Alcides Fernandes

Effects of muscarinic cholinergic receptor antagonists on postnatal eye growth of rhesus monkeys.

PURPOSE To study a potential role for muscarinic receptors in the inhibition of deprivation-induced excessive axial elongation and myopia in a monkey model. METHODS The right eyes of 20 newborn rhesus monkeys were occluded with a black contact lens. In seven monkeys each, either atropine or pirenzepine was topically applied daily to the occluded eyes. The nonoccluded fellow eyes and both the occluded and nonoccluded fellow eyes of another six monkeys were treated with vehicle solution. RESULTS After 33 to 39 weeks, in 5 monkeys of the vehicle group, occluded eyes were longer and the myopic shift significantly greater than in the nonoccluded fellow eyes. In six atropine-treated monkeys, axial length and reduction of the initial hyperopia of occluded and nonoccluded fellow eyes were not different statistically. The myopic shift of the occluded eyes was significantly smaller than in the vehicle-treated occluded eyes. In the pirenzepine-treated group, axial length of the occluded eyes was similar to the nonoccluded eyes of controls and the occluded eyes of atropine-treated monkeys. There was a trend of pirenzepine to reduce the myopic shift of the occluded eye. No effect of atropine or pirenzepine was noted on muscarinic receptor density in retina, brain, or heart, but a small increase was observed in iris + ciliary body. CONCLUSIONS The drug treatment results implicate muscarinic receptors in postnatal eye growth regulation. Because of interanimal differences our data do not indicate whether nonselective or selective muscarinic blockade is more effective in reducing deprivation-induced myopia.

High Susceptibility to Experimental Myopia in a Mouse Model with a Retinal ON Pathway Defect

PURPOSE Nob mice share the same mutation in the Nyx gene that is found in humans with complete congenital stationary night blindness (CSNB1). Nob mutant mice were studied to determine whether this defect resulted in myopia, as it does in humans. METHODS Refractive development was measured in unmanipulated wild-type C57BL/6J (WT) and nob mice from 4 to 12 weeks of age by using an infrared photorefractor. The right eye was form deprived by means of a skull-mounted goggling apparatus at 4 weeks of age. Refractive errors were recorded every 2 weeks after goggling. The content of dopamine and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by HPLC with electrochemical detection (HPLC-ECD) in retinas of nob and WT mice under light- and dark-adapted conditions. RESULTS The nob mice had greater hyperopic refractive errors than did the WT mice under normal visual conditions, until 12 weeks of age when both strains had similar refractions. At 6 weeks of age, refractions became less hyperopic in the nob mice but continued to become more hyperopic in the WT mice. After 2 weeks of form deprivation (6 weeks of age), the nob mice displayed a significant myopic shift (~4 D) in refractive error relative to the opposite and control eyes, whereas WT mice required 6 weeks of goggling to elicit a similar response. As expected with loss of ON pathway transmission, light exposure did not alter DOPAC levels in the nob mice. However, dopamine and DOPAC levels were significantly lower in the nob mice compared with WT. CONCLUSIONS Under normal laboratory visual conditions, only minor differences in refractive development were observed between the nob and WT mice. The largest myopic shift in the nob mice resulted after form deprivation, suggesting that visual pathways dependent on nyctalopin and/or abnormally low dopaminergic activity play a role in regulating refractive development. These findings demonstrate an interaction of genetics and environment in refractive development.

Transduction of photoreceptors with equine infectious anemia virus lentiviral vectors: safety and biodistribution of StarGen for Stargardt disease.

PURPOSE StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. METHODS Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. RESULTS Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. CONCLUSIONS In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

Possible sources of neuroprotection following subretinal silicon chip implantation in RCS rats

Current retinal prosthetics are designed to stimulate existing neural circuits in diseased retinas to create a visual signal. However, implantation of retinal prosthetics may create a neurotrophic environment that also leads to improvements in visual function. Possible sources of increased neuroprotective effects on the retina may arise from electrical activity generated by the prosthetic, mechanical injury due to surgical implantation, and/or presence of a chronic foreign body. This study evaluates these three neuroprotective sources by implanting Royal College of Surgeons (RCS) rats, a model of retinitis pigmentosa, with a subretinal implant at an early stage of photoreceptor degeneration. Treatment groups included rats implanted with active and inactive devices, as well as sham-operated. These groups were compared to unoperated controls. Evaluation of retinal function throughout an 18 week post-implantation period demonstrated transient functional improvements in eyes implanted with an inactive device at 6, 12 and 14 weeks post-implantation. However, the number of photoreceptors located directly over or around the implant or sham incision was significantly increased in eyes implanted with an active or inactive device or sham-operated. These results indicate that in the RCS rat localized neuroprotection of photoreceptors from mechanical injury or a chronic foreign body may provide similar results to subretinal electrical stimulation at the current output evaluated here.

