Visual and electrical evoked response recorded from subdural electrodes implanted above the visual cortex in normal dogs under two methods of anesthesia.

Sensitive methods are required to record electrical evoked potentials over the visual cortex to evaluate the efficacy and safety of a retinal prosthesis before it can be implanted on the retinal surface of patients afflicted by outer retinal diseases. This study was designed to examine subdural electrodes as a mean to evaluate cortical evoked potentials in response to light and electrical stimulation of the retina in three dogs under two methods of anesthesia-halothane and propofol. Results showed that subdural electrodes could be stabilized over the visual cortex for several (3-5) months, and that they were 6.95 times more sensitive than subdermal electrodes in recording cortical visual evoked potentials (VEPs) and 4.31 times more sensitive in recording cortical electrical evoked potentials under both methods of anesthesia. The waveforms' shape changed for each electrode in the subdural array during 6/6 (100%) and 20/38 (52%) multi-channel recording sessions under halothane and propofol, respectively. This change could point to a cortical retinotopic organization versus hierarchical organization of different cortical areas for a given retinal stimulus. In summary, subdural electrodes show promising results for recording visual and electrical evoked responses (EERs) and thus for evaluation of the retinal prosthesis.

Optic nerve transection in monkeys may result in secondary degeneration of retinal ganglion cells.

PURPOSE: Interest in neuroprotection for optic neuropathies is, in part, based on the assumption that retinal ganglion cells (RGCs) die, not only as a result of direct (primary) injury, but also indirectly as a result of negative effects from neighboring dying RGCs (secondary degeneration). This experiment was designed to test whether secondary RGC degeneration occurs after orbital optic nerve injury in monkeys. METHODS: The superior one third of the orbital optic nerve on one side was transected in eight cynomolgus monkeys (Macaca fascicularis). Twelve weeks after the partial transection, the number of RGC bodies in the superior and inferior halves of the retina of the experimental and control eyes and the number and diameter of axons in the optic nerve were compared by detailed histomorphometry. Vitreous was obtained for amino acid analysis. A sham operation was performed in three additional monkeys. RESULTS: Transection caused loss of 55% +/- 13% of RGC bodies in the superior retina of experimental compared with fellow control eyes (mean +/- SD, t-test, P < 0.00,001, n = 7). Inferior RGCs, not directly injured by transection, decreased by 22% +/- 10% (P = 0.002). The loss of superior optic nerve axons was 83% +/- 12% (mean +/- SD, t-test, P = 0.0008, n = 5) whereas, the inferior loss was 34% +/- 20% (P = 0.02, n = 5). Intravitreal levels of glutamate and other amino acids in eyes with transected nerves were not different from levels in control eyes 12 weeks after injury. Fundus examination, fluorescein angiography, and histologic evaluation confirmed that there was no vascular compromise to retinal tissues by the transection procedure. CONCLUSIONS: This experiment suggests that primary RGC death due to optic nerve injury is associated with secondary death of surrounding RGCs that are not directly injured.

Absence of ocular effects after either single or repeated exposure to 10 mW/cm(2) from a 60 GHz CW source.

This study was designed to examine ocular effects associated with exposure to millimeter waves (60 GHz). Rabbits served as the primary experimental subjects. To confirm the results of the rabbit experiments in a higher species, the second phase of the study used nonhuman primates (Macaca mulatta). First, this study used time-resolved infrared radiometry to assess the field distribution patterns produced by different antennas operating at 60 GHz. These results allowed us to select an antenna that produced a uniform energy distribution and the best distance at which to expose our experimental subjects. The study then examined ocular changes after exposure at an incident power density of 10 mW/cm(2). Acute exposure of both rabbits and nonhuman primates consisted of a single 8 h exposure, and the repeated exposure protocol consisted of five separate 4 h exposures on consecutive days. One eye in each animal was exposed and the contralateral eye served as the sham-exposed control. After postexposure diagnostic examinations, animals were euthanized and the eyes were removed. Ocular tissue was examined by both light and transmission electron microscopy. Neither microscopic examinations nor the diagnostic procedures performed on the eyes of acute and repeatedly exposed rabbits found any ocular changes that could be attributed to millimeter-wave exposure at 10 mW/cm(2). Examination of the primates after comparable exposures also failed to detect any ocular changes due to exposure. On the basis of our results, we conclude that single or repeated exposure to 60 GHz CW radiation at 10 mW/cm(2) does not result in any detectable ocular damage.

