Threshold and retreatment parameters of NPe6 photodynamic therapy in retinal and choroidal vessels.

BACKGROUND AND OBJECTIVE: To determine the threshold fluence for producing choroidal and retinal vascular occlusion with mono-L-aspartyl chlorin e6 (NPe6) photodynamic therapy (PDT) during primary treatment and the effect of retreatment. METHODS: Primary treatment: Rats, rabbits, and monkeys underwent NPe6 PDT to determine the threshold fluences for choroidal and retinal vessel occlusion. The threshold was determined by analyzing fluorescein angiograms for areas of nonperfusion. Retreatment: Dutch-belted rabbits underwent NPe6 PDT followed by fluorescein angiography. Rabbits were retreated one week later at the same parameters. RESULTS: Fluence levels and vascular damage thresholds were always higher for retinal than for choroidal vascular occlusion. Retreatment caused choroidal vessel closure at all tested fluences but retinal capillaries closed only at a fluence > 17.7 J/cm2. CONCLUSION: NPe6 PDT has a lower threshold to occlude choroidal vessels than retinal vessels. The cumulative effect of retreatment does not damage retinal vessels unless the threshold is exceeded during a single retreatment session.

Threshold power levels for NPe6 photodynamic therapy.

OBJECTIVE: To determine the threshold power levels for producing retinal and choroidal vascular occlusion using mono-L-aspartyl chlorin e6 (NPe6) photodynamic therapy; to evaluate its efficacy with longer intervals between photosensitizer injection and laser application; to determine the elapsed time between light application and appearance of angiographic changes. METHODS: Pigmented and nonpigmented rabbits were injected intravenously with 2 mg/kg of NPe6 before laser irradiation of the retina-choroid. Group 1 was treated at increasing power levels; fluorescein angiograms were obtained at each fluence. Group 2 animals were exposed to laser irradiation at 5 minutes, and 1 and 3 hours postinjection to determine (by fluorescein angiography 24 hours post-treatment) if increasing the interval affected outcome. Group 3 animals underwent fluorescein angiography at 30 minutes, 1 hour, 2 hours, and 24 hours posttreatment to document the time between laser application and subsequent vessel closure. RESULTS: Choroidal vessel occlusion was angiographically evident in all lesions at fluences of > or = 2.65 J/cm2 in pigmented rabbits and at > or = 0.88 J/cm2 in nonpigmented rabbits. Lesion diameter decreased as the time between injection and treatment increased. Vessel occlusion was documented at least 2 hours after treatment. CONCLUSION: Choroidal vessel occlusion can occur at very low fluence.

Problems with and pitfalls of photodynamic therapy.

OBJECTIVE: To delineate the various factors that may influence the outcome of photodynamic therapy of the retina and choroid. DESIGN: Experimental animal study. ANIMALS: Pigmented and nonpigmented rabbits; rhesus monkeys. INTERVENTION: The hydrophilic photosensitizer, mono-L-aspartyl chlorin e6, which is maximally activated at 664 nm, was studied after intravenous injection into pigmented and nonpigmented rabbits and rhesus monkeys. Laser light was supplied by a red diode laser coupled to a modified slit-lamp biomicroscope and delivered to the ocular fundus after passing through a standard fundus contact lens. Standard photodynamic parameters were used. The effects of fundus pigmentation, intraocular pressure, spot focus and defocus, region of fundus treated, equivalent fluence, and retreatment were observed in the different animal species. MAIN OUTCOME MEASURES: Slit-lamp biomicroscopy, fluorescein angiography, light and transmission electron microscopy. RESULTS: Fundus pigmentation appeared to be a factor only at the lowest fluence level tested, where only 4 of 12 lesions attempted in pigmented fundi were noted on fluorescein angiography, compared with 12 of 12 lesions in albino rabbits. At normal intraocular pressures and a given fluence, 10 of 10 lesions were fully manifested on fluorescein angiography, compared with 4 of 10 at 30 mmHg and 0 of 10 at pressures sufficient to blanch the optic nerve (>60 mmHg). For laser spots either focused or defocused, there were 6 of 6 lesions that were fully manifested on fluorescein angiography for each of the parameters. Lesions treated in the fovea resulted in larger spots on fluorescein angiography. The fluence of 5 mW for 10 seconds resulted in a larger lesion on angiography than the equivalent fluence of 10 mW for 5 seconds. Areas of retreatment in rabbits demonstrated more thinning of the neurosensory retina and loss of photoreceptor outer segments and nuclei than corresponding areas receiving one treatment. CONCLUSIONS: Photodynamic therapy results varied, depending on intraocular pressure, region of fundus treated, ocular pigmentation, and the total time of exposure to the photosensitizer. Retreatment resulted in progressive thinning of the neurosensory retina with loss of photoreceptor outer segments and nuclei in the rabbit eye.

