LASER SAFETY THRESHOLDS FOR CETACEANS AND PINNIPEDS

An increase in the use of oceanographic lidar has raised concern over laser safety for marine mammals. We were able to address some of these concerns by combining information about current laser safety standards, retinal damage mechanisms for humans, and research on eye anatomy for humans, cetaceans, and pinnipeds. To estimate the irradiance at the retina, the image size at the retina and pupil diameter must be known. We estimate the smallest spot size using retinal resolution or visual acuity for six species of cetaceans and five species of pinnipeds. A sensitivity ratio was calculated for each species using the ratio of the irradiance at the retina of the marine mammal to the irradiance at the retina of humans. The sensitivity ratio was used to suggest exposure limits for the various species. Because the human eye is more sensitive than either the cetacean or pinniped eye, we conclude that laser energies that are eye-safe for humans will also be safe for marine mammals, and higher laser irradiances may be permissible if illumination of humans is avoided.     

c-Met modulates RPE migratory response to laser-induced retinal injury

Retinal laser injuries are often associated with aberrant migration of the retinal pigment epithelium (RPE), which can cause expansion of the scar beyond the confines of the original laser burn. In this study, we devised a novel method of laser-induced injury to the RPE layer in mouse models and began to dissect the mechanisms associated with pathogenesis and progression of laser-induced RPE injury. We have hypothesized that the proto-oncogene receptor, c-Met, is intimately involved with migration of RPE cells, and may be an early responder to injury. Using transgenic mouse models, we show that constitutive activation of c-Met induces more robust RPE migration into the outer retina of laser-injured eyes, while abrogation of the receptor using a cre-lox method reduces these responses. We also demonstrate that retinal laser injury increases expression of both HGF and c-Met, and activation of c-Met after injury is correlated with RPE cell migration. RPE migration may be responsible for clinically significant anatomic changes observed after laser injury. Abrogation of c-Met activity may be a therapeutic target to minimize retinal damage from aberrant RPE cell migration.     

Optical radiation and the eyes with special emphasis on children

The Sun is the most abundant source of optical radiation for the child eye. New hand-held visible lasers are a threat to the child eye. Some scientific data suggest that near infrared radiation may cause cumulative damage in the ocular lens. The child eye usually is exposed to ambient solar radiation, gazing at the horizon. Ambient Sun ultraviolet radiation (UVR) exposure to the child is complex due to atmospheric scattering and strong dependence on background reflection. Solar exposure causes biological damage, only by photochemical mechanisms. UVR exposure to a child eye is mainly a threat to the anterior segment of the eye, but also age dependently to the retina. Above threshold exposure to UVR, for short delay onset of damage, causes a toxic reaction on the surface of the eye, snow blindness, and cataract. Sub-threshold daily exposure to UVR over decades is associated with several ocular surface pathologies and eye lid cancer. Visible radiation is a threat to the retina. A single above threshold exposure, for short delay onset of damage to the retina causes immediate photochemical Type II retinal damage, Sun blindness. A single exposure of the retina to a very high intensity laser beam may cause thermal or thermo-mechanical damage in the retina. In environments with high irradiance of optical radiation, the child eye should be protected. Legislation and public information is required for avoidance of damage from high intensity laser systems. More research is urgently needed to exclude the potential hazard of near infrared radiation.     

Effects of retinal dopamine depletion on the growth of the fish eye

We investigated the suitability of fishes as animal models to study the involvement of the retinal dopaminergic system in the visually guided control of eye growth (emmetropization). Advantages of such a model system are (i) that all dopaminergic cells in the retina can be destroyed without apparent damage to other neurons, (ii) simple optical design and short depth of field of the eye, and (iii) continuous growth throughout life. Depleting the retina of dopamine in Aequidens pulcher (Cichlidae) had no apparent effect on refractive state, since size and focal length of the eye were reduced by the same amount. Furthermore, imposed defocus was compensated at a normal rate in spite of the absence of retinal dopamine. In A. pulcher, the dopaminergic system of the retina thus appears not to have an essential role in emmetropization. Our results furthermore suggest that in eyes of more complicated optical design, manipulation of the retinal dopaminergic system may lead to unrelated effects indistinguishable from direct interference with emmetropization. A major disadvantage of the fish model was that refractive state of the eye could not be measured accurately in vivo with standard methods. Accepted: 9 January 1999     

