Cone cell dysfunction attenuates retinal neovascularization in oxygen-induced retinopathy mouse model.

Aberrant neovascularization is the most common feature in retinopathy of prematurity (ROP), which leads to the retinal detachment and visual defects in neonates with a low gestational age eventually. Understanding the regulation of inappropriate angiogenic signaling benefits individuals at-risk. Recently, neural activity originating from the specific neural activity has been considered to contribute to retinal angiogenesis. Here, we explored the impact of cone cell dysfunction on oxygen-induced retinopathy (OIR), a mouse model commonly employed to understand retinal diseases associated with abnormal blood vessel growth, using the Gnat2cpfl3 (cone photoreceptor function loss-3) strain of mice (regardless of the sex), which is known for its inherent cone cell dysfunction. We found that the retinal avascular area, hypoxic area, and neovascular area were significantly attenuated in Gnat2cpfl3 OIR mice compared to those in C57BL/6 OIR mice. Moreover, the HIF-1α/VEGF axis was also reduced in Gnat2cpfl3 OIR mice. Collectively, our results indicated that cone cell dysfunction, as observed in Gnat2cpfl3 OIR mice, leads to attenuated retinal neovascularization. This finding suggests that retinal neural activity may precede and potentially influence the onset of pathological neovascularization.

Astrocytic expression of Yes-associated protein (YAP) regulates retinal neovascularization in a mouse model of oxygen-induced retinopathy.

Pathological neovascularization is the hallmark of many vascular oculopathies. There is still a great deal of uncertainty surrounding retinal neovascularization research. A working hypothesis that astrocytic Yes-associated protein (YAP) act as a key factor in retinal neovascularization was proposed. And our study was conducted to verified this hypothesis. In vivo, we successfully generated mice deficient in YAP in astrocytes (YAPf/f GFAP-Cre mice) and set up oxygen-induced retinopathy (OIR) model. Pathological neovascularization was evaluated by immunofluorescence staining and western blotting. In vitro, cultured retinal astrocytes were transfected with YAP siRNA. Enzyme-linked immunosorbent assay (ELISA) and western blot were used to determine the proteins in the supernatants and cells. The results showed that YAP was upregulated and activated in the OIR mice retinas. Conditional ablation of YAP aggravated pathological neovascularization, along with the upregulation of vascular endothelial growth factor A (VEGF-A) and monocyte chemoattractant protein-1 (MCP-1). Studies in vitro confirmed that the knockdown of YAP in astrocytes lead to increases in VEGF-A and MCP-1 levels, thus enhancing pro-angiogenic capability of YAP-deficit astrocytes. In conclusion, astrocytic YAP alleviates retinal pathological angiogenesis by inhibiting the over-activation of astrocytes, which suppresses excessive VEGF-A production and neuroinflammation.

Differences in Oxygen-Induced Retinopathy Susceptibility Between Two Sprague Dawley Rat Vendors: A Comparison of Retinal Transcriptomes.

PURPOSE: To determine the retinal transcriptomic differences underlying the oxygen-induced retinopathy phenotypes between Sprague Dawley rat pups from two commonly used commercial vendors. This will allow us to discover genes and pathways that may be related to differences in disease severity in similarly aged premature babies and suggest possible new treatment approaches. METHODS: We analyzed retinal vascular morphometry and transcriptomes from Sprague Dawley rat pups from Charles River Laboratories and Envigo (previously Harlan). Room air control and oxygen-induced retinopathy groups were compared. Oxygen-induced retinopathy was induced with the rat 50/10 model. RESULTS: Pups from Charles River Laboratories developed a more severe oxygen-induced retinopathy phenotype, with 3.6-fold larger percent avascular area at P15 and twofold larger % neovascular area at P20 than pups from Envigo. Changes in retinal transcriptomes of rat pups from both vendors were substantial at baseline and in response to oxygen-induced retinopathy. Baseline differences centered on activated pathways of neuronal development in Charles River Laboratories pups. In response to oxygen-induced retinopathy, during the neovascular phase, retinas from Charles River Laboratories pups exhibited activation of pathways regulating necrosis, neuroinflammation, and interferon signaling, supporting the observed increase of neovascularization. Conversely, retinas from Envigo pups showed decreased necrosis and increased focal adhesion kinase signaling, supporting more normal vascular development. Comparing oxygen-induced retinopathy transcriptomes at P15 to those at P20, canonical pathways such as phosphate and tensin homolog, interferon, and coordinated lysosomal expression and regulation element signaling were identified, highlighting potential novel mechanistic targets for future research. CONCLUSION: Transcriptomic profiles differ substantially between rat pup retinas from Charles River Laboratories and Envigo at baseline and in response to oxygen-induced retinopathy, providing insight into vascular morphologic differences. Comparing transcriptomes identified new pathways for further research in oxygen-induced retinopathy pathogenesis and increased scientific rigor of this model.