Fluorescence properties of a hydrophilic sensitizer in pigmented rats, rabbits, and monkeys.

BACKGROUND AND OBJECTIVE: To evaluate fluorescence properties of mono-L-aspartyl chlorin e6 (NPe6; Meija Seika Kaisha, Ltd., Tokyo, Japan) photodynamic therapy, which allows real-time simultaneous imaging of choroidal and retinal vasculature during treatment without the addition of another dye. MATERIALS AND METHODS: Four pigmented rabbits, 4 pigmented rats, and 2 African green monkeys were administered intravenous injections of the NPe6 dye. The animals were immediately placed in front of the scanning laser ophthalmoscope and the fundus was viewed with the helium-neon laser. The resulting fluorescence was viewed and recorded on super-VHS videotape. RESULTS: Fluorescence demonstrated clearly that NPe6 entered the retinal and choroidal circulation within seconds of intravenous injection. The concentration of NPe6 was diminished over a period of 1.5 hours in the monkey and 5 hours in the rat, as evidenced by considerable diminution of the intensity of fluorescence. CONCLUSION: NPe6 fluorescence allows evaluation of drug availability within the retinal and choroidal circulation and visualization of pathological lesions before commencement of photodynamic therapy.

Fluorescent labeling of blood cells for evaluation of retinal and choroidal circulation.

BACKGROUND AND OBJECTIVE: To develop a method of staining and tracking cells in vivo with a scanning laser ophthalmoscope (SLO) to evaluate the retinal and choroidal circulations. MATERIALS AND METHODS: Twelve pigmented male rats were used. Staining of leukocytes. Two commercially available nucleic acid stains SYTO 16 and SYTO 59 (Molecular Probes, Eugene, OR) were used to stain leukocytes. Blood (0.5 mL) was withdrawn from the subject animal and placed in a heparinized tube to which phosphate buffered solution was added (1.0 mL buffer/0.5 mL blood). Ten microliters of solution of SYTO 16 or SYTO 59 in alcohol (5 mM) was added. Staining of Erythrocytes. The lipophilic carbocyanine dye indocarbocyanine (D-307, Molecular Probes) was used to stain erythrocytes. Blood (0.1 mL) was withdrawn from the subject and handled as above; plasma and leukocytes were removed with a pipette. HEPES buffer was added to the remaining erythrocytes (1.4 mL buffer added to 0.1 mL blood). Ten microliters of D-307 in alcohol (5 mM) was added to the 1.5 mL buffered blood. The final concentration of dyes in the buffered blood mixtures was 0.03 mM. The stained cells were injected intravenously in the subject animal. The eyes were observed with SLO; the resident helium-neon laser was used to excite the SYTO 59 and the resident argon laser for SYTO 16. The He-Ne laser was used to excite the D-307-stained erythrocytes. RESULTS: The movement of the leukocytes and erythrocytes in the retinal and choroidal vessels was clearly distinguishable from the surrounding tissue. CONCLUSION: By selecting different dyes for staining leukocytes or erythrocytes and choosing appropriate laser wavelengths, the hemodynamics of blood cells can be observed and evaluated in the retina and choroid.

