Antiangiogenic activity of photobiomodulation in experimental model using chorioallantoic embryonic membrane of chicken eggs / Atividade antiangiogênica da fotobiomodulação em modelo experimental usando membrana embrionária corioalantóide de ovos de galinhas

ABSTRACT Purpose: The purpose of this study was to investigate the vascular effects of photobiomodulation using a light-emitting diode on the chorioallantoic embryonic membrane of chicken eggs grouped into different times of exposure and to detect the morphological changes induced by the light on the vascular network architecture using quantitative metrics. Methods: We used a phototherapy device with light-emitting diode (670 nm wavelength) as the source of photobiomodulation. We applied the red light at a distance of 2.5 cm to the surface of the chorioallantoic embryonic membrane of chicken eggs in 2, 4, or 8 sessions for 90 s and analyzed the vascular network architecture using AngioTool software (National Cancer Institute, USA). We treated the negative control group with 50 μl phosphate-buffered-saline (pH 7.4) and the positive control group (Beva) with 50 μl bevacizumab solution (Avastin, Produtos Roche Químicos e Farmacêuticos, S.A., Brazil). Results: We found a decrease in total vessel length in the Beva group (24.96% ± 12.85%) and in all the groups that received 670 nm red light therapy (2× group, 34.66% ± 8.66%; 4× group, 42.42% ± 5.26%; 8× group, 38.48% ± 6.96%), compared with the negative control group. The fluence of 5.4 J/cm2 in 4 sessions (4×) showed more regular vessels. The number of junctions in the groups that received a higher incidence of 670 nm red light (4× and 8×) significantly decreased (p<0.0001). Conclusion: Photo-biomodulation helps reduce vascularization in chorioallantoic embryonic membrane of chicken eggs and changes in the network architecture. Our results open the possibility of future clinical studies on using this therapy in patients with retinal diseases with neovascular components, especially age-related macular degeneration.

RESUMO Objetivo: investigar os efeitos vasculares da foto-biomodulação com diodo emissor de luz utilizando membrana embrionária corioalantóide de ovos de galinhas em grupos com diferentes tempos de exposição e detectar as alterações morfológicas por meio de métricas quantitativas promovidas pela luz na arquitetura da rede vascular. Métodos: Um aparelho de fototerapia com diodo emissor de luz no comprimento de onda de 670 nm foi usado como fonte de fotobiomodulação. A luz vermelha foi aplicada a uma distância de 2,5 cm da superfície da membrana embrionária corioalantóide em 2, 4 ou 8 sessões de 90 s a arquitetura da rede vascular foi analisada por meio do software AngioTool (National Cancer Institute, USA). Usamos um grupo controle negativo tratado com 50 µL de solução salina tamponada com fosfato (PBS) pH 7,4 e um grupo controle positivo (Beva) tratado com 50 µL de solução de bevacizumabe (Avastin, Produtos Roche Químicos e Farmacêuticos S.A., Brasil). Resultados: Uma diminuição no comprimento total do vaso foi detectada para o grupo Beva (24,96 ± 12,85%), e para todos os grupos que receberam terapia de luz vermelha de 670 nm, 34,66 ± 8,66% (2x), 42,42 ± 5,26% (4x) e 38,48 ± 6,96% (8x) em comparação ao grupo controle. A incidência de 5,4 J/cm2 em 4 sessões (4x) mostrou vasos mais regulares. A redução foi mais intensa nos grupos que receberam maior incidência de luz vermelha de 670 nm (4x e 8x). Conclusão: A fotobiomodulação contribui para a redução da vascularização nos vasos da membrana embrionária corioalantóide de ovos de galinhas e mudanças na arquitetura da rede. Os achados deste experimento abrem a possibilidade de considerar um estudo clínico usando esta terapia em pacientes com doenças retinais com componentes neovasculares, especialmente degeneração macular relacionada à idade.