Long-term histological and electrophysiological results of an inactive epiretinal electrode array implantation in dogs.

PURPOSE: Short-term pattern electrical stimulation of the retina via multielectrode arrays in humans blind from photoreceptor loss has shown that ambulatory vision and limited character recognition is possible. To develop an implantable retinal prosthesis that would provide useful vision, these results need to be sustained over a prolonged period of retinal electrical stimulation. As a first step toward this goal, the biocompatibility and the feasibility of surgically implanting an electrically inactive electrode array onto the retinal surface was tested. METHODS: A 5 x 5 electrode array (25 platinum disc-shaped electrodes in a silicone matrix) was implanted onto the retinal surface using retinal tacks in each of the 4 mixed-breed sighted dogs. Color fundus photography, fluorescein angiography, electroretinography, and visual evoked potentials were obtained preoperatively, at 1-week intervals for 2 weeks postoperatively, then at 2-week intervals up to 2 months postoperatively, and thereafter at 1-month intervals. One dog was killed at 2 months after implantation and a second dog after 3 months of implantation. Histologic evaluation of the retinas was performed. The remaining two dogs continue to be followed beyond 6 months after the implantation surgery. RESULTS: No retinal detachment, infection, or uncontrolled intraocular bleeding occurred in any of the animals. Retinal tacks and the retinal array remained firmly affixed to the retina throughout the follow-up period. Hyperpigmentation of the retinal pigment epithelium was observed only around the site of retinal tack insertion. No fibrous encapsulation of the implant or intraocular inflammation was visible. A- and b-wave amplitudes of the electroretinogram were depressed at the first postoperative week testing but recovered over the ensuing 1 week and were not statistically different from the normal unoperated fellow eye throughout the postoperative period. N1 and P1 wave amplitudes of the visual evoked potentials were not significantly different from the normal fellow eyes at any of the postoperative test intervals. Fluorescein angiography showed that the entire retina including the area under the electrode array remained well perfused. Similarly, histologic evaluation revealed near total preservation of the retina underlying the electrode array. CONCLUSIONS: Implantation of an electrode array on the epiretinal side (i.e., side closest to the ganglion cell layer) is surgically feasible, with insignificant damage to the underlying retina. The platinum and silicone arrays as well as the metal tacks are biocompatible. With the success of implanting an electrically inactive device onto the retinal surface for prolonged periods, the effects of long-term retinal electrical stimulation are now ready to be tested as the next step toward developing a prototype retinal prosthesis for human use.

Clinical and histopathologic features of canine oxygen-induced proliferative retinopathy.

PURPOSE: In previous studies the morphologic features of the acute vaso-obliterative and vasoproliferative stages of oxygen-induced retinopathy (OIR) were quantified and described in the dog model of retinopathy of prematurity (ROP). In the present study the sequelae of these events were examined using fluorescein angiography and histologic, enzyme, and immunohistochemical techniques. METHODS: Thirty newborn animals were exposed to 95% to 100% oxygen for 4 days and returned to room air until they were 22 to 45 days of age. Before death some animals were anesthetized, and fluorescein angiography was performed. Retina and vitreous from some animals were processed for adenosine diphosphatase (ADPase) flat-embedding. In other cases, eyes were prepared for full-thickness eyewall sectioning or frozen for histochemical analysis. RESULTS: Fluorescein angiography, funduscopic examination, and ADPase preparations showed dilated and tortuous retinal vessels, pigmentary changes, incomplete vascularization of peripheral retina, vitreous hemorrhage, and persistence of massive intravitreal neovascularization. Full-thickness eyewall sections showed tractional retinal folds, tented intravitreal vascularized membranes, and vitreous synchysis. Immunohistochemical analysis showed inner retinal astrogliosis. Enzyme histochemistry showed high alpha glycerophosphate dehydrogenase activity in poorly differentiated neovascular formations and low activity in formations with mature pericytes and endothelial cells. CONCLUSIONS: End-stage OIR in the neonatal dog shares many features with the chronic human disease. These results provide additional support for the use of this model in experimental studies of ROP.

Clinicopathological correlation of technetium bone scan in vascularization of hydroxyapatite implants. A primate model.

