting pituitary adenoma, respectively. Chronic isotretinoin, anabolic androgenic steroids, and prolactin-secreting pituitary adenoma or, in this last case, its inhibitory treatment, can cause lacrimal gland atrophy, sicca syndrome, and dry eye syndrome, and a differential diagnosis of Sjögren's syndrome. Further studies on doses, time, and other susceptibilities to the long-lasting adverse effects of retinoic acid, anabolic androgenic steroids, and the repercussions of prolactin-secreting pituitary adenoma are necessary to confirm and expand upon these associations.

Corneal blindness in Plato's cave: the acting forces to prevent and revert corneal opacity. Part I: epidemiology and new physiopathological concepts.

The burden of corneal blindness and visual deficiency can be felt worldwide. Its association with several endemic diseases such as childhood blindness, trauma, infectious keratitis (including variants caused by herpes, hanseniasis, and fungi), vitamin A deficiency, diabetes mellitus, and other dry eye syndromes reflects its poorly understood underlying mechanisms and suggests that the actual frequency of the disease is underestimated. The low effectiveness of preventive and therapeutic strategies against corneal scarring or deformity predicts a high frequency of patients with corneal blindness in the future. Corneal blindness is associated with environmental factors and socioeconomic limitations that restrain health assistance and maintain a modest efficiency of the current therapeutic strategies for resolving corneal diseases in large-scale programs. We present here a critical review of the concepts associated with corneal blindness that need to be considered when planning strategies to prevent and treat corneal blindness worldwide (to be able to leave Plato's cave, where corneal blindness is encaged.

Anti-fibrotic effects of rosmarinic acid on Tenon's capsule fibroblasts stimulated with TGF-ß: therapeutic potential in ocular surgery.

PURPOSE: Collagen deposition and myofibroblast differentiation are critical factors related to excessive scarring in ocular surgeries. This study evaluated the anti-fibrotic activity of rosmarinic acid on rabbit Tenon's capsule fibroblasts stimulated with transforming growth factor- ß2. METHODS: Primary cultures of rabbit Tenon's capsule fibroblasts were treated with various concentrations of rosmarinic acid for 12 h, in the presence and absence of transforming growth factor-ß2. After 48 h, the proliferation index of rabbit Tenon's capsule fibroblasts and the differentiation of myofibroblasts were investigated through immunofluorescence staining for proliferating cell nuclear antigen and alpha smooth muscle actin. An automated cell counter and colorimetric metabolic activity assay were used to evaluate cell number and viability. Collagen expression and production were determined by quantitative real-time polymerase chain reaction and hydroxyproline assay, respectively. RESULTS: Unstimulated rabbit Tenon's capsule fibroblasts treated with any concentration of rosmarinic acid exhibited diminished collagen expression (p<0.01) but showed no differences in proliferation index. Transforming growth factor-ß2 exposure induced myofibroblast differentiation and increased collagen production. Exposure to rosmarinic acid at 1.0 and 3.0 µM concentrations reduced the proliferation index (p<0.02), as well as the collagen expression and hydroxyproline content (p<0.05). Exposure to 3.0 µM rosmarinic acid reduced viability (p=0.035) in unstimulated rabbit Tenon's capsule fibroblasts and cell numbers (p=0.001) in both stimulated and unstimulated rabbit Tenon's capsule fibroblast cultures. CONCLUSIONS: Exposure to 1.0 µM rosmarinic acid was noncytotoxic and led to reduced collagen expression and proliferation of stimulated rabbit Tenon's capsule fibroblasts. These findings suggest that rosmarinic acid is a relatively non-injurious anti-fibrotic compound to rabbit Tenon's capsule fibroblasts, with potential application as an adjunctive agent in ocular procedures, particularly in glaucoma surgeries.