Intravitreal injection of peptides PnPa11 and PnPa13, derivatives of Phoneutria nigriventer spider venom, prevents retinal damage

PnPa11 and PnPa13 are synthetic peptides derived from Phoneutria nigriventer spider venom, which display antinociceptive and neuroprotective properties. In this work, we evaluated the safety of intravitreal use and the neuroprotective effect of these peptides. Methods: The cytotoxicity and the antiangiogenic activity of these peptides were evaluated by the sulforhodamine-B method and chicken chorioallantoic membrane (CAM) assay, respectively. The in vivo safety was analyzed in Wistar rats that were intravitreally injected with different doses (0.50; 1.25; 2.50; 3.75 and 5.00 µg/mL) of these peptides (right eye, n = 6). The retinal function was assessed by electroretinography exams (ERG), intraocular pressure (IOP), and histological analyzes. In order to investigate the neuroprotective effect, Wistar rats received intravitreal injections (right eye, n = 6) of peptides at 1.25 µg/mL and then were exposed to blue LED light. In addition, the visual function and the retinal microstructure were verified. Results: Cytotoxicity analyses demonstrated that the peptides did not present any toxicity over ARPE-19 (adult retinal pigmented epithelial) cell line and the antiangiogenic study highlighted that the peptides promoted the reduction of blood vessels. The intravitreal injection did not cause major changes, neither induced any irreversible damage. In the retinal degeneration assay, the ERG records demonstrated that the prior treatment with PnPa11 and PnPa13 protected the retina from damage. Morphological analyses confirmed the ERG findings. Immunoblotting analyses revealed that PnPa11 increased Erk1/2, NR2A, and NR2B retinal expression after the light stress model, but did not cause Akt1 activation, while PnPa13 prevented Erk1/2 and Akt1 dephosphorylation. Conclusions: The intraocular administration of these peptides was well tolerated and presented protective activity against retinal degeneration, suggesting the potential use of these peptides as neuroprotectors in the ophthalmological field.(AU)

Intravitreal injection of the synthetic peptide LyeTx I b, derived from a spider toxin, into the rabbit eye is safe and prevents neovascularization in a chorioallantoic membrane model

The great diversity of molecules found in spider venoms include amino acids, polyamines, proteins and peptides, among others. Some of these compounds can interact with different neuronal receptors and ion channels including those present in the ocular system. To study potential toxicity and safety of intravitreal injection in rabbits of LyeTx I b, a synthetic peptide derived from the toxin LyeTx I found in venom from the spider Lycosa eritrognatha and to evaluate the angiogenic activity on a CAM model. Methods: ARPE-19 cells were treated with LyeTx I b (0.36; 0.54; 0.72; 2.89; 4.34 or 9.06 µM). In this study, New Zealand rabbits were used. LyeTx I b (2.89 µM) labeled with FITC dissolved in PBS, or only PBS, were injected into vitreous humor. Electroretinogram (ERG) was recorded 1 day before injection and at 7,14 and 28 days post-injection. Clinical examination of the retina was conducted through tonometer and eye fundus after ERG. Eyes were enucleated and retinas were prepared for histology in order to assess retinal structure. CAMs were exposed to LyeTx I b (0.54; 0.72; 2.17 or 2.89 µM). Results: ARPE-19 cells exposed to LyeTx I b showed cell viability at the same levels of the control. The fluorescence of LyeTx I b labeled with FITC indicated its retinal localization. Our findings indicate ERG responses from rats injected in the eye with LyeTx I b were very similar to the corresponding responses of those animals injected only with vehicle. Clinical examination found no alterations of intraocular pressure or retinal integrity. No histological damage in retinal layers was observed. CAM presented reduced neovascularization when exposed to LyeTx I b. Conclusions: Intravitreal injection of LyeTx I b is safe for use in the rabbit eye and prevents neovascularization in the CAM model, at Bevacizumab levels. These findings support intravitreal LyeTx l b as a good candidate to develop future alternative treatment for the retina in neovascularization diseases.(AU)