Diffuser contact lenses retard axial elongation in infant rhesus monkeys

In each of five monkeys, one eye was fitted with a diffuser lens at birth. This lens allowed pattern vision, but also reduced contrast by about 1 log unit. In four out of five monkeys, the treated eyes were shorter and more hyperopic than the untreated fellow eyes. At 25 weeks of age, interocular differences (OD -- OS) of the experimental group were significantly greater than interocular differences of age-matched normal monkeys for both axial length (P < 0.05) and refractive error (P < 0.02). In addition, while the treated eyes were significantly different from normal eyes for both axial length measurements (P < 0.01) and refractive error (P < 0.01), there were no significant differences between the untreated fellow eyes and normal eyes. In primates less severe pattern deprivation appears to produce an effect on eye growth that is opposite to that of severe pattern deprivation (little or no pattern vision), which typically results in axial myopia.

Nicotinic Acetylcholine Receptor Subunits in Rhesus Monkey Retina

PURPOSE The purpose of this study was to detect and establish the cellular localizations of nicotinic acetylcholine receptor (nAChR) subunits in Rhesus monkey retina. METHODS Retinas were dissected from the eyes of monkeys killed after unrelated experiments. RNA was extracted and analyzed by RT-PCR, using primers designed against human sequences of alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits. The RT-PCR products were separated by gel electrophoresis and sequenced. Frozen sections of postmortem fixed monkey eyes were immunolabeled with well-characterized and specific monoclonal antibodies against the alpha3, alpha4, alpha6, alpha7, beta2, or beta4 nAChR subunits and visualized with fluorescence labeling. RESULTS Products of the predicted size for the alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits were detected by RT-PCR in Rhesus monkey retina. Homology between transcripts from monkey retina and human nucleotide sequences ranged from 93 to 99%. Immunohistochemical studies demonstrated that neurons in various cell layers of monkey retina expressed alpha3, alpha4, alpha7, or beta2 nAChR subunits and cells with the morphology of microglia were immunoreactive for the alpha6 or beta4 nAChR subunits. CONCLUSIONS nAChR subunits are expressed in the monkey retina and localize to diverse retinal neurons as well as putative microglia. Besides mediating visual processing, retinal nAChRs may influence refractive development and ocular pathologies such as neovascularization.

The refractive development of untreated eyes of rhesus monkeys varies according to the treatment received by their fellow eyes.

To determine the extent to which the visual experience of one eye may influence the refractive development of its fellow eye, we analyzed the data of untreated (UT) eyes of monkeys that received different types of unilateral pattern deprivation. Subjects were 15 juvenile rhesus monkeys, with five monkeys in each of three treatment groups: aphakic eyes with optical correction (AC), aphakic eyes with no correction (ANC), and eyes that were occluded with an opaque contact lens (OC). Under general anaesthesia, refractive error (D) was determined by cycloplegic retinoscopy and axial length (mm) was determined with A-scan ultrasonography. For measurements of refractive error of the UT eyes, there was a significant main effect of groups according to the treatment of the fellow eyes, F(2, 12) = 6.6. While UT eyes paired with AC fellow eyes (mean = +4.2 D) were significantly more hyperopic than the eyes of age-matched normal monkeys (mean = +2.4 D), t(25), = 2.5, UT eyes paired with OC fellow eyes (mean = -0.5 D) were significantly more myopic than the eyes of normal monkeys, t(25) = -9. UT eyes paired with ANC fellow eyes (mean = +1.9 D) were not significantly different from normal eyes. For measurements of axial length there was also a significant main effect of groups, F(2, 12) = 6.9. While UT eyes paired with AC fellow eyes (mean = 16.9 mm) were significantly shorter than the eyes of age-matched normal monkeys (mean = 17.5 mm), t(25) = 2.3, UT eyes paired with OC fellow eyes (mean = 18.1 mm) were significantly longer than the eyes of normal monkeys, t(25) = 2.3. UT eyes paired with ANC fellow eyes (mean = 17.5 mm) were not significantly different from the eyes of normal monkeys. The measurements of axial length and of refractive error of the UT eyes were also significantly correlated with one another, probably indicating that the differences in refractive error were due to differences in axial length, r = -0.8. The present data reveal that despite normal visual experience, UT eyes can have their refractive development altered, systematically, simply as a function of the type of pattern deprivation received by their fellow eyes. These data add to the growing evidence that there is an interocular mechanism that is active during emmetropization. As a consequence, future models of eye growth will need to consider both: (1) the direct influence of visual input on the growing eye; as well as (2) the indirect influence coming from the fellow eye.

