Comparative Study Of Retinal Damage Induced By Laser Irradiation At Wavelengths Of 650 Nm And 450 Nm And The Role Of PARP-1 In Blue Light Damage

Electromagnetic radiation usually refers to all electromagnetic waves with electromagnetic radiation properties,including radio waves,microwaves,infrared,visible light,ultraviolet,X-rays and gamma rays.It spread throughout the universe as a carrier of energy and information.Visible light is the part of electromagnetic waves that can be perceived by the human eye.The human eye can generally perceive electromagnetic waves with frequency:380～750THz,wavelength:400～760 nm.Different wavelengths of visible light can be recognized by the human eye in the form of different colors such as red,orange,yellow,green,blue,indigo and violet.The retina,the innermost layer of the eye,is the neural tissue responsible for converting light signals into visual information and a part of the central nervous system originates in the brain during development.Retinal neurons include photoreceptor cells,bipolar cells,horizontal cells,anaplastic cells and ganglion cells.Linked by synapses,these neurons are responsible for converting light signals into neuroelectrical signals,transmitting to the visual centers of the cerebral cortex,ultimately forming vision.In addition to being an essential information carrier for the formation of vision,light of different wavelengths can also play a variety of biomodulatory roles through the retina.Studies have shown that blue light at a wavelength of around 480nm stimulates intrinsically photosensitive retinal ganglion cells in the retina,transmits light stimuli to the supraoptic nucleus biorhythmic center which in turn affects the secretion of pineal melatonin,thereby regulates circadian rhythms and sleep conditions.In addition,In the process of emmetropization of the eye,light plays an important role in the development of refractive state.Recently,repetitive irradiation with a low-energy red laser has become an emerging therapy in the field of youth myopia prevention and control.A one-year multicenter clinical study has shown that repeated irradiation of myopic children with a low-energy 650 nm red laser（2 m W,3min/session,2 sessions/day）can significantly slow down the growth of the eye axis,depress the myopia growth curve and control the progression of myopia in adolescents.The use of the biomodulatory properties of light has also led to the emergence of a variety of commercial phototherapy products.However,prolonged exposure of the eye to excessive light can trigger photochemical damage to the retina,photooxidation is considered to be an important molecular mechanism for triggering retinal photochemical damage.A large quantity of endogenous photosensitive group exists in the retina.When photosensitive groups absorb light radiation energy at specific wavelengths,they can break chemical bonds in other molecules through direct electron exchange or direct hydrogen exchange,resulting in the formation of reactive oxygen species（ROS）radicals such as singlet oxygen（¹O₂）,superoxide radicals（O₂^-）,hydrogen peroxide（H₂O₂）and hydroxyl radicals（-OH）.Excess intracellular ROS cause oxidative damage such as lipid peroxidation,inactivation of enzymes and proteins,and nuclear DNA breakage.The high oxygen consumption of retinal tissues,rich in high concentrations of polyunsaturated fatty acids and prolonged exposure to light radiation also significantly increase the susceptibility of the retina to photo-oxidative damage.Poly（ADP-ribose）polymerases are nucleases responsible for DNA damage repair in the nucleus of eukaryotic cells.To this day,18 isoforms of the PARP family have been identified.PARP-1 is the most abundant in eukaryotic cells,its structure and function have been most intensively studied.The activity of PARP-1 is basically low,but it can be activated rapidly when senses nuclear DNA damage.Activated PARP-1 catalyzes the transfer of ADP-ribosyl units from nicotinamide adenine dinucleotide（NAD⁺）to target genes regulating protein functions,such as histones and transcription factors,a post-translational protein modification known as poly（ADP-ribosyl）action.PARP-1 binds to DNA damage sites（mostly single-stranded DNA breaks）and catalyzes the synthesis of poly（ADP-ribosyl）action on protein substrates,recruits other DNA repair proteins to the damage site and co-repairs the DNA damage.However,PARP-1 is over-activated when DNA damage is severe,leading to excessive intracellular NAD+depletion,which in turn leads to ATP depletion and ultimately to parthanatos cell death.In this study,we compared the differences in retinal photochemical damage induced by different wavelengths（650nm red laser vs 450nm blue laser）,different irradiance intensities（high and low irradiance）and different irradiation modes（intermittent and continuous）.In addition,we also investigated the role of PARP-1 in the molecular mechanisms of blue light-induced damage to the retinal cells.This is expected to provide an experimental basis for the safety of photobiomodulatory commodities and the development of the retinal photodamage protective drugs.Objectives1.To demonstrate the difference in photoreceptor cell（661W）damage induced by different wavelengths（650nm red laser vs 450nm blue laser）in vitro.2.To demonstrate the difference in photochemical damage to the retina induced by different wavelengths（650nm red laser vs 450nm blue laser）,different irradiance intensities（high and low irradiance）and different irradiation modes（intermittent and continuous）in vivo.3.To investigate the role of PARP-1 in the molecular mechanism on photoreceptor cell（661W）damage induced by 450 nm blue laser irradiation by establishing an in vitro model.4.To demonstrate protective effect of inhibiting PARP-1 pathway on retinal damage induced by blue light in mice.Methods1.Differences in photoreceptor cells（661W cells）damage induced by 650 nm red and 450 nm blue laser（2.55 m W/cm²）irradiation in.