OBJECTIVES: To report the histopathological and bone scan characteristics of the stages of hydroxyapatite fibrovascular integration and to consider the implications for the timing of peg drilling in a primate model. DESIGN: Three monkeys received hydroxyapatite implants covered only anteriorly with a fascia lata button to which the rectus muscles were sutured. Weekly bone scans were evaluated quantitatively and qualitatively. The orbits were harvested at 2, 4, and 8 weeks and examined histopathologically. RESULTS: Quantitatively, the implant's technetium uptake increased, then reached a plateau by 4 weeks. Peripheral uptake was present on the images and histologically at 2 weeks. When bone scan images suggested complete vascularization by the fourth week, the implant was 99% vascularized histologically. Completion of vascularization was ascertained at 8 weeks, without further discernible changes in the bone scans. CONCLUSIONS: The technetium bone scan is sensitive to the vascularization of the hydroxyapatite implant and discerns when complete vascularization is approached. This primate study models closely the clinical findings we have recently reported. We advocate at least a 4-week interval between the time the bone scan suggests full vascularization and peg drilling.

Endothelin receptor-binding subtypes in the human retina and choroid.

PURPOSE: Endothelins (ETs) are a family of vasoconstrictive peptides produced in part by vascular endothelial cells. They are synthesized in the eye, particularly in the uveal tract, and can markedly constrict the iris and preretinal vessels in the rabbit eye. Two ET receptor subtype families have been characterized and cloned, termed ETA and ETB. To better understand the distribution of ocular ET receptors, we localized ET receptor-binding sites in human and rabbit eyes. METHOD: Autoradiography with iodine 125-labeled ET-1 and varying concentrations of unlabeled ET-3 was used to localize ET receptor-binding subtypes in the posterior pole of human and rabbit eyes. RESULTS: The ETA-like receptor-binding sites are localized to the retinal and choroidal blood vessels, whereas the ETB-like receptor-binding sites are localized to the neural and glial substance of the retina. CONCLUSIONS: Endothelin receptor-binding subtypes are differentially located in the posterior pole of human and rabbit eyes. Endothelin peptides released within the retina and choroid may participate in the ocular manifestations of a variety of systemic disorders, including systemic hypertension.

Increased sensitivity of the non-human primate eye to microwave radiation following ophthalmic drug pretreatment.

Previous studies in our laboratory have established that pulsed microwaves at 2.45 GHz and 10 mW/cm2 are associated with production of corneal endothelial lesions and with disruption of the blood-aqueous barrier in the non-human primate eye. In the study reported here we examined ocular damage in monkeys (M. mulatta and M. fascicularis) following topical treatment with one of two ophthalmic drugs (timolol maleate and pilocarpine) that preceded exposure to pulsed microwaves. Anesthetized monkeys were sham exposed or exposed to pulsed, 2.45 GHz microwaves (10 microseconds, 100 pps) at average power densities of 0.2, 1, 5, 10, or 15 mW/cm2 4 h a day for 3 consecutive days (respective SARs were 0.052, 0.26, 1.3, 2.6, and 3.9 W/kg). Immediately before microwave exposure, one or both eyes were treated topically with one drop of 0.5% timolol maleate or of 2% pilocarpine. Following administration of a drug, we observed a significant reduction in the power-density threshold (from 10 to 1 mW/cm2) for induction of corneal endothelial lesions and for increased vascular permeability of the iris. Diagnostic procedures (in vivo specular microscopy and fluorescein iris angiography) were performed following each exposure protocol. In addition, increased vascular permeability was confirmed with horseradish peroxidase tracer techniques. Although we did not measure intraocular temperatures in experimental animals, the results suggest that a mechanism other than significant heating of the eye is involved. Our data indicate that pulsed microwaves at an average SAR of 0.26 W/kg, if administered after pretreatment with ophthalmic drugs, can produce significant ocular effects in the anesthetized primate.

A primate model of macular cyst.

Perfluoropropane (C3F8) was injected into nine eyes of six cynomolgus monkeys. In all nine eyes, macular changes consistent with the development of a macular cyst were noted within 3 months after injection. On fluorescein angiography, there was no evidence of retinal vascular leakage associated with the macular cysts. Histopathologic evaluation of six eyes demonstrated microcystic changes throughout the perifoveal inner nuclear layer. These coalesced into larger cystic spaces in the outer plexiform layer of the central macula. These macular changes are previously unreported in patients or monkeys undergoing intravitreal injection of expansile gases. This model for macular cysts may be helpful in evaluating the pathogenesis and role of interventional strategies in patients with cystoid macular edema.

