The effects of glaucoma filtering surgery on the variability of diurnal intraocular pressure.

The results of this study indicate that filtration surgery reduces the mean diurnal IOP, the range of diurnal variation, and the day-to-day variability. The effect on the range is proportionally greater than on the other two parameters. Further study will be required to determine whether this selective effect makes surgical reduction of IOP any more effective in the prevention of further glaucomatous visual field damage than other therapeutic methods that lower mean IOP to the same degree.

Feasibility of targeted drug delivery to selective areas of the retina.

A new method was developed to deliver locally a bolus dose of a drug to the retinal vasculature. The targeted delivery system was based on encapsulating the drug in heat-sensitive liposomes, which are injected intravenously and lysed in the retinal vessels by a heat pulse generated by a laser. To test if substances delivered in the vessels could also penetrate into the surrounding tissue, 6-carboxyfluorescein was encapsulated in liposomes and used as a marker for drug penetration. Moderate argon laser pulses were applied to the retinal vessels of Dutch pigmented rabbits to induce breakdown of the blood-retinal barrier (BRB). A suspension of liposomes at a dose of 2 ml/kg body weight, corresponding to a carboxyfluorescein dose of 12 mg/kg, was injected into the ear vein. The dye was released from the liposomes proximal to the damaged portion of the vessel. Fundus fluorescein angiograms were recorded with a video camera and digitized for subsequent image analysis. The penetration of carboxyfluorescein into the retinal tissue was evaluated by comparing the fluorescence intensity of the area around the damaged vessel with that of an adjacent control area. The dye penetration increased with the numbers of laser applications (P less than 0.001). The leakage was localized distally to the released site and was restricted to areas with a disrupted BRB. The mass of carboxyfluorescein that penetrated gradually spread with time. Both veins and arteries could be used for the targeted delivery. These results indicated that this delivery system, which is fully controllable by laser through the pupil, can deliver drugs inside the vasculature and into the retinal tissue wherever the BRB is disrupted.

Association between intraocular pressure peaks and progression of visual field loss.

Little is known about the relation between diurnal variations of intraocular pressure (IOP) and prognosis for glaucomatous visual field damage. The association between apparent progressive loss of visual field and the occurrence of IOP peaks was studied. Pressure peaks were detected by a self-tonometer in the natural environment of patients with glaucoma. The study groups consisted of patients with and without a strong indication of progressive visual field losses, all with IOPs of 22 mm Hg or less obtained in the ophthalmologist's office. Patients apparently undergoing progressive visual field loss were found to have significantly more frequent IOP peaks than patients with stable visual fields. A statistical evaluation indicated that, in a population with a 30% prevalence of progressive loss of visual field, 75% of the patients with peaks have progressive loss and 75% of those without peaks do not have visual field progression. Intraocular pressure peaks were thus shown to have an association with the apparent progression of vision loss independent of the mean IOP. Home tonometry appeared to be a promising tool for identifying patients at increased risk of developing visual field loss who may require intensified follow-up and an alteration in clinical management. However, the present study must be complemented by a prospective study.

Quantitative analysis of retinal hemodynamics using targeted dye delivery.

A new method designed to allow repeated mapping of retinal hemodynamics on a macro- and microcirculatory level was evaluated in the primate eye. The method, called "targeted dye delivery," consists of encapsulating a fluorescent dye in temperature-sensitive liposomes, injecting the liposomes systemically, and using a light pulse from an argon laser to release a bolus of dye in a targeted retinal vessel. The follow-up of the well-defined dye front thus generated allows calculation of the blood flow and capillary transit time. Evaluation of targeted dye delivery in a monkey indicated that centerline blood velocity and the vessel diameter can be measured with a reproducibility of 10% and 4%, respectively, in vessels that are 40 microns and larger. These measurements yielded flow values that had a reproducibility of 10% on the same day and 13% on different days. The normalization of flow rate by the vessel diameter was consistent with theoretic estimates and promises to be a circulation indicator independent of variations between individual and species. The transit time across capillary beds at different locations was found to be similar, thus indicating that the method could be used to evaluate the local viability of the microcirculation.

Topography of the retinal thickness in normal subjects.

A noninvasive method has been developed that is capable of providing quantitative thickness profiles of the retina. The method was used to map the retinal thickness in five normal human volunteers and determine the reproducibility of the measurements. The reproducibility or equivalent sensitivity of the measurements to detect changes was found to be 5% or 19 microns on the same day and 8% or 31 microns on different days. By averaging the values obtained in five normal subjects, ranging in age from 21 to 43 years (mean, 34 years), a preliminary normal baseline was derived for the thickness profile at the fovea and the thickness cross-section from the optic disc to the fovea. The results of the study indicated that this noninvasive method promises to be of clinical use in diagnosing ocular diseases that produce changes in the thickness of the retinal as well as in monitoring the effectiveness of therapy.