Ocular vascular thrombosis following tin ethyl etiopurpurin (SnET2) photodynamic therapy: time dependencies.

BACKGROUND AND OBJECTIVES: To evaluate the optimal time from the end of photosensitizer injection to the commencement of light application for creating characteristic fundus lesions and the time to vascular occlusion following photodynamic therapy (PDT) with tin ethyl etiopurpurin (SnET2). MATERIALS AND METHODS: Following intravenous injection of SnET2 0.5 mg/kg or lipid emulsion alone, the fundus of rabbits was irradiated at different times (5 to 240 minutes) after photosensitizer injection using 664 +/- 7-nm laser light with an irradiance of 354 mW/cm2 and fluence of 20 J/cm2. Ophthalmoscopy and fluorescein angiography were performed 1 day after SnET2 PDT. In separate groups of rabbits, treated areas of the fundus were imaged within 30 minutes following PDT using fluorescein vesicle and microsphere angiography with scanning laser ophthalmoscopy to document time of vascular occlusion. All animals were killed 1 day following treatment and eyes were examined by histopathology. RESULTS: Areas of hypofluorescence (indicating vascular occlusion) were seen when activating laser light was applied 5 to 20 minutes after SnET2 injection. Retinal vessels remained perfused in all cases. The time to vascular occlusion was 70 to 120 and 40 to 90 minutes in nonpigmented and pigmented rabbits, respectively. No safety issues were seen. CONCLUSION: PDT with SnET2 was effective in occluding the choriocapillaris. Activating light needs to be applied within a specific time frame after photosensitizer injection to achieve vascular occlusion.

Reversal of blood flow in experimental branch retinal vein occlusion.

BACKGROUND AND OBJECTIVE: To demonstrate that the obstructed vascular lumen of the experimentally induced branch retinal vein occlusion (BRVO) induces retrograde blood flow, resulting in flow from the occluded vein to the feeder arterioles. MATERIALS AND METHODS: Photocoagulation was used to create occlusion of the branch retinal vein in a monkey model (n = 2; 1 cynomolgus, 1 rhesus). Twenty-four hours following photocoagulation, the eyes were examined for evidence of vascular occlusive disease. Vascular occlusion was proven by fluorescent vesicle angiography with scanning laser ophthalmoscopy; these results were recorded to SVHS videotape. The images were then serially analyzed frame by frame to track individual microsphere movement. RESULTS: The authors observed retrograde flow proximal to the point of vessel obstruction and extending backward into the arterial system. CONCLUSIONS: This demonstrates the existence of retrograde flow in an experimental model of BRVO and might explain vascular complications seen in this disease process.

Blood velocity in an experimental iris tumor.

BACKGROUND AND OBJECTIVE: Greene strain melanoma was implanted into the irides of eight nonpigmented rabbits to evaluate the blood flow in tumor vasculature. MATERIALS AND METHODS: Conventional scanning laser ophthalmoscopy was used in conjunction with fluorescent microsphere angiography (FMA). Changes were documented on SVHS videotape for later analysis. Individual microsphere movement was tracked through the tumor vessels. Subsequently, blood velocity measurements were taken. RESULTS: The tumor vessels were poorly organized and inefficient. Tumor blood velocity was up to 2.5 times slower compared with normal blood velocity in the unaffected iris of the same eye of the same rabbit (P = .05). Tumor blood flow could be qualitatively visualized in real time in the liver rabbit model. CONCLUSION: The ability to visualize fluorescent microspheres within the poorly organized tumor vasculature coupled with the reduced blood velocity in the tumor helps to explain the success of hyperthermic tumoricidal therapy, and may allow for development of more efficient and selective drug delivery systems and tumoricidal agents.

Fluorescent microsphere imaging: a particle-tracking approach to the hemodynamic assessment of the retina and choroid.