（1）661W cells were exposed to 650nm red and 450nm blue laser irradiation in vitro..（2）The activity changes of 661W cells induced by 650nm red and 450nm blue laser irradiation were detected using CCK-8.（3）Hochest/PI staining was used to detect the death of 661W cells induced by650nm red and 450nm blue laser irradiation.（4）The expression of reactive oxygen species（ROS）and 8-hydroxy-2’-deoxyguanosine（8-OHd G）,a marker of oxidative DNA damage,was detected by cellular immunofluorescence staining in 661W cells induced by 650nm red and450nm blue laser irradiation.2.Retinal damage induced by intermittent repetitive irradiation with low irradiance（2.55 m W/cm2）650 nm red and 450 nm blue lasers in mice.（1）A mouse eye model was established with intermittent repetitive irradiation with low irradiance（2.55 m W/cm²）650 nm red and 450 nm blue laser（2）The structural and ultrastructural changes of the retina after irradiation with650nm red and 450nm blue lasers were observed by HE staining and transmission electron microscopy,and the changes in the content of Rhodopsin were detected by Western Blot and tissue immunofluorescence staining.（3）The functional changes of mouse retina after 650nm red and 450nm blue laser irradiation were assessed by electroretinography（ERG）.3.Retinal damage induced by continuous irradiation of low irradiance（2.55m W/cm²）650 nm red and 450 nm blue lasers in mice.（1）A mouse model was established with continuous irradiation with low irradiance（2.55 m W/cm²）650 nm red and 450 nm blue laser（2）The structural and ultrastructural changes of the retina after irradiation with650nm red and 450nm blue lasers were observed by HE staining and transmission electron microscopy,and the changes in the content of Rhodopsin were detected by Western Blot and tissue immunofluorescence staining.（3）The functional changes of mouse retina after 650nm red and 450nm blue laser irradiation were assessed by electroretinography（ERG）.4.Retinal damage induced by intermittent repetitive irradiation with high irradiance（12.74 m W/cm²）650 nm red and 450 nm blue lasers in mice.（1）A mouse eye model was established with intermittent repetitive irradiation with low irradiance（12.74 m W/cm²）650 nm red and 450 nm blue laser（2）The structural and ultrastructural changes of the retina after irradiation with650nm red and 450nm blue lasers were observed by HE staining and transmission electron microscopy,and the changes in the content of Rhodopsin were detected by Western Blot and tissue immunofluorescence staining.（3）The functional changes of mouse retina after 650nm red and 450nm blue laser irradiation were assessed by electroretinography（ERG）.5.Retinal damage induced by continuous irradiation of high irradiance（12.74m W/cm²）650 nm red and 450 nm blue lasers in mice.（1）A mouse model was established with continuous irradiation with high irradiance（12.74 m W/cm²）650 nm red and 450 nm blue laser（2）The structural and ultrastructural changes of the retina after irradiation with650nm red and 450nm blue lasers were observed by HE staining and transmission electron microscopy,and the changes in the content of Rhodopsin were detected by Western Blot and tissue immunofluorescence staining.（3）The functional changes of mouse retina after 650nm red and 450nm blue laser irradiation were assessed by electroretinography（ERG）.6.To investigate the potential mechanism of 450nm blue laser-induced retinal photoreceptor cell damage.（1）To establish an in vitro model of photoreceptor cell（661W cells）photodamage induced by 450nm blue laser.（2）Western Blot was applied to detect PARP-1 protein expression in 661W cells after blue light damage.（3）Preparation of a lentiviral knockdown PARP-1 cell line.（4）Assess the protective effect of PARP-1 knockdown on blue light-induced cell damage using Hochest/PI staining.7.To verify the protective effect of PARP-1 inhibition on 450nm blue laser-induced retinal damage in mice.（1）A 450nm blue laser-induced retinal photodamage model in mice was established（2）The protective effects of PARP-1 inhibitor on blue light-induced retinal structural and ultrastructural damage in mice were assessed by HE staining and transmission electron microscopy,and the changes in the content of Rhodopsin were detected by Western Blot and tissue immunofluorescence staining methods.