Visualization of the retinal microvasculature by targeted dye delivery.

Although fluorescein angiography has proven to be an important tool in the diagnosis and management of retinal vascular diseases, it is subject to certain limitations, namely the presence of the choroidal background, which usually precludes a detailed examination of the retinal microvasculature. Moreover, the inability to repeat the bolus reduces the chance of obtaining high-quality photographs of early phases, and does not allow for a complete binocular examination or for testing the response to induced physiologic changes. We have developed a method of targeted dye delivery that consists of encapsulating the dye in lipid vesicles, injecting them intravenously, and causing them to release their contents locally when a short heat pulse is induced in a retinal artery by a laser. This method was applied in the rhesus monkey in order to visualize the retinal microvasculature. A well-defined bolus and absence of background fluorescence permitted both following of the dye front through the vasculature and clear imaging of the capillary network over the whole posterior pole. The bolus delivery could be repeated as many as 100 times in 45 min without significant loss of contrast. The comparison of these results with conventional fluorescein angiography illustrated the advantage of the new method. The examination of the safety of the delivery system indicates that there is no major obstacle to the eventual application to humans.

Presence and rapid decline of early morning intraocular pressure peaks in glaucoma patients.

A group of nine glaucoma patients in whom home tonometry, with the self-tonometer, had previously demonstrated elevated intraocular pressure (IOP) peaks upon waking were asked to measure their IOPs when they awoke in the morning, 30 minutes later, and 1 hour later. This was repeated for 4 days and medication was taken only after the third measurement. Eight of the nine patients demonstrated a reproducible decrease in IOP ranging from 2 to 18 mmHg within 30 minutes. There are indications that the momentary IOP elevation is associated with wakening. This phenomenon raises interesting questions regarding IOP variations. It indicates that in certain cases significant IOP peaks disappear before the patient can reach the clinic and may have an effect on loss of vision.

Feasibility test of a new method to measure retinal thickness noninvasively.

There are many devastating ocular diseases that are directly related to an alteration of the retinal or nerve fiber layer thickness, such as glaucoma and macular edema. To diagnose these diseases earlier and to monitor their therapy more sensitively, an accurate measurement of the tissue thickness is needed. Since no clinical method is currently available, we developed and tested a new method capable of measuring noninvasively the retinal thickness. The separation between the images of the anterior and posterior intersections is quantitated by an optoelectronic system. The theoretical performance of the method has been calculated. Tests of the method in a model eye indicated that the measurements were basically diffraction limited, their reproductivity was +/- 9 microns, and their accuracy was 5.5 microns. Tests performed in vivo indicated that two intersections between the laser and the retina are present and correspond to the anterior and posterior surfaces of the retina. These intersections can be resolved and analyzed to yield quantitative data. These encouraging results indicate that this method is feasible and could yield sensitive measurements of the retinal thickness.

The relation between glaucomatous damage and optic nerve head mechanical compliance.

Histologic studies seem to imply that the mechanical compliance of the optic nerve head, namely, its displacement under a given pressure, may be altered in glaucoma. We have developed a method to noninvasively measure the optic nerve head displacement. In postmortem human glaucomatous eyes, the optic nerve head compliance decreased as the visual field worsened (n = 15, r = -.34). The mean difference between the optic nerve head displacement of the two eyes in subjects with symmetric clinical findings differed significantly from the mean in subjects with asymmetry. In the latter group, the lower value was always measured in the more affected eye. No significant correlation was found between age and the optic nerve head compliance. Overall, the results indicate that, in glaucoma, there is a stiffening of the mechanical support of the optic nerve head.

In vivo evaluation of a noninvasive method to measure the retinal thickness in primates.

To diagnose certain macular diseases earlier and monitor their therapy more sensitively, we are developing a noninvasive method to measure the retinal thickness. The new instrument, which is an extension of slit-lamp biomicroscopy, was used to obtain the data, which were analyzed with an algorithm to yield thickness measurements. The measurements performed in monkeys indicated that the retinal thickness can be visualized in a region extending from the optic disc to the fovea and that quantitative results can be obtained. The retinal thickness reproducibility was 6% for the same location on the same day, 15% for the same location on different days, and 12% for the same location in different eyes. The average retinal thickness in these areas was 335 microns, indicating that the reproducibility was between 20 and 50 microns. Measurements across the foveola illustrated that retinal thicknesses as low as 80 microns could be obtained.