BACKGROUND AND OBJECTIVES: Quantitative assessment of choriocapillaris circulation has proven difficult. Although the fluorescent vesicle system provides a means of quantifying the retinal circulation, the attempts at imaging fluorescent liposomes in the choroidal microcirculation have been largely unsuccessful. The authors introduce a new tool, fluorescent microsphere imaging, and examine its utility for evaluating the hemodynamics of the retina and choroidal microcirculation. The usefulness of fluorescent microsphere imaging is demonstrated through the examination of the retinal and choroidal circulations of three rhesus monkeys. MATERIALS AND METHODS: Fluorescent microsphere imaging uses polystyrene latex microspheres that incorporate one or more dyes. These microspheres are injected intravenously into an animal and are excited in the eye through the resident lasers of a scanning laser ophthalmoscope. The excited particles are detected by the ophthalmoscope, and its output is then digitized directly or recorded on a videocassette recorder for subsequent image analysis. Multiple-dye microspheres use the principle of resonance energy transfer for the activation of the final dye in a non-radiative cascade. These microspheres enable the investigator to tailor the excitation and emission spectra of the particles for the investigation of different ocular tissues. RESULTS: Using 488/515 microspheres (excitation and emission peaks at 488 nm and 515 nm, respectively), the authors captured images of particles circulating in the perimacular retinal circulation. Shifting excitation and emission spectra toward the red and infrared enabled the imaging of blood flow in progressively deeper tissue. Using 633/825 microspheres, the authors recorded and tracked particles in the microcirculation of the choroid. CONCLUSION: The authors' findings suggest that fluorescent microsphere imaging provides images useful for studying the retinal circulation and for evaluating previously inaccessible choroidal hemodynamics.

Photodynamic therapy for choriocapillaris using tin ethyl etiopurpurin (SnET2).

BACKGROUND AND OBJECTIVE: To investigate the use of photodynamic therapy (PDT) using tin ethyl etiopurpurin (SnET2) for occluding the choriocapillaris in the eyes of pigmented rabbits. MATERIALS AND METHODS: Following intravenous injection of SnET2 (0.5 and 1 mg/kg) or lipid emulsion alone, the fundus of pigmented rabbits (n = 21) was irradiated starting 15 to 45 minutes after photosensitizer injection using 664-nm light at a fluence of 300 mW/cm2 and light doses of 5 to 20 J/cm2. Funduscopy, fluorescein angiography, and light and electron microscopy were performed at 1, 14, and 28 days after PDT. RESULTS: Following SnET2 and PDT, closure of the choriocapillaris was achieved with light doses as low as 5 J/cm2 (17 seconds) and a drug dose of 0.5 mg/kg of SnET2. Vascular occlusion was documented by fluorescein angiography and histology. Photodynamic damage was noted in the choriocapillary endothelial cells. Retinal pigment epithelial damage and outer retinal damage were also observed. No funduscopic, angiographic, or histologic findings were present in the eyes of pigmented control rabbits. CONCLUSIONS: PDT with SnET2 was effective in this animal model, using low levels of activating light for the occlusion of the choriocapillaris. This has clinical implications for the treatment of choroidal neovascularization and could be a more selective therapy than thermal laser photocoagulation.

A fluorescent vesicle system for the measurement of blood velocity in the choroidal vessels.

BACKGROUND AND OBJECTIVE: Evaluation of the pathophysiologic mechanisms involved in the choroidal circulation is difficult because of the presence of the retinal pigment epithelium. The authors have developed a technique for measuring blood velocity in individual choroidal vessels using the scanning laser ophthalmoscope and intravenously injected indocyanine green encapsulated in multilamellar vesicles. In this report, the authors describe their attempts to measure choroidal blood velocity in three different regions of the choroid of the primate eye. MATERIALS AND METHODS: A video-to-digital processor was used to digitize the video images of the choroid of a monkey at a sample rate of 1/30 of a second. The blood velocity in the selected choroidal vessel segments was calculated by measuring the distance the liposome traveled in a given time. RESULTS: The average blood velocities were 5.16 mm/s under the macula, 4.04 mm/s in the vortex vein system, and 2.03 mm/s in the choroidal circulation nasal to the optic nerve. CONCLUSION: This technique is an effective method of measuring blood velocity in a primate model and has several important advantages compared with the dye dilution technique.