Management of extended-wear contact lenses in infant rhesus monkeys

Infant rhesus monkeys were fitted with extended-wear contact lenses (EWCLs) to correct surgically induced aphakia. The protocol that produced the most favorable experimental outcome, including good lens-wear compliance, involved: (1) custom-made EWCLs with parameters adjusted for each animal, (2) selection for study of neonates who seemed to tolerate EWCLs most naturally, (3) individual housing of monkeys, (4) lens check at 2-h intervals around the clock by trained personnel, (5) a large inventory of EWCLs for each animal, (6) periodic ophthalmologic examinations, and (7) darkroom housing of monkeys during treatment of lens-wear-related incidents. The EWCL is a new tool for studies of the visual system and is a useful complement to existing experimental techniques.

Multifocal Versus Monofocal Correction of Neonatal Monocular Aphakia

We compared the efficacy of monofocal and multifocal intraocular lenses (IOLs) as a means of optically rehabilitating eyes with monocular congenital cataracts using a primate model. A congenital cataract was simulated in the right eye of 20 monkeys from birth to 2 weeks of age with an opalescent contact lens. A lensectomy was then performed and a monofocal IOL was implanted in 11 eyes and a multifocal IOL in 9 eyes. Any residual refractive error was then corrected with an extended-wear contact lens. The fellow eyes were then randomized to no treatment or 70% occlusion therapy. Grating acuity was assessed using forced-choice preferential looking with Teller acuity cards at regular intervals. At 32 weeks of age, the pseudophakic eyes were mildly amblyopic, but the grating acuities were not significantly different between the pseudophakic eyes with multifocal versus monofocal IOLs or between subjects who had received occlusion therapy versus no treatment of the fellow eye. We conclude that at least through 32 weeks of age, multifocal and monofocal IOLs are of similar efficacy and occlusion therapy may be less important to prevent amblyopia from developing in pseudophakic eyes compared to contact-lens corrected aphakic eyes.

Heterochromia after pediatric cataract surgery

PURPOSE Changes in iris color have been noted anecdotally after cataract surgery in infants, but they have not been studied systematically. The mechanism for these iris color changes has not previously been reported in the biomedical literature. METHODS Photographs were taken of both eyes of 15 children and 11 rhesus monkeys who had undergone unilateral cataract surgery. Masked examiners reviewed the photographs and compared the iris color of the eyes that were operated on with the eyes that were not operated on. Between 4 and 6 weeks postoperatively, the level of prostaglandin F(2alpha) in the aqueous humor (n = 4) and vitreous humor (n = 2) was measured in both the operated and nonoperated eyes of 4 monkeys that had undergone a neonatal lensectomy during the first 5 days of life. RESULTS Thirteen of 15 children had a darker iris color in the operated eye in relation to the nonoperated (control) eye. Four of 11 monkeys had a uniformly darker iris in the operated eye; the other 7 monkeys had regional darkening or patches of darker iris in the eye that was operated on. The prostaglandin F(2alpha) levels in neonatal monkeys were higher in the aqueous humor and in the vitreous humor of the operated eye in relation to the nonoperated eye. CONCLUSION In some children, cataract surgery is associated with a darkening of the iris color in the operated eye. We speculate that this darkening results from an exuberant prostaglandin release stimulated by the cataract surgery and may occur through the same or a similar mechanism by which latanoprost causes the darkening of iris color.