Feasibility of blood flow measurement by externally controlled dye delivery.

We are developing a new method of delivering substances locally and repeatedly in the retinal vasculature under external control. This delivery system is based on encapsulating the substance in heat-sensitive lipsomes, which are injected intravenously and lysed by a heat pulse delivered by a laser. The feasibility of using this system with dyes and creating a sharp dye front was tested in vitro and in vivo. The results indicate that the background fluorescence of intact liposomes is minimal but in contrast a dramatic increase in fluorescence is achieved where the dye is released. In vivo tests indicated that only the selected vascular branch fluoresced. Moreover, a sharp dye front could be obtained repeatedly and preserved over significant distances. The presence of a sharp dye front allowed measurements, in vitro, of blood velocity which correlated well (r = 0.985, P less than 0.001) with the average blood velocity values calculated from the known flow rate.

A potential method for local drug and dye delivery in the ocular vasculature.

We propose a new drug and dye delivery system that would allow repeated release of substances in the ocular vasculature by an externally controlled mechanism. The substances are encapsulated in heat-sensitive liposomes, which are lysed by locally applying a heat pulse produced by an argon laser. The system was tested by investigating the release of carboxyfluorescein encapsulated in the liposomes. The liposome suspension was incubated at 37 degrees or 38.5 degrees C and irradiated at different powers and pulse durations. The amount of dye released was monitored by fluorophotometry and compared with the concentration obtained when the liposomes were lysed at their transition temperature of 41 degrees C. The results showed that 85% of the encapsulated substance can be released. Moreover, a dramatic contrast was observed between the fluorescence before and after the lysis. Presently the energy density is higher than but close to the maximal permissible exposure for humans. The release mechanism with the short laser pulse appeared to be similar to that present when liposomes were heated slowly.

An interactive model eye for use with ophthalmic instruments.

A model eye was found to be a practical and versatile simulator of conditions present in the application of different surgical and diagnostic ophthalmic instruments. It realistically simulated the thermal and acoustic effects of lasers on tissues and thus could be used for teaching and practicing laser therapeutics. The geometric optics were similar to those of the human eye, and realistic conditions of scatter and fluorescence could be created.

Evaluation of an objective method for the in vivo measurement of changes in light transmittance of the human crystalline lens.

Because the results of various vision tests and diagnostic methods are influenced by the amount of light transmitted by the lens, it would be useful to quantify changes in transmittance. We have developed a new method capable of following these changes objectively and quantitatively. The method is based on an increasing loss of fluorescence emitted by the lens substance as the probing volume is located more posteriorly along the lens axis. The loss of fluorescence is quantified by a parameter called the fluorescence reading ratio (FRR). In the present study, performed on postmortem human lenses, we evaluated the validity of our proposed in vivo method by comparing it with measurements of lens transmittance by spectrophotometry. The correlation of FRR with the transmittance between 420- and 500 nm (r greater than 0.7, P less than 0.015) indicates that the measurement of FRR can provide information on the transmittance at these wavelengths. In addition, a correlation was found between age and transmittance at 440 nm (r = 0.93, P less than 0.001) and FRR (r = 0.88, P less than 0.001).

Comparison of a noninvasive measurement of optic nervehead mechanical compliance with an invasive method.

We have postulated that abnormal mechanical support of the optic nervehead at the level of the lamina cribrosa could be the precursor of glaucomatous damage. Recent studies have shown deformations of the lamina cribrosa to be among the earliest changes in glaucoma. To evaluate the support of the nervehead, we have developed a noninvasive optical method to measure the optic nervehead compliance, namely, the displacement of the optic nervehead induced by an artificial increase in intraocular pressure. To test the validity of the method, we have compared noninvasive measurements obtained in post-mortem enucleated human eyes with those recorded using an invasive technique. Both methods had a reproducibility better than 6 microns and induced no damage capable of interfering with the results. The displacements measured by both methods were similar, thus indicating that our optical method is capable of measuring bulk motion of the optic nervehead. Our results were identical with those obtained by other authors using a third method. The data obtained also established the normal range of optic nervehead displacements induced by a range of intraocular pressure increments.

Self-tonometry to manage patients with glaucoma and apparently controlled intraocular pressure.