Epithelial Membrane Protein 2 (EMP2) Blockade Attenuates Pathological Neovascularization in Murine Oxygen-Induced Retinopathy.

Purpose: Retinopathy of prematurity (ROP) results from postnatal hyperoxia exposure in premature infants and is characterized by aberrant neovascularization of retinal blood vessels. Epithelial membrane protein-2 (EMP2) regulates hypoxia-inducible factor (HIF)-induced vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line and genetic knock-out of Emp2 in a murine oxygen-induced retinopathy (OIR) model attenuates neovascularization. We hypothesize that EMP2 blockade via intravitreal injection protects against neovascularization. Methods: Ex vivo choroid sprouting assay was performed, comparing media and human IgG controls versus anti-EMP2 antibody (Ab) treatment. In vivo, eyes from wild-type (WT) mice exposed to hyperoxia from postnatal (P) days 7 to 12 were treated with P12 intravitreal injections of control IgG or anti-EMP2 Abs. Neovascularization was assessed at P17 by flat mount imaging. Local and systemic effects of anti-EMP2 Ab treatment were assessed. Results: Choroid sprouts treated with 30 µg/mL of anti-EMP2 Ab demonstrated a 48% reduction in vessel growth compared to control IgG-treated sprouts. Compared to IgG-treated controls, WT OIR mice treated with 4 µg/g of intravitreal anti-EMP2 Ab demonstrated a 42% reduction in neovascularization. They demonstrated down-regulation of retinal gene expression in pathways related to vasculature development and up-regulation in genes related to fatty acid oxidation and tricarboxylic acid cycle respiratory electron transport, compared to controls. Anti-EMP2 Ab-treated OIR mice did not exhibit gross retinal histologic abnormalities, vision transduction abnormalities, or weight loss. Conclusions: Our results suggest that EMP2 blockade could be a local and specific treatment modality for retinal neovascularization in oxygen-induced retinopathies, without systemic adverse effects.

Transcriptional Comparison of Human and Murine Retinal Neovascularization.

Purpose: Retinal neovascularization (RNV) is the leading cause of vision loss in diseases like proliferative diabetic retinopathy (PDR). A significant failure rate of current treatments indicates the need for novel treatment targets. Animal models are crucial in this process, but current diabetic retinopathy models do not develop RNV. Although the nondiabetic oxygen-induced retinopathy (OIR) mouse model is used to study RNV development, it is largely unknown how closely it resembles human PDR. Methods: We therefore performed RNA sequencing on murine (C57BL/6J) OIR retinas (n = 14) and human PDR RNV membranes (n = 7) extracted during vitrectomy, each with reference to control tissue (n=13/10). Differentially expressed genes (DEG) and associated biological processes were analyzed and compared between human and murine RNV to assess molecular overlap and identify phylogenetically conserved factors. Results: In total, 213 murine- and 1223 human-specific factors were upregulated with a small overlap of 94 DEG (7% of human DEG), although similar biological processes such as angiogenesis, regulation of immune response, and extracellular matrix organization were activated in both species. Phylogenetically conserved mediators included ANGPT2, S100A8, MCAM, EDNRA, and CCR7. Conclusions: Even though few individual genes were upregulated simultaneously in both species, similar biological processes appeared to be activated. These findings demonstrate the potential and limitations of the OIR model to study human PDR and identify phylogenetically conserved potential treatment targets for PDR.

Effects of oxygen in lungs of rats

PURPOSE: To analyze microscopically the effects of different concentrations of oxygen in the lungs of rats. METHODS: There were 20 rats distributed in three experimental groups (concentration of oxygen to 40%, 70% and 100%) and a control group. The animals were exposed to the oxygen in a chamber of acrylic during three days and after exposition, the animals were submitted to median thoracotomia to remove the lungs. The lung tissue of all of the animals was analyzed as regards presence of acute and chronic inflammation, capillary congestion, alveolar walls thick, interstitial and alveolar edema, alveolar hemorrhage, denudation capillary and alveolar endothelium areas and atelectasis. RESULTS: The analysis histopathologic revealed significant statistics difference for acute and chronic inflammation, capillary congestion, alveolar walls thick, interstitial and alveolar edema, alveolar hemorrhage, denudation capillary and alveolar epithelium areas. CONCLUSIONS: Exposition to the oxygen during 72 hours in the concentration of 40% does not produce significant histopathologic alterations in the lung tissue; in the concentration of 70%, can promotes the alveolar walls thick and capillary congestion and in the concentration of 100% can cause death and originate diffuse pulmonary lesion. .