In vitro evaluation of polymeric matrix and porous biodegradable reservoir devices for slow-release drug delivery.

BACKGROUND AND OBJECTIVE: Biodegradable polymeric devices were evaluated in vitro for intravitreal drug delivery. MATERIALS AND METHODS: The matrix (short-term drug delivery) and the porous (longer-term drug delivery) reservoir devices were made from polycaprolactone of two molecular weights (30,000 and 56,000). Matrix devices were loaded with 5-fluorouracil or ganciclovir. Porous reservoirs were loaded with 5-fluorouracil, ganciclovir, 5-carboxyfluorescein, or foscarnet. The release was monitored in phosphate-buffered solution using ultraviolet spectrophotometry. RESULTS: Release from the matrix devices was characterized by an initial burst, followed by a nonlinear release. The porous reservoirs demonstrated zero order linear release of drugs, sustained up to 250 days in this experiment. CONCLUSIONS: The matrix device is capable of sustained release over several months; the porous reservoir can deliver drugs for over 1 year. Further studies are needed to evaluate in vivo biodegradation behavior and toxicity of drugs used for sustained release.

Fluorescent vesicle angiography with sodium fluorescein and indocyanine green.

BACKGROUND AND OBJECTIVE: The authors evaluated the feasibility of merging free-dye angiography and the fluorescent vesicle technique to achieve the best characteristics of both. MATERIALS AND METHODS: Fluorescent vesicles encapsulated with either indocyanine green or carboxyfluorescein were mixed with free indocyanine green or free sodium fluorescein, respectively, and imaged with a scanning laser ophthalmoscope in both an in vitro model and primate and rabbit models. RESULTS: In the in vitro model of the sodium fluorescein combination, optimal viewing of vesicleen capsulated dye and free dye was at a ratio of 150:1; for indocyanine green, the ratio was 50:1. In vivo, high-quality fluorescent vesicle angiograms were obtained that demonstrated leakage of free dye from choroidal laser spots. CONCLUSIONS: Free dye and fluorescent vesicles can be combined to obtain an angiogram with all of the advantages of a traditional angiogram, while allowing the operator to assess the changes in retinal or choroidal circulation directly.

Study of lymphocyte dynamics in the ocular circulation: technique of labeling cells.

The purpose of this experiment was to study in vivo the dynamic behavior of the lymphocyte in the retinal circulation. We developed a new technique capable of visualization of lymphocyte motion in the retinal and choroidal vessels using a rat model. Live cells freshly removed on a donor animal were labeled by a simple method using fluorescein isothiocyanate. Labeled cells were injected systemically into another animal. Retinal images were reconstituted on a video screen with a scanning laser ophthalmoscope (SLO) utilizing the argon green laser excitation wavelength (514.5 nm) to detect cell fluorescence. Lymphocytes were clearly seen and followed in the retinal vessels. Some slowed down in the capillary system, or even stopped for a few seconds, or were definitively caught in it. Labeled cells remained visible after circulating several times. A method was developed for in vivo visualization of lymphocytes in the retinal circulation. This method has the potential for application in the study of lymphocyte cell behavior under physiological as well as pathological conditions.

Experimental retinopathy by hyperbaric oxygenation.