A primate model of anterior segment ischemia after strabismus surgery. The role of the conjunctival circulation.

Fornix conjunctival incisions in strabismus surgery may provide partial protection against anterior segment ischemia by preserving the perilimbal conjunctival-Tenon's circulation, which is disrupted with limbal conjunctival incisions. Six adult cynomolgus monkeys underwent tenotomies of three or four rectus muscles by making limbal conjunctival incisions in one eye and fornix incisions in the other. Iris fluorescein angiography and slit-lamp biomicroscopy were used to monitor changes. The eyes that had limbal incisions exhibited more severe anterior segment ischemic changes than the eyes that had fornix incisions in every instance of four rectus muscle surgery.

Effect of single-dose ivermectin therapy on human Onchocerca volvulus infection with onchocercal ocular involvement.

Ivermectin has shown promise as a potentially safe and effective microfilaricidal drug for the treatment of onchocerciasis. Several limited studies have shown it to have fewer side effects, especially ocular complications, than the currently available drug, diethylcarbamazine. The detailed ocular findings in 200 moderately to heavily infected Liberians who were enrolled in a safety and dose-finding study are presented. They received either 0, 100, 150, or 200 micrograms/kg of ivermectin and were followed up for 12 months. In clinical studies so far carried out ivermectin in a dose of 100, 150, or 200 micrograms/kg has not been associated with any major adverse reactions nor were there any sight-threatening effects even in the presence of severe ocular disease. Each of these doses significantly reduced the ocular microfilaria load for at least 12 months when compared with either the placebo (p less than 0.05) or pretreatment values (p less than 0.001). However, the 100 and 150 micrograms/kg doses caused fewer minor side effects than the higher dose. These results confirm that ivermectin in a single oral dose may be a safe and effective microfilaricidal drug for the treatment of onchocerciasis and that it appears to be free of major ocular side effects.

Controlled trial and dose-finding study of ivermectin for treatment of onchocerciasis.

Ivermectin, given as a single oral dose, has shown considerable promise as a new treatment for onchocerciasis. We assessed the safety and efficacy of ivermectin and tried to determine the optimal dose. Two hundred Liberians received 100, 150, or 200 micrograms of ivermectin/kg or placebo and were followed up for 12 months. Therapy was associated with only minimal systemic and ocular side effects. A 200-micrograms/kg dose was associated with greater systemic reaction than was a 100-micrograms/kg dose. Each treatment group had significant reduction in skin microfilaria counts by day 3 and a decrease of approximately 95% at three months. At three months the proportion of persons with no microfilariae in a specimen of skin was significantly less in the 100-micrograms/kg group than in the 150-micrograms/kg or 200-micrograms/kg groups. At 12 months, the level of microfilariae in skin was still reduced approximately 80%. Significant reduction in ocular involvement was seen in all groups. These results confirm that single-dose ivermectin is effective and well-tolerated and suggest that 150 micrograms of ivermectin/kg may be the optimal dose for initial therapy.

Ocular fluorescein phototoxicity.

Fluorescein angiography is one of the most commonly used diagnostic techniques in modern ophthalmology. Prior to this study, recommendations on the phototoxicity of this technique were based on light levels alone (i.e., without fluorescein present). Using a rabbit model to demonstrate retinal damage, intravenous sodium fluorescein was found to reduce the amount of blue light needed to cause a phototoxic retinal lesion almost a log unit (from 1.6 to 0.2 W/cm(2)). Intravenous fluorescein also reduced the threshold for light damage to iris and cornea in the rabbit. The results suggest that blue light exposure should be minimized when sodium fluorescein is present in ocular tissue.

Treatment of onchocerciasis. The ocular effects of ivermectin and diethylcarbamazine.

The ocular changes that occur with diethylcarbamazine treatment of onchocerciasis seriously restrict its usefulness. Ivermectin, a newly developed antifilarial drug, was compared with diethylcarbamazine for treatment of onchocerciasis in a double-masked, placebo-controlled trial. Thirty men with moderate to severe infection and ocular involvement were randomly assigned to receive ivermectin as a single oral dose (200 micrograms/kg), diethylcarbamazine (administered for eight days), or placebo. Detailed ocular examinations were performed serially over a 12-month period. Diethylcarbamazine treatment caused a marked increase in living and dead microfilariae in the cornea, punctate opacities, and limbitis during the first week of therapy. Ivermectin had no such effect. However, ivermectin therapy resulted in a long-term reduction in intraocular microfilariae comparable to that seen with diethylcarbamazine. Ivermectin appears to have few ocular complications and be a better-tolerated and more effective microfilaricidal agent than diethylcarbamazine for the treatment of onchocerciasis.