Alterações da histoarquitetura pulmonar de camundongos neonatos expostos à hiperóxia / Alterations in the pulmonary histoarchitecture of neonatal mice exposed to hyperoxia

OBJETIVOS: Analisar os efeitos da exposição à hiperóxia (100% de oxigênio) sobre a histoarquitetura pulmonar de camundongos neonatos. MÉTODOS: Camundongos neonatos da linhagem Balb/c foram expostos à hiperóxia (GH) (100% de oxigênio) (n = 10) em uma câmara (15 x 20 x 30 cm) por 24 horas, com fluxo de 2 L/min. O grupo controle (GC) (n = 10) foi exposto a normóxia em um mesmo tipo de câmara e pelo mesmo tempo. Após a exposição, os animais foram sacrificados por decapitação, os pulmões foram removidos para análise histológica e processados de acordo com a rotina do laboratório. Cortes de 3 µm de espessura foram corados com hematoxilina e eosina (H&E). A análise morfométrica foi realizada com o objetivo de analisar macrófagos presentes na luz alveolar, densidade de superfície (Sv) de trocas gasosas, densidade de volume (Vv) de parênquima pulmonar e áreas de atelectasias. RESULTADOS: Foi verificada diminuição do número de macrófagos alveolares (MØ) no GH (GH = 0,08±0,01 MØ/mm²; GC = 0,18±0,03 MØ/mm²; p = 0,0475), Sv de troca gasosa no GH (GH = 8,08 ± 0,12 mm² /mm³; GC = 8,65 ± 0,20 mm² /mm³; p = 0,0233), Vv de parênquima pulmonar no GH (GH = 54,7/33,5/83,5 %/mm²; GC = 75/56,7/107,9 %/mm²; p < 0.0001) quando comparado com o GC. Entretanto, houve aumento de áreas de atelectasias no GH (GH = 17,5/11,3/38,4 atelectasia/mm²; GC = 14/6,1/24,4 atelectasia/mm²; p = 0,0166) quando comparado com o GC. CONCLUSÃO: Nossos resultados indicam que a hiperóxia promoveu alterações na histoarquitetura pulmonar, aumentando áreas de atelectasia e hemorragia alveolar difusa.

OBJECTIVES: To analyze the effects of exposure to hyperoxia (100% oxygen) on the lung histoarchitecture of neonatal mice. METHODS: Neonatal Balb/c mice were exposed to hyperoxia (HG) (100% oxygen) (n = 10) in a chamber (15 x 20 x 30 cm) for 24 horas ours with a flow of 2 L/min. The control group (CG) (n = 10) was exposed to normoxia in the same type of chamber and for the same time. After exposure, the animals were euthanized by decapitation; the lungs were removed and processed for histological examination according to the laboratory routine. Three-mm thick sections were stained with hematoxylin and eosin (H&E). The morphometric analysis was performed with in order to analyze the macrophages present in the alveolar lumen, surface density (Sv) of gas exchange, volume density (Vv) of lung parenchyma, and areas of atelectasis. RESULTS: A decrease in the number of alveolar macrophages (MØ) was observed in the HG (HG = 0.08±0.01 MØ/mm², CG = 0.18±0.03 MØ/mm², p = 0.0475), Sv of gas exchange in HG (HG = 8.08±0.12 mm² /mm³, CG = 8.65±0.20 mm² /mm³, p = 0.0233), Vv of lung parenchyma in HG (HG = 54.7/33.5/83.5%/ mm²; CG = 75/56.7/107.9%/mm², p < 0.0001) when compared with the CG. However, there was an increase in areas of atelectasis in HG (HG = 17.5/11.3/38.4 atelectasis/mm², CG = 14/6.1/24.4 atelectasis/mm², p = 0.0166) when compared with the CG. CONCLUSION: The present results indicate that hyperoxia caused alterations in lung histoarchitecture, increasing areas of atelectasis and diffuse alveolar hemorrhage.