Retinal oxidative stress induced by high intraocular pressure

Glaucoma is an optic neuropathy in which retinal ganglion cells die probably through an apoptotic process. Apoptosis is known to involve free radicals in several systems including the retina. In this context, the aim of the present work was to analyze retinal oxidative damage in rats with glaucoma induced by the chronic injection of hyaluronic acid in the eye anterior chamber. The results showed a significant decrease in total retinal superoxide dismutase and catalase activities after 6 and 3 weeks of treatment with hyaluronic acid, respectively. Also, although GPX activity increased after 10 weeks of ocular hypertension, GSH levels significantly decreased at 6 weeks of treatment with hyaluronic acid. Moreover, retinal lipid peroxidation significantly increased in a time-of-hypertension-dependent manner. On the other hand, a significant decrease in both diurnal and nocturnal retinal melatonin content was detected at 3, 6, or 10 weeks of treatment with hyaluronic acid. The present results suggest that retinal oxidative stress may be involved in glaucomatous cell death. Thus, manipulation of intracellular redox status using antioxidants may be a new therapeutic tool to prevent glaucomatous neurodegeneration.     

Oxidative damage-induced inflammation initiates age-related macular degeneration

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.     

Topical administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema

Age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions are complicated by neovascularization and macular edema. Multi-targeted kinase inhibitors that inhibit select growth factor receptor tyrosine kinases and/or components of their down-stream signaling cascades (such as Src kinases) are rationale treatment strategies for these disease processes. We describe the discovery and characterization of two such agents. TG100572, which inhibits Src kinases and selected receptor tyrosine kinases, induced apoptosis of proliferating endothelial cells in vitro. Systemic delivery of TG100572 in a murine model of laser-induced choroidal neovascularization (CNV) caused significant suppression of CNV, but with an associated weight loss suggestive of systemic toxicity. To minimize systemic exposure, topical delivery of TG100572 to the cornea was explored, and while substantial levels of TG100572 were achieved in the retina and choroid, superior exposure levels were achieved using TG100801, an inactive prodrug that generates TG100572 by de-esterification. Neither TG100801 nor TG100572 were detectable in plasma following topical delivery of TG100801, and adverse safety signals (such as weight loss) were not observed even with prolonged dosing schedules. Topical TG100801 significantly suppressed laser-induced CNV in mice, and reduced fluorescein leakage from the vasculature and retinal thickening measured by optical coherence tomography in a rat model of retinal vein occlusion. These data suggest that TG100801 may provide a new topically applied treatment approach for ocular neovascularization and retinal edema.     

Evaluation of a 345 nm Femtosecond Laser for Corneal Surgery with Respect to Intraocular Radiation Hazard

Purpose

We report our findings from a preclinical safety study designed to assess potential side effects of corneal ultraviolet femtosecond laser treatment on lens and retina.

Methods

Refractive lenticules (-5 dpt) with a diameter of 6 mm were created in the right cornea of eight Dutch Belted rabbits. Radiant exposure was 0.5 J/cm² in two animals and 18 J/cm² in six animals. The presence of lens opacities was assessed prior to and up to six months following laser application using Scheimpflug images (Pentacam, Oculus) and backscatter analysis (Opacity Lensmeter 702, Interzeag). Ganzfeld flash and flicker electroretinogram (ERG) recordings were obtained from both eyes prior to and up to six weeks following laser application. At the study endpoint, retinas were examined by light microscopy.

Results

Independent of energy dose applied, no cataract formation could be observed clinically or with either of the two objective methods used. No changes in ERG recordings over time and no difference between treated and untreated eye were detected. Histologically, retinal morphology was preserved and retinal pigment epithelium as well as photoreceptor inner and outer segments appeared undamaged. Quantitative digital image analysis did not reveal cell loss in inner or outer nuclear layers.