（3）The protective effects of PARP-1 inhibitors on blue light-induced retinal function in mice were evaluated by ERG.Results1、In vitro studies showed that under the same irradiance and irradiation duration,650 nm red laser irradiation did not cause a significant increase in ROS and 8-OHd G content in 661W cells,and no significant increase in cell mortality was observed,while 450 nm blue laser irradiation significantly induced an increase in ROS and8-OHd G production in 661W cells,resulting in a significant increase in cell mortality.2.The intermittent repeated irradiation of 650nm red and 450nm blue laser at low irradiance（2.55m W/cm²）did not lead to a significant decrease in the thickness of the outer nuclear layer of the mouse retina,the outer segments of the photoreceptor cells were neatly arranged,the photoreceptor cell bodies were intact and tightly arranged,the content of the Rhodopsin did not change significantly,and the ERG amplitude of the mouse retina did not decrease significantly.3.Continuous irradiation of 650 nm red laser at low irradiance（2.55 m W/cm²）did not cause any significant decrease in the thickness of the outer nuclear layer of the mouse retina,the outer segments of the photoreceptor cells were neatly arranged,the photoreceptor cell bodies were intact and tightly arranged,the content of the Rhodopsin did not change significantly,and the ERG amplitude of the mouse retina did not decrease significantly.In contrast,the 450nm blue laser caused the thickness of the outer nuclear layer of the mouse retina to be thinned,the number of cells to be reduced,the outer disc membrane of the photoreceptor cells to be swollen and disorganized,the photoreceptor cell bodies to be smaller,sparsely arranged,the envelope to be ruptured,the nucleus to be fixed,the content of Rhodopsin to be reduced significantly,and the ERG amplitude of the mouse retina to be reduced significantly.4.Intermittent repeated irradiation of 650 nm red laser with high irradiance（12.74 m W/cm²）did not cause any significant decrease in the thickness of the outer nuclear layer of the mouse retina,the outer segments of the photoreceptor cells were neatly arranged,the photoreceptor cell bodies were intact and tightly arranged,the content of the Rhodopsin did not change significantly,and the ERG amplitude of the mouse retina did not decrease significantly.the 450nm blue laser did not cause any significant decrease in the thickness of the outer nuclear layer of the mouse retina,the outer disc membrane of the photoreceptor cells to be swollen and disorganized,the photoreceptor cell bodies to be smaller,sparsely arranged,the envelope to be ruptured,the nucleus to be fixed,the content of the Rhodopsin did not change significantly,and the ERG amplitude of the mouse retina to be reduced significantly.5.Continuous irradiation of 650 nm red laser with high irradiance（12.74m W/cm²）did not cause any significant decrease in the thickness of the outer nuclear layer of the mouse retina,the outer segments of the photoreceptor cells were neatly arranged,the photoreceptor cell bodies were intact and tightly arranged,the content of Rhodopsin to be reduced significantly,and the ERG amplitude of the mouse retina did not decrease significantly.In contrast,the 450nm blue laser caused the thickness of the outer nuclear layer of the mouse retina to be thinned,the number of cells to be reduced,the outer disc membrane of the photoreceptor cells to be swollen and disorganized,the photoreceptor cell bodies to be smaller,sparsely arranged,the envelope to be ruptured,the nucleus to be fixed,the content of Rhodopsin to be reduced significantly,and the ERG amplitude of the mouse retina to be reduced significantly.6.450 nm blue laser irradiation induced an increase in PARP-1 protein expression in 661W cells,while PARP-1 knockdown could inhibit 450 nm blue laser-induced damage in 661W cells.7.The application of PARP-1 inhibitor significantly protected against 450 nm blue laser-induced retinal damage in mice,maintained the thickness of the outer nuclear layer of the retina,kept the outer segmental disc membrane of photoreceptor cells aligned neatly and the photoreceptor cell bodies aligned tightly,maintained the retinal expression content of Rhodopsin,and significantly increased the ERG a and b wave amplitudes.Conclusions1.Retinal photodamage induced by monochromatic laser irradiation of different wavelengths was variable,with 450 nm blue laser more likely to cause retinal photodamage than 650 nm red laser at the same irradiance and the same irradiation duration.2.There were differences in light-induced retinal damage under different irradiation intensities（high vs.low irradiance）and different irradiation modes（intermittent vs.continuous irradiation）of the same wavelength monochromatic laser;the retinal photodamage gradually increased with increasing irradiance;intermittent repetitive irradiation was safer than continuous irradiation when the total irradiation duration was the same.3、The PARP-1 pathway plays an important role in the molecular mechanism of450 nm blue laser-induced photoreceptor cell death,and inhibition of the PARP-1pathway significantly increased the survival rate of 661W cells under 450 nm blue laser irradiation and significantly protected against blue light-induced retinal damage in mice.