Lung morphometry, collagen and elastin content: changes after hyperoxic exposure in preterm rabbits

INTRODUCTION: Elastic and collagen fiber deposition increases throughout normal lung development, and this fiber network significantly changes when development of the lung is disturbed. In preterm rats and lambs, prolonged hyperoxic exposure is associated with impaired alveolization and causes significant changes in the deposition and structure of elastic fibers. OBJECTIVES: To evaluate the effects of hyperoxic exposure on elastic and collagen fiber deposition in the lung interstitial matrix and in alveolarization in preterm rabbits. METHODS: After c-section, 28-day preterm New-Zealand-White rabbits were randomized into 2 study groups, according to the oxygen exposure, namely: Room air (oxygen = 21 percent) or Oxygen (oxygen > 95 percent). The animals were killed on day 11 and their lungs were analyzed for the alveolar size (Lm), the internal surface area (ISA), the alveoli number, and the density and distribution of collagen and elastic fibers. RESULTS: An increase in the Lm and a decrease in the alveoli number were observed among rabbits that were exposed to hyperoxia with no differences regarding the ISA. No difference in the density of elastic fibers was observed after oxygen exposure, however there were fewer collagen fibers and an evident disorganization of fiber deposition. DISCUSSION: This model reproduces anatomo-pathological injuries representing the arrest of normal alveolar development and lung architecture disorganization by just a prolonged exposition to oxygen. CONCLUSIONS: In the preterm rabbit, prolonged oxygen exposure impaired alveolization and also lowered the proportion of collagen fibers, with an evident fiber network disorganization.

Ultraestructura de la retinopatía causada por la hiperoxia en ratas en desarrollo / Ultra structure of retinopathy induced by hyperoxia in developing rats

In order to evaluate the effect of postnatal hyperoxia on retinal structure, newborn rats were exposed to different oxygenation intervals (80 ± 1%) with three interruptions of 21% (30 min each). Four groups of rats were exposed from birth to the 6th, 9th, 12th and 14th postnatal day, respectively and another group was placed under normoxia. After this period all oxygenated groups and the controls remained under normoxia until they were 30 days old for the structural analysis of retina. Retinal histology was carried out using conventional techniques for transmission electron microscopy (TEM). In the ganglion cell layer of the retina from rats exposed for 9 days to hyperoxia, capillaries with large projections toward the lumen, were observed as a possible consequence of cellular edema of endothelium. The most severe damage was observed in rats exposed to hyperoxia during 12 and 14 days, showing mitochondrias swollen up and without crests in the areas surrounding the capillaries, necrosis and apoptosis processes, dense bodies, cells with swollen cytoplasms and rupture of the plasmatic membrane. The results suggest that postnatal hyperoxia causes severe damages to the retina in developing rats with a direct relationship between the time exposed to oxygen and ultra structural damages.

Con el propósito de evaluar el efecto de la hiperoxia posnatal sobre la estructura retiniana se analizaron retinas de ratas recién nacidas expuestas a diferentes periodos de oxigenación (80 ±1%), con tres interrupciones de 21% (30 min c/u). Cuatro grupos de ratas fueron expuestas desde su nacimiento hasta el 6to, 9no, 12mo y 14to días de vida y otro grupo fue mantenido en normoxia. Después de este periodo tanto los grupos expuestos a la hiperoxia como los controles permanecieron en normoxia hasta una edad de 30 días para el análisis estructural de la retina. La histología se hizo usando técnicas convencionales para microscopía electrónica de transmisión (MET). En la capa de células ganglionares de la retina de ratas expuestas a nueve días de hiperoxia, se observaron capilares con notables proyecciones hacia la luz, posiblemente como consecuencia de edema celular del endotelio. El daño más intenso fue observado en las ratas expuestas a hiperoxia durante 12 y 14 días, mostrando mitocondrias hinchadas y sin crestas en las áreas circundantes a los capilares, procesos de necrosis y apoptosis, cuerpos densos, células con citoplasmas hinchados y con ruptura de la membrana plasmática. Los resultados sugieren que la hiperoxia posnatal causa graves daños a la retina en las ratas en desarrollo, con una relación directa entre el tiempo de exposición al oxígeno y los daños ultraestructurales.