Comparison of ivermectin and diethylcarbamazine in the treatment of onchocerciasis.

We compared ivermectin with diethylcarbamazine for the treatment of onchocerciasis in a double-blind, placebo-controlled trial. Thirty men with moderate to heavy infection and ocular involvement were randomly assigned to receive ivermectin in a single oral dose (200 micrograms per kilogram of body weight), diethylcarbamazine daily for eight days, or placebo. Diethylcarbamazine caused a significantly more severe systemic reaction than ivermectin (P less than 0.001), whereas the reaction to ivermectin did not differ from the reaction to placebo. Diethylcarbamazine markedly increased the number of punctate opacities in the eye (P less than 0.001), as well as the number of dead and living microfilariae in the cornea over the first week of therapy. Ivermectin had no such effect. Both ivermectin and diethylcarbamazine promptly reduced skin microfilaria counts, but only in the ivermectin group did counts remain significantly lower (P less than 0.005) than in the placebo group at the end of six months of observation. Analysis of adult worms isolated from nodules obtained two months after the start of therapy showed no effect of either drug on viability. Ivermectin appears to be a better tolerated, safer, and more effective microfilaricidal agent than diethylcarbamazine for the treatment of onchocerciasis.

Effects of 2.45-GHz microwaves on primate corneal endothelium.

Both eyes of anesthetized cynomolgus monkeys (Macaca fascicularis) were irradiated with 2.45-GHz microwaves, either pulsed or continuous wave. In vivo corneal endothelial abnormalities were observed by specular microscopy and confirmed through histologic techniques after a 16- to 48-hour postexposure period. Pulsed microwaves with an average power density of 10 mW/cm2 (equivalent to a specific absorption rate (SAR) = 2.6 W/kg) produced these effects, while levels of 20-30 mW/cm2 (equivalent to a SAR = 5.3 to 7.8 W/kg) with continuous wave irradiation were required to produce similar changes.

Cross-polarization photography of the nerve fiber layer.

Cross-polarized photography was used to enhance the visibility of the arcuate bundles of the retinal nerve fiber layer ( NFL ). A fundus camera (Zeiss) was modified to accept a linear polarizing filter in front of the flash and another polarizing filter in front of the film plane. The azimuths of the two filters were kept at right angles to one another. Cross-polarized photographs were taken of the NFL of seven normal eyes and 18 eyes with ocular hypertension. The polarizers were rotated together to obtain four different orientations, equally spaced through 90 degrees. The manner in which visibility of the arcuate fibers was enhanced varied consistently with the orientation of the polarizing filters. Cross-polarization accentuated apparent defects in the NFL when compared with standard red-free illumination.

High-resolution photography of the retinal nerve fiber layer.

Abnormalities in the appearance of the retinal nerve fiber layer can indicate the presence of optic nerve atrophy, but a major problem has been the difficulty of visualizing the nerve fiber layer in some individuals. By selectively illuminating the white nerve fiber layer in comparison with the red retinal pigment epithelium and choroidal background, red-free light increases the visibility of the nerve fiber layer. Compared with the standard red-free photographic technique, a 560-nm short-pass cut-off filter, used with extended-range, high-resolution, extremely fine-grain film (Kodak No. 2415 Technical Pan film), considerably enhanced the ability to resolve nerve fiber bundles.

Fluorescein angiography of the heavily pigmented iris and new dyes for iris angiography.

An inexpensive adapter mounted in front of the objective lens of a fundus camera permits routine fluorescein angiography of the heavily pigmented iris. Angiograms of these patients whose iris pigment was dense enough that standard methods of iris angiography would disclose little or no information were performed with the use of the adapter. For use in the study of the iris vasculature in the research laboratory, three dyes with spectral characteristics different from those of fluorescein allow angiography of the iris at different wavelengths. Angiograms using these dyes were performed on the eye of a cynomolgus monkey. The angiograms were each taken as close as possible to the same time sequence and show dramatically different filling and staining patterns.