Retinopathy of prematurity (ROP) usually occurs after a prolonged exposure to normobaric hyperoxia in newborn mammals and infants. We hypothesized that experimental ROP also could develop after acute exposures to hyperbaric oxygenation (HBO), providing that a severe and maintained retinal vasoconstriction occurred during HBO exposure. Five- to seven-day-old, Long Evans Sprague-Dawley rats were exposed for 5 h either to 5 atm abs oxygen or to 5 atm abs O2 with 190 mmHg inspired PCO2 (hypercapnia). Control rats breathed air at atmospheric pressure. Two months after exposures, rats were anesthetized, perfused intraventricularly with India ink, and retinal images were obtained. Retinal vascular density (RVD) in each image was calculated as the number of pixels in the retinal vessel area divided by the total number of pixels in the image (retinal tissue and vessels). The RVD was significantly increased from 0.0112 +/- 0.004 in the air-exposed controls to 0.0417 +/- 0.029 in the HBO-exposed rats (mean +/- SD; n = 4 in each group). HBO with hypercapnia produced a nonsignificant increase in RVD (0.0255 +/- 0.007; n = 4), reducing the HBO-induced increase in RVD by 39%. These results are consistent with the hypothesis that a sustained HBO-induced retinal vasoconstriction in newborn rats, followed by a hypoxic-ischemic injury, might result in vascular proliferation, thereby initiating ROP development on return to air. Hypercapnia does not completely prevent HBO-induced retinal vasoproliferation, probably because possible vasodilation, induced by hypercapnia, can greatly elevate retinal tissue PO2 and promote oxidative damage.

Noninvasive monitoring of intraocular pharmacokinetics of daunorubicin using fluorophotometry.

PURPOSE: Daunorubicin is a cytotoxic drug, which, in nontoxic doses, is effective in preventing cellular proliferation in experimental vitreoretinopathy. We studied dose and clearance of daunorubicin in various ocular tissues using fluorophotometry techniques. METHODS: In vitro tests: The emission of fluorescence from the daunorubicin solution having a concentration range of 0.1 to 10 micrograms/mL in phosphate buffer was measured using an excitation wavelength range of 489 +/- 10 nm. The emission of fluorescence was measured at 514 nm; the linearity of the response was determined using linear regression analysis. There is a fluorescence peak of daunorubicin at 485 nm. The validity and reproducibility of the method were examined. In vivo tests: The rabbits were randomized into three groups and daunorubicin concentrations of 4, 6, or 8 micrograms/mL were injected into the vitreous. Fluorophotometry scanning from the retina to the anterior chamber was performed with a commercially available fluorophotometer at various times up to 48 hours after injection to quantify fluorescence emission of daunorubicin. RESULTS: The standard curve of fluorescence versus concentration of daunorubicin was linear in the range of 0.1 to 8 micrograms/mL. It was sensitive up to 0.1 microgram. The daunorubicin time concentration profile showed a dose response relationship over the 48-hour period studied. The half-life of daunorubicin in the vitreous was about 5 hours. CONCLUSION: We performed fluorophotometry using a fluorophotometer whose exciter emits light at 489 nm, which is very close to an absorption peak of daunorubicin. These two values are close enough to obviate the need for modifying the commercial fluorophotometer. Therefore the concentration of daunorubicin in the vitreous cavity can be measured noninvasively.

Fluorescent vesicle system. A new technique for measuring blood flow in the retina.

PURPOSE: To measure blood flow in the retinal circulation and optic nerve head capillaries with an innovative fluorescent vesicle system. METHODS: Carboxyfluorescein was encapsulated into liposomes; the vesicles ranged from 0.1 to 2 microns in diameter. After intravenous injection of the liposome suspension, the fundus was viewed using a scanning laser ophthalmoscope. Images of the fundus showing circulating liposomes were stored on videotape. An image analyzing system was used to digitize the captured video frames and transfer them to a computer's hard disk for permanent storage. Software developed in the authors' laboratory allowed them to overlay multiple video frames to create a single image that provided a visible record of the path taken by a particular vesicle in a given time period. The information on this image was used to calculate the velocity of the vesicle, and hence the velocity of the blood flow in the vessel. RESULTS: With this system, individual liposomes as small as 100 nm were visible in all retinal vessels (arteries, capillaries, and veins). Quantitative analysis of vesicle movement in the major retinal vessels of the cynomolgus monkey yielded an average velocity of 9.33 +/- 1.67 mm/second in a large vein (diameter, 130 microns) and 16.10 +/- 5.7 mm/second in a large retinal artery (diameter, 64 microns). The average velocity in the macular capillaries was 0.76 mm/second (range, 0.45-1.33 mm/second), whereas the average velocity in the optic nerve head capillaries was 1.39 mm/second (range, 0.96-2.25 mm/second). CONCLUSION: The fluorescent vesicle system can be used for simultaneous measurement of blood flow in the retinal arteries, veins, and capillaries of the macula and optic nerve.