Conclusions

Our analysis confirms theoretical considerations suggesting that ultraviolet femtosecond laser treatment of the cornea is safe for intraocular tissues. Transmitted light including stray light induces no photochemical effects in lens or retina at energy levels much higher than required for the clinical purpose. These conclusions cannot be applied to eyes with pre-existing retinal damage, as these may be more vulnerable to light.     

Virtual retinal display system with nodal point image of a laser beam: construction example and evaluation of subjective brightness perception]

We investigated the perception of brightness for red monochromatic laser light. For this purpose, a modified virtual retinal display (VRD) was constructed. The modification involved projecting the laser beam into the eye. In our VRD, the laser beam pivots in the nodal point of the eye (badal system). The displayed image therefore does not depend on the refractive state of the eye. Brightness perception was assessed by means of psychophysical experiments. The results of these experiments indicate that perception of brightness at 652 nm increases more rapidly with increasing physical stimulus than does the perception of white light (colour temperature 2'935 K). At a wavelength of 652 nm, an optical power of 79 nW is required to produce an image subtending 2 degrees of equal brightness, as is perceived with an image of about 2'900 cd/m2 subtending 2 degrees.     

In vivo electroporation of morpholinos into the adult zebrafish retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection. In all cases, resident Müller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days. Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.     

578.2 nm激光照射对兔视网膜的损伤效应研究

研究578.2 nm激光照射对兔视网膜的作用特点,以新西兰白兔5只10眼为实验对象,铜蒸汽激光(578.2 nm)通过裂隙灯照射兔视网膜后极部,照射时间为100 s,光斑直径为2 mm,照射剂量分别为60 J/cm2、80 J/cm2、100 J/cm2、120 J/cm2、160 J/cm2、200 J/cm2,每组4个光斑。照后1 h及24 h进行眼底照相及光镜观察。照光后可见,随激光功率密度的增加,兔视网膜的损伤也逐渐加重,并且照后24 h的损伤要重于照后1h。80 J/cm2和60 J/cm2在照后1 h和24 h均未发现明显改变。578.2 nm激光照射白兔后的主要病理学改变位于脉络膜。因此,以578.2 nm激光作为光动力治疗眼底疾病的光源时,照射剂量不宜超过80 J/cm2。     

Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.     

A computational modeling for the detection of diabetic retinopathy severity

Prolonged diabetes ultimately leads to Diabetic Retinopathy (DR) which is one of the leading causes of preventable blindness in the world. Through advanced image analysis techniques are used for abnormalities detection in retina that define and correlate the severity of DR. A thorough study is done in this area in recent past years and on the basis of these studies we have developed a computer based prediction model that is used to determine the severity of DR. To identify severity DR, we have analyzed the human eye image. We have extracted some important features from human eye image i.e. Blood Artery, Optical disc, Exudates. Based on these image and data we have designed an automated system for the determination of DR severity. This automated DR severity assessment methods can be used to predict the clinical case and conditions when young clinicians would agree or disagree with their more experienced fellow members. The algorithms described in this study may be used in clinical practice to validate or invalidate the diagnoses. Algorithms or method developed here may also be used for pooling diagnostic knowledge for serving mankind. Here we have described a computational based low cost retinal diagnostic approach which can aid an ophthalmologist to quickly diagnose the various stages of DR. This system can accept retinal images and can successfully detect any pathological condition associated with DR.     

Injury risk prediction from computational simulations of ocular blast loading

A predictive Lagrangian–Eulerian finite element eye model was used to analyze 2.27 and 0.45 kg trinitrotoluene equivalent blasts detonated from 24 different locations. Free air and ground level blasts were simulated directly in front of the eye and at lateral offset locations with box, average, less protective, and more protective orbital anthropometries, resulting in 96 simulations. Injury risk curves were developed for hyphema, lens dislocation, retinal damage, and globe rupture from experimental and computational data to compute risk from corneoscleral stress and intra-ocular pressure computational outputs. Corneoscleral stress, intra-ocular pressure, and injury risks increased when the blast size was larger and located nearer to the eye. Risks ranged from 20–100 % for hyphema, 1–100 % for lens dislocation, 2–100 % for retinal damage, and 0–98 % for globe rupture depending on the blast condition. Orbital geometry affected the stresses, pressures, and associated ocular injury risks of the blast conditions simulated. Orbital geometries that more fully surrounded the eye such as the more protective orbit tended to produce higher corneoscleral stresses and compression of the eye against the surrounding rigid orbit contributing to high stresses as the blast wave propagated. However, the more protective orbit tended to produce lower intra-ocular pressures in comparison with the other three orbital geometries which may indicate that the more protective orbit inhibits propagation of the blast wave and reduces ocular loading. Results of this parametric computational study of ocular blast loading are valuable to the design of eye protection equipment and the mitigation of blast-related eye injuries.     

