RESUMO
Purpose: Subthreshold, nanosecond pulsed laser treatment shows promise as a treatment for age-related macular degeneration (AMD); however, the safety profile needs to be robustly examined. The aim of this study was to investigate the effects of laser treatment in humans and mice. Methods: Patients with AMD were treated with nanosecond pulsed laser at subthreshold (no visible retinal effect) energy doses (0.15-0.45 mJ) and retinal sensitivity was assessed with microperimetry. Adult C57BL6J mice were treated at subthreshold (0.065 mJ) and suprathreshold (photoreceptor loss, 0.5 mJ) energy settings. The retinal and vascular responses were analyzed by fundus imaging, histologic assessment, and quantitative PCR. Results: Microperimetry analysis showed laser treatment had no effect on retinal sensitivity under treated areas in patients 6 months to 7 years after treatment. In mice, subthreshold laser treatment induced RPE loss at 5 hours, and by 7 days the RPE had retiled. Fundus imaging showed reduced RPE pigmentation but no change in retinal thickness up to 3 months. Electron microscopy revealed changes in melanosomes in the RPE, but Bruch's membrane was intact across the laser regions. Histologic analysis showed normal vasculature and no neovascularization. Suprathreshold laser treatment did not induce changes in angiogenic genes associated with neovascularization. Instead pigment epithelium-derived factor, an antiangiogenic factor, was upregulated. Conclusions: In humans, low-energy, nanosecond pulsed laser treatment is not damaging to local retinal sensitivity. In mice, treatment does not damage Bruch's membrane or induce neovascularization, highlighting a reduced side effect profile of this nanosecond laser when used in a subthreshold manner.
Assuntos
Cegueira/prevenção & controle , Terapia com Luz de Baixa Intensidade , Degeneração Macular/radioterapia , Neovascularização Retiniana/prevenção & controle , Idoso , Animais , Cegueira/fisiopatologia , Proteínas do Olho/genética , Feminino , Angiofluoresceinografia , Humanos , Imuno-Histoquímica , Lasers de Estado Sólido/uso terapêutico , Degeneração Macular/fisiopatologia , Masculino , Melanossomas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pessoa de Meia-Idade , Fatores de Crescimento Neural/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Retina/fisiopatologia , Neovascularização Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Serpinas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Acuidade Visual/fisiologia , Testes de Campo VisualRESUMO
Long-lived proteins exist in a number of tissues in the human body; however, little is known about the reactions involved in their degradation over time. Lens proteins, which do not turn over, provide a useful system to examine such processes. Using a combination of Western blotting and proteomic methodology, age-related changes to a major protein, γS-crystallin, were studied. By teenage years, insoluble intact γS-crystallin was detected, indicative of protein denaturation. This was not the only change, however, because blots revealed evidence of significant cross-linking as well as cleavage of γS-crystallin in all adult lenses. Cleavage at a serine residue near the C terminus was a major reaction that caused the release of a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. Several other crystallin-derived peptides with double basic residues also lodged in the cell membrane fraction. Model studies showed that once cleaved from γS-crystallin, SPAVQSFRRIVE adopts a markedly different shape from that in the intact protein. Further, the acquired helical conformation may explain why the peptide seems to affect water permeability. This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may therefore yield peptides with untoward biological activity.