Calcein fluorophotometry in streptozocin-induced diabetic rats.

Calcein was used in vitreous fluorophotometry (VF) to study the blood-retinal barrier (BRB) in rats with streptozocin-induced diabetes. Calcein was injected intravenously in 18 diabetic rats and sodium fluorescein (NaF) in 12 diabetic rats. VF was performed at 1, 2, 4, 6, 9, and 21 hours following the single injection in these diabetic rats; it was also performed at these times in 24 normal (control) rats. On another day, the same experiment was performed using NaF. There was no significant difference between the two dyes in the diabetic and nondiabetic rats at 1 hour. However, at 4 hours and at later measurement points, there was dye leakage into the vitreous, peaking at 9 hours; the leakage was significantly greater in the diabetic rats. Also, the leakage was significantly greater with calcein than it was with NaF. The data demonstrate that calcein circulates longer and at higher levels than NaF and thus is probably more useful for BRB integrity studies.

Microwave-induced retinal destruction with sparing of sclera and choriocapillaris.

We studied the effect of short-term hyperthermia on sclera, choroid, and retina by delivering microwave radiation (2.45 GHz) for 1 minute to 12 eyes of Dutch belted pigmented rabbits. Four eyes each were treated with 43 degrees C, 45 degrees C, and 47 degrees C and followed for 4 weeks. The 43 degrees C group showed minimal disruption of retinal pigment epithelium and outer retina, with pigment migration; the 45 degrees C and 47 degrees C groups showed complete retinal and RPE damage, pigment migration, and glial proliferation. At the same time, the sclera and choroid in all of the eyes remained essentially unchanged. We conclude that microwave-induced hyperthermia can create retinal scarring without significant damage to sclera and choriocapillaris. The next experimental step will be to refine the microwave delivery system to ensure predictable and reproducible lesions.

Bioavailability of microsphere-entrapped cyclosporine A in the cornea and aqueous of rabbits.

Extended release of immunosuppressive drugs and sustained drug levels are desirable for the treatment of corneal graft rejection and other ocular immune disorders. This experiment was conducted with biodegradable microspheres containing cyclosporine A to assess their suitability for achieving this goal. Microspheres containing cyclosporine A were prepared using a solvent evaporation process. A mixture of poly(lactic) and poly(glycolic) acid polymers (50:50) and cyclosporine A was dissolved in a mixture of chloroform and acetone. The solution was then emulsified in an aqueous solution of polyvinyl alcohol and stirred for 24 hours to evaporate the organic solvent. The final assayed concentration of cyclosporine A was 15.38 mg/mL. A 0.13 mL aliquot (2.0 mg) of the suspension of the microspheres was injected subconjunctivally in 24 eyes of white New Zealand rabbits. The concentration of cyclosporine A in the aqueous and cornea was measured at 6, 12, 24, 48, 72, and 144 hours after injection (n = 4 for each group). Corneal levels of cyclosporine A ranged from 2392 +/- 70 ng/mL at 6 hours to 1297 +/- 459 ng/mL at 144 hours. The aqueous levels ranged from 110 +/- 0 ng/mL at 6 hours to 62 +/- 11 ng/mL at 144 hours. These data indicate that a microsphere drug delivery system is an effective means of delivering cyclosporine A to the cornea and anterior chamber, and may provide an alternative for the treatment of ocular immune disorders.