Retinal detachment following hockey injury.

Thirty-three cases of retinal detachment following hockey injury were seen during a 15-year period at the retina clinic of Maisonneuve-Rosemont Hospital, Montreal. Most injuries occurred in adolescents, the average age being 18 years. The mean interval between injury and preoperative examination was 3 years. Almost half of these young hockey players remained legally blind in the affected eye even after a successful operation. A helmet with a protective face visor is suggested as the best prevention against severe ocular damage such as retinal detachment.     

Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina.     

Glutamate-Induced Excitotoxicity in Retina: Neuroprotection with Receptor Antagonist,Dextromethorphan, but Not with Calcium Channel Blockers

The purpose of our studies was to evaluate different strategies for possible neuroprotection in glutamate-induced neurotoxicity in the retina. In a first set of experiments we attempted to determine if dextrorphan antagonism of glutamate action on NMDA receptors would protect against excitotoxic injury associated with secondary damage seen after surgical laser treatment in retina. In a second set of experiments, the effects of different calcium channel blockers in an in-vitro model of N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell excitotoxicity that utilized rabbit retinal explants were evaluated. Dextrorphan infusion prior to laser treatment of rabbit retina produced a significant decrease in the area of neural retinal damage. We attribute the apparent dextrorphan protection to attenuation of glutamate mediated excitotoxicity secondary to laser induced cell death. Preincubation of rabbit retinal explants with verapamil, nimodipine or -conotoxin MVIIA did not cause a significant change in NMDA induced cell death in the ganglion cell layer.     

Ischemic tolerance protects the rat retina from glaucomatous damage

Glaucoma is a leading cause of acquired blindness which may involve an ischemic-like insult to retinal ganglion cells and optic nerve head. We investigated the effect of a weekly application of brief ischemia pulses (ischemic conditioning) on the rat retinal damage induced by experimental glaucoma. Glaucoma was induced by weekly injections of chondroitin sulfate (CS) in the rat eye anterior chamber. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 min; this maneuver started after 6 weekly injections of vehicle or CS and was weekly repeated in one eye, while the contralateral eye was submitted to a sham procedure. Glaucoma was evaluated in terms of: i) intraocular pressure (IOP), ii) retinal function (electroretinogram (ERG)), iii) visual pathway function (visual evoked potentials, (VEPs)) iv) histology of the retina and optic nerve head. Retinal thiobarbituric acid substances levels were assessed as an index of lipid peroxidation. Ischemic conditioning significantly preserved ERG, VEPs, as well as retinal and optic nerve head structure from glaucomatous damage, without changes in IOP. Moreover, ischemia pulses abrogated the increase in lipid peroxidation induced by experimental glaucoma. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies in glaucoma treatment.     

A possible role for the vascular membrane in retinal regeneration in Rana catesbienna tadpoles

We have studied the process of retinal regeneration in Rana catesbienna tadpoles using a recently developed monoclonal antibody (2D3) directed against frog neurons and germinitive neuroepithelium. We have found that, following retinal degeneration induced by devascularization, new retina is generated in the posterior eye from transdifferentiating pigment epithelial (RPE) cells and in the anterior eye from increased proliferation at the normal growth zone in the ora serrata. This demonstrates that the anuran retina regenerates in a manner similar to that observed previously in urodeles. In addition, the use of MAb-2D3 has allowed us to study the process of RPE transdifferentiation more accurately than was previously possible, and consequently we have found a high degree of association of migratory pigment cells with the retinal vascular membrane at the time of the initial RPE transdifferentiation to retinal neuroblasts.     

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch’s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.