We tested a self-tonometer than can be used by the patient alone to monitor intraocular pressure (IOP) in a normal environment. The instrument is safely and easily used after training. Normal subjects and patients with glaucoma who had an IOP of 22 mm Hg or less at three consecutive visits were referred for diurnal monitoring. After successful training, they received a self-tonometer and instructions to obtain five measurements daily between awakening and bedtime for three to six days. More than half the patients had one or more readings above 22 mm Hg. About half of the IOP peaks occurred at times outside of normal office hours. Interestingly, more elevated IOP readings were recorded in patients with suspected or documented progression of glaucomatous damage than in patients thought to be stable or in normal subjects. In eyes that have already sustained glaucomatous damage, the progression may be explained by the presence of previously undocumented IOP peaks. Valuable additional clinical information can be gained by diurnal IOP monitoring, and the self-tonometer is a practical tool for its measurement.

Evaluation of vitreous body integrity in retinitis pigmentosa by vitreous fluorophotometry.

We performed vitreous fluorophotometry on 23 eyes of 15 normal individuals and on 29 eyes of 15 patients with retinitis pigmentosa one hour after the systemic administration of sodium fluorescein. The concentration gradient of fluorescein in the posterior vitreous was evaluated from the results of the testing. Patients with retinitis pigmentosa showed a significantly flat gradient compared with normal subjects, implying that fluorescein is distributed more rapidly in the vitreous body of the patients. It is suggested that patients with retinitis pigmentosa have an abnormal vitreous gel structure that causes more rapid distribution of fluorescein. If verified, this would indicate that vitreous fluorophotometry could be useful to evaluate changes in the integrity of the vitreous body in vivo.

Fluorescein and fluorescein glucuronide pharmacokinetics after intravenous injection.

The permeability of the blood-retinal and blood-aqueous barriers to fluorescein (F) and the rate of aqueous flow can be estimated by measurements of F in the vitreous, aqueous, and plasma after systemic administration. F is commonly measured by fluorescence, but fluorescein glucuronide (FG), a metabolite of F, also fluoresces. To assess the influence of FG on the quantitation of F by fluorescence, we studied the pharmacokinetics of F and FG for 38 hr in the plasma of five normal subjects given 14 mg/kg of sodium fluorescein intravenously. The plasma and the plasma ultrafiltrate were measured by fluorescence and by high performance liquid chromatography. In our fluorophotometer, FG was 0.124 times as fluorescent as F. F was rapidly converted to FG, and within 10 min the concentration of unbound FG exceeded that of unbound F. The terminal half-lives of F and FG in the plasma ultrafiltrate were 23.5 and 264 min, respectively, so that FG contributed almost all of the plasma fluorescence after 4-5 hr. Because FG was less bound in the plasma than F, the ratio of the fluorescence of the plasma ultrafiltrate to that of the plasma increased with time. The greatest proportion of the total F available to penetrate into the ocular compartments occurred shortly after injection. We concluded that FG is an important contributor to the fluorescence of the plasma ultrafiltrate after intravenous injection and that accurate quantitation of physiologic parameters calculated from the plasma F requires taking this factor into account.

Ocular fluorophotometry in the normal- and diabetic monkey.

Normal- and diabetic rhesus monkeys without retinopathy demonstrable by ophthalmoscopy or fluorescein angiography were examined with ocular fluorophotometry to detect alterations in their blood-ocular barriers. All vitreous fluorophotometry values were corrected for fluorescence attributable to background levels and then normalized to a blood fluorescein level of 10 micrograms ml-1. Reproducibility studies demonstrated an average coefficient of variation of 0.17 for all animals combined. Insulin-dependent monkeys, both pancreatectomized and streptozotocin-treated, demonstrated significantly higher posterior vitreous fluorescence levels than either control animals or monkeys treated with streptozotocin that were not insulin-dependent. These results cannot be attributed to differences in fluorescein binding or to vitreous abnormalities. However, 14 out of 24 (58%) of the insulin-dependent animals exhibited posterior vitreous fluorescence values within two standard deviations of the control mean. No correlation was apparent between the vitreous values and age or duration of treatment. No difference in anterior chamber concentrations was found between groups after correction. Our results indicate that alterations in blood-retinal barrier can occur in insulin-dependent diabetic monkeys before development of retinopathy.

Application of a self-tonometer to home tonometry.

Although home tonometry has been advocated in the management and diagnosis of glaucoma, the lack of proper instrumentation has precluded its routine performance. We have developed a self-tonometer that can be used by the patient at home and at work and have evaluated the performance of the instrument under these conditions. The results show that reproducibility of self-tonometer measurements is +/- 1.4 mm Hg and is independent of the user. When the readings of the self-tonometer in a given individual were compared with those of the Goldmann tonometer, they varied by +/- 2.4 mm Hg. Patients who could be trained in the clinic performed well and safely on their own. An average of fewer than five attempts was necessary to obtain four readings at each time of day. Our preliminary findings obtained during home tonometry demonstrated some of the benefits of repeated diurnal curves.