Anti-fibrotic effects of rosmarinic acid on Tenon's capsule fibroblasts stimulated with TGF-ß: therapeutic potential in ocular surgery / Efeito antifibrótico do Ácido Rosmarínico sobre os fibroblastos da Cápsula de Tenon estimulados com TGF-beta2: potencial terapêutico na cirurgia ocular

ABSTRACT Purpose: Collagen deposition and myofibroblast differentiation are critical factors related to excessive scarring in ocular surgeries. This study evaluated the anti-fibrotic activity of rosmarinic acid on rabbit Tenon's capsule fibroblasts stimulated with transforming growth factor- β2. Methods: Primary cultures of rabbit Tenon's capsule fibroblasts were treated with various concentrations of rosmarinic acid for 12 h, in the presence and absence of transforming growth factor-β2. After 48 h, the proliferation index of rabbit Tenon's capsule fibroblasts and the differentiation of myofibroblasts were investigated through immunofluorescence staining for proliferating cell nuclear antigen and alpha smooth muscle actin. An automated cell counter and colorimetric metabolic activity assay were used to evaluate cell number and viability. Collagen expression and production were determined by quantitative real-time polymerase chain reaction and hydroxyproline assay, respectively. Results: Unstimulated rabbit Tenon's capsule fibroblasts treated with any concentration of rosmarinic acid exhibited diminished collagen expression (p<0.01) but showed no differences in proliferation index. Transforming growth factor-β2 exposure induced myofibroblast differentiation and increased collagen production. Exposure to rosmarinic acid at 1.0 and 3.0 µM concentrations reduced the proliferation index (p<0.02), as well as the collagen expression and hydroxyproline content (p<0.05). Exposure to 3.0 µM rosmarinic acid reduced viability (p=0.035) in unstimulated rabbit Tenon's capsule fibroblasts and cell numbers (p=0.001) in both stimulated and unstimulated rabbit Tenon's capsule fibroblast cultures. Conclusions: Exposure to 1.0 µM rosmarinic acid was noncytotoxic and led to reduced collagen expression and proliferation of stimulated rabbit Tenon's capsule fibroblasts. These findings suggest that rosmarinic acid is a relatively non-injurious anti-fibrotic compound to rabbit Tenon's capsule fibroblasts, with potential application as an adjunctive agent in ocular procedures, particularly in glaucoma surgeries.

RESUMO Objetivo: A deposição de colágeno e a diferenciação de miofibroblastos são fatores chaves relacionados à cicatrização excessiva em cirurgias oculares. Este estudo avaliou a atividade anti-fibrótica do ácido rosmarínico nos fibroblastos da cápsula de Tenon de coelhos estimulados com o fator de crescimento transformador-β2. Métodos: Culturas primárias de fibroblastos da cápsula de Tenon de coelhos foram tratadas com várias concentrações de ácido rosmarínico por 12h, na presença e na ausência do fator de crescimento transformador-β2. Após 48h, o índice de proliferação dos fibroblastos da cápsula de Tenon de coelhos e a diferenciação dos miofibroblastos foram investigados por coloração por imunofluorescência para proliferação de antígeno nuclear celular e α-actina de músculo liso, respectivamente. Um contador automático de células e um ensaio de atividade metabólica colorimétrica foram utilizados para avaliar o número e a viabilidade das células. A expressão e produção do colágeno foram determinadas por reação quantitativa em cadeia da polimerase em tempo real e ensaio de hidroxiprolina, respectivamente. Resultados: Fibroblastos da cápsula de Tenon de coelhos não estimulados tratados com qualquer concentração de ácido rosmarínico exibiram diminuição de colágeno (p<0,01), mas não mostraram diferenças no índice de proliferação. A exposição ao fator de crescimento transformador-β2 induziu a diferenciação de miofibroblastos e aumentou a produção de colágeno. A exposição ao ácido rosmarínico nas concentrações de 1,0 e 3,0 µM reduziu o índice de proliferação (p<0,02), bem como a expressão de colágeno e a quantificação de hidroxiprolina (p<0.05). A exposição a 3,0 µM de ácido rosmarínico reduziu a viabilidade (p=0,035) de fibroblastos da cápsula de Tenon de coelhos não estimulados e o número de células (p=0,001) em culturas de fibroblastos da cápsula de Tenon de coelhos estimuladas e não estimuladas. Conclusões: A exposição ao ácido rosmarínico 1,0 µM foi não citotóxica e levou à expressão reduzida de colágeno e menor proliferação de fibroblastos da cápsula de Tenon estimulados pelo fator de crescimento transformador-β2. Esses achados sugerem que o ácido rosmarínico é um composto antifibrótico relativamente não lesivo aos fibroblastos da cápsula de Tenon de coelhos, com potencial aplicação como agente adjuvante em procedimentos oculares, particularmente em cirurgias de glaucoma.