Oxígeno toxicidad evidenciada por ciitología de lavado bronquial / Oxigen-toxicity identify by citology of bronchial lavage

Los objetivos del trabajo son: Identificar la presencia de alteraciones celulares en la vías aéreas producidas por oxigenoterapia e intentar determinar en los casos de oxigenotoxicidad la relación con FiO2, presión en las vías aéreas, PEEP y tiempo de exposición. El análisis reporta 14 pacientes con signos de oxigenotoxicidad (78 por ciento) siendo el hallazgo más significativo a nivel microscópico: hipoplasia de células alveolares tipo I, metaplasia escamosa y células reactivas. 43 por ciento de los pacientes evidenciaron signos de oxigenotoxicidad en las primeras 48 horas de oxigenoterapia, no lográndose establecer relación con Fio2, PEEP y PIM. En todos los pacientes en etapa neonatal (22 por ciento) se evidenció toxicidad por oxígeno, llamándose a la reflexión en relación al uso indiscriminado de oxigenoterapia

Toxicidade do oxigênio: implicaçöes clínicas / Toxicity of oxygen: clinical implications

O uso de altas concentraçöes de oxigênio tem aumentado na medicina moderna. Säo discutidas as alteraçöes bioquímicas, anatomopatológicas bem como os efeitos fisiológicos, os principais sinais clínicos e sintomas da exposiçäo a altas fraçöes inspiradas de oxigênio. O tratamento profilático e o limite de segurança do uso de oxigênio säo apresentados

Açäo de íons Fe+++ no sistema de óxido-reduçäo do glutation e na lipoperoxidaçäo de membrana em eritrócitos humanos / Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to ion Fe+++

Várias säo as doenças que estäo relacionadas ao desequilíbrio entre a açäo de agentes oxidantes e o sistema de defesa do órgäo acometido. Dentre elas destaca-se a hemocromatose transfusional, na qual a funçäo de vários órgäos está comprometida pelo excesso de ferro. Nesta condiçäo, pouco é conhecido sobre o papel e as consequências da sobrecarga deste metal no eritrócito. A presença de ácidos graxos polinsaturados na membrana, do ferro ligado à hemoglobina, e de altas tensöes de oxigênio em seu interior permite supor que o eritrócito é, intrinsicamente, uma célula vulnerável ao estresse oxidativo. Alguns autores, que utilizam a sobrecarga de ferro com o intuito de provocar estresse oxidativo eritrocitário, verificaram diminuiçäo dos níveis de GSH e aumento de lipoperoxidaçäo mensurada pelo MDA. Com a finalidade de estudar a ocorrência da lipoperoxidaçäo e as possíveis alteraçöes do sistema anti-oxidante do glóbulo vermelho em funçäo da exposiçäo ao Fe+++, o presente trabalho analisou as variáveis glutation reduzido (GSH total), glutation oxidado (GSSG), índice de estresse oxidativo (R=GSSG/GSHtotal-GSSG), CATALASE, glutation redutase (GSH-Rd), glutation peroxidase (GSH-Px), superóxido dismutase (SOD) e malonaldeído (MDA) de eritrócitos normais submetidos a diferentes concentaçöes de ferro. O estudo foi realizado em duas fases, sendo que na primeira foram estudadas todas as variáveis eritrocitárias citadas acima, em funçäo de seis concentraçöes de ferro (0, 1, 5, 10, 50 e 100 uM Fe+++). Na segunda fase, foi analisado o GSH total de hemolisados e sobrenadanantes em 50, 100, 150, 200, 250 e 300 segundos de leitura espectrofotométrica. Nesta fase, foi também avaliada a interferência do tampäo EDTA no nível de GSH total de hemácias submetidas a quatro concentraçös de Fe+++ (0, 5, 10 e 100 uM Fe+++). Os resultados da Fase I näo mostraram diferenças significativas quando as variáveis foram analisadas em funçäo do aumento da concentraçäo de Fe+++. Entretanto, em relaçäo aos outros grupos, os resultados sugerem que eritrócitos incubados com 5 e 10 uM Fe+++ estäo sob estresse oxidativo máximo, documentado pelo alto índice R de 0,07 M/M, e a incubaçäo com 5, 10 e 50 uM Fe+++ provoca lipoperoxidaçäo de membrana, mensurada pelo MDA, maior que nos outros grupos. Apesar de näo termos observado alteraçöes enzimáticas eritrocitárias (GSH-Rd, GSH-Px, CATALASE e SOD), pode ser inferido que com as incubaçöes de 5 e 10 uM Fe+++ ocorre entrada de ferro no eritrócito...