Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates.

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

Telemedical diagnosis of anterior segment eye diseases: validation of digital slit-lamp still images.

PURPOSE: This study compared digital images from a portable slit-lamp camera with 35 mm slit-lamp photographs and traditional ophthalmic assessments in anterior segment disorder's detection. METHODS: A total of 196 patients (392 eyes) were recruited from an anterior segment ophthalmology clinic. Each patient underwent an examination by an anterior segment ophthalmologist. Two to three standardized views of 640 x 480 pixels digital images (portable digital slit-lamp camera) and 35 mm photographic slides (Zeiss slit-lamp camera) were taken after the examination. The same ophthalmologist reviewed these images in a masked fashion. Two other masked graders also assessed the digital images. The presence or absence of 33 specific findings was noted at each examination. RESULTS: Digital images showed moderate to excellent agreement to clinical findings (kappa 0.45-0.82) in areas other than lid pathologies. Lens findings from digital images had moderate to good agreement with the clinical gold standard (unweighted kappa 0.43-0.65, sensitivity 59-77%, specificity 86-94%). Gross cornea signs were well detected with digital images, (kappa 0.72-0.85, sensitivity 67-100%, specificity 98-99). More subtle corneal, conjunctival and lid abnormalities were not identified well. The statistical figures were very similar to the above-mentioned figures when the 35-mm film results were compared to clinical diagnoses. The two image formats showed better agreement when compared to each other than when either is compared with clinical findings. CONCLUSION: Diagnoses using digital slit-lamp images were comparable to diagnosis using 35 mm photographic slides for some anterior segment abnormalities.

Emergency eye care in rural Australia: role of internet.

PURPOSE: Significant differences exist in the utilization of emergency eye care services in rural and urban Australia. Meanwhile, influence of internet-based technology in emergency eye care service utilization has not been established. This study aims to demonstrate, from a health provider perspective, an internet-based service's impact on emergency eye care in rural Australia. METHODS: The teleophthalmology service was initiated in the Carnarvon Regional Hospital (CRH) of the Gascoyne region in Western Australia. A digital, slit lamp and fundus camera were used for the service. Economic data was gathered from the Department of Health of Western Australia (DOHWA), the CRH and the Lions Eye Institute. RESULTS: During the study period (January-December, 2003) 118 persons took part in teleophthalmology consultations. Emergency cases constituted 3% of these consultations. Previous year, there were seven eye-related emergency evacuations (inter-hospital air transfers) from the Gascoyne region to City of Perth. CONCLUSIONS: Analysis demonstrates implementation of internet-based health services has a marked impact on rural emergency eye care delivery. Internet is well suited to ophthalmology for the diagnosis and management of acute conditions in remote areas. Integration of such services to mainstream health care is recommended.

Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

Corneal replacement using a synthetic hydrogel cornea, AlphaCor: device, preliminary outcomes and complications.

PURPOSE: Clinical assessment of outcome of corneal replacement with a synthetic cornea, AlphaCor, in patients considered at too high risk for conventional penetrating keratoplasty with donor tissue to be successful, but excluding indications such as end-stage dry eye that might be suited to traditional prosthokeratoplasty. METHODS: All patients in the multicentre clinical trial were managed according to an approved protocol, with Ethics Committee approval in each centre. Preoperative visual acuity ranged from perception of light (PL) to 6/60 (20/200). Implantation was by means of an intralamellar technique, with a conjunctival flap in most cases. Tissues anterior to the optic were removed as a secondary procedure. RESULTS: Up to 30 November 2001, 40 AlphaCor devices had been implanted in 38 patients, of mean age 60 years. Follow-up ranged from 0.5 months to 3 years. There had been one extrusion (2.5%) and four cases (10%) where a device had been removed due to melt-related complications. All five of these cases received a donor corneal graft after the device was removed, with these grafts remaining anatomically satisfactory and epithelialised to date. Corneal melts in AlphaCor recipients were found to be strongly associated with a history of ocular herpes simplex infection. Two further devices (5%) were removed owing to reduced optic clarity after presumed drug-related deposition, and have been successfully replaced with second devices. Mean preoperative best-corrected visual acuity was hand movements. Visual acuities after surgery ranged from PL to 6/6(-2) (20/20(-2)). CONCLUSIONS: Early results suggest that the AlphaCor, previously known as the Chirila keratoprosthesis (Chirila KPro), has a low incidence of the complications traditionally associated with keratoprostheses and can be effective in restoring vision in patients considered untreatable by conventional corneal transplantation. Importantly, the device can be replaced with a donor graft in the event of development of a significant complication. A history of ocular herpes simplex is a contraindication to AlphaCor implantation. Ongoing monitoring of clinical outcomes in all patients will allow the indications for AlphaCor, as opposed to donor grafts, to be determined.

Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy.

Neovascularisation (NV) within the eye often results in visual loss. Vascular endothelial growth factor (VEGF) has been implicated in the development of ocular NV. Previous studies have shown that VEGF antagonists successfully suppressed retinal and choroidal NV in animal models. However, the systemic approach and transient nature of the delivery systems used in these studies hinder therapeutic application. To achieve stable and localised ocular anti-angiogenic therapy, we explored the use of recombinant adeno-associated virus (rAAV)-mediated secretion gene therapy (SGT). In this study, we generated a rAAV vector encoding soluble VEGF receptor 1, sFlt-1 (AAV-CMV.sflt) and determined its ability to inhibit cautery-induced corneal NV and laser-induced choroidal NV. Delivery of AAV-CMV.sflt into the anterior chamber resulted in transgene expression in the iris pigment epithelium and corneal endothelium, which reduced the development of corneal NV in the stroma of cauterised rats by 36% compared with cauterised control groups (P = 0.009). Subretinal delivery of AAV-CMV.sflt near the equator of the eye also suppressed choroidal NV at the laser lesions around the optic nerve by 19% (P = 0.002), indicating that there was diffusion of the secreted anti-angiogenic protein across the retina. Both results suggest that the long-term suppression of ocular NV is possible through the use of stable rAAV-mediated SGT.

Online eye care in prisons in Western Australia.

In prisons, prison medical officers provide general medical care. However, if specialist care is needed then the prisoner is transported to a specialist medical centre. This is a costly procedure and prison escapes occur during transportation. We have tested our Internet-based eye care system in prisons in Western Australia. Medical and ophthalmic history, visual acuity and intraocular pressure were stored in a browser-based multimedia database. Digital images of the retina and the external eye were recorded and transmitted to a central server. Based on the medical data and the digital images, the specialist ophthalmologist could provide a diagnosis within 24 h. Eleven patients (mean age 48, range 30-82 years) were reviewed during two separate visits to a maximum-security prison in Western Australia. Our main aim was to train prison medical officers and nurses to operate the portable ophthalmic imaging instruments and to use the Internet-based eye care system. The outcome of the pilot study indicated that considerable savings could be made in transport costs and the security risk could be reduced. The Ministry of Justice in Western Australia has decided to implement telemedicine services to provide regular ophthalmic consultation to its prisons.

Working toward a portable tele-ophthalmic system for use in maximum-security prisons: a pilot study.

The medical care of prisoners is a difficult and often costly process. Basic medical needs are serviced by prison medical officers. However, specialized care often means transport to specialist centers with the attendant cost and safety. We examined portable ophthalmic equipment in a prison environment to screen 11 prisoners who were scheduled for specialist ophthalmic assessment. Medical and ophthalmic histories were documented, visual acuity was tested, digital images were taken of the external eye and retina, and intraocular pressures taken. The data were sent via modem to a specialist ophthalmologist, and the reports were dictated via e-mail at the end of each session. Of the 11 patients who were scheduled to travel for ophthalmic assessment, only 2 were still required to travel to a specialist ophthalmic center. This pilot study showed that there were considerable cost savings to be made by this screening process with the attendant increase in community safety. The prototype equipment requires refinement by further experimentation, but showed the potential as an adjunct to current examination and assessment techniques when applied to a prison population.

Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization.

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina.

OBJECTIVES: To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. METHODS: An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. RESULTS: The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage. CONCLUSIONS: Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. CLINICAL RELEVANCE: The intraocular safety of rAd should be carefully considered before clinical trials are performed.

Controlled production of active cathepsin D in retinal pigment epithelial cells following adenovirus-mediated gene delivery.

The transduction of a low cathepsin D-producing retinal pigment epithelial cell line with a recombinant adenovirus, Ad.proCatD, carrying a viral promoter and the precursor form of the lysosomal enzyme cathepsin D, procathepsin D, led to the upregulation of proCatD expression. However, the resultant aspartic protease activity did not exceed that observed in normal primary human retinal pigment epithelial cells. Following the injection of Ad. proCatD into rat eyes, immunohistochemistry and Western blot analysis localized the expression of procathepsin D to the retinal pigment epithelial cell layer and to the sclera/choroid/retinal epithelial cell layers, respectively. This upregulation of procathepsin D expression was accompanied by a limited increase in aspartic protease activity. The injected eyes did not demonstrate any of the retinal changes that have been associated with the overproduction and secretion of active cathepsin D. Immunoelectronmicroscopy of Ad.proCatD-transduced retinal pigment epithelial cells demonstrated the presence of cathepsin D not only in cytoplasmic vesicles and lysosomes but also in the nucleoli and, less strongly, elsewhere in euchromatic regions of some 10% of cells. In spite of the upregulated expression of procathepsin D, the production of active cathepsin D in Ad.proCatD-transduced retinal pigment epithelial cells was strictly controlled. It is proposed that active cathepsin D production is controlled at the point of posttranslational modification by an intranuclear feedback mechanism initiated by the relative excess of procathepsin D in Ad. proCatD-transduced retinal pigment epithelial cells.

Calcification of poly(2-hydroxyethyl methacrylate) hydrogel sponges implanted in the rabbit cornea: a 3-month study.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels have been used in the past as ocular implants. In a recent development, PHEMA sponges have shown suitable properties as materials for the peripheral component of an artificial cornea (keratoprosthesis). However, the propensity of PHEMA to calcify could threaten the long-term stability of the implanted devices. In an attempt to improve the understanding of the calcification mechanism, the dynamics, extent, and nature of calcified deposits within PHEMA sponges implanted in the cornea were investigated in this study, and the possible correlation between necrosis of cells and calcification was critically examined. Samples of a PHEMA sponge were implanted in rabbit corneas and explanted at predetermined time points (2, 4, and 12 weeks). The samples were examined by microscopy (light, transmission, scanning) and energy dispersive analysis of X-rays. Histological assessment and semiquantitative analysis of the amount of calcium deposited was performed using image analysis. An in vitro experiment was also performed by incubating sponge samples for 2 weeks in a solution of calcium and phosphate ions at a ratio similar to that in hydroxyapatite, in the absence of cells. Calcification was not seen in the 2- and 4-week explants, however, small deposits were detected in two of the 12-week explants, both within and on the sponge's constituent polymer particles. The deposit volumes represented 0.094% and 0.21%, respectively, of the total sponge volumes. Calcium deposits were present in large amounts both within the constituent polymer particles and on the surface of the sponges incubated in the abiotic calcifying solution. Cooperative mechanisms are suggested for the calcification of PHEMA sponges in vivo. The initial event may occur at a molecular level, when plasma proteins are adsorbed onto the polymer surface and bound through chelation to the calcium ions present in the medium. After their natural degradation, these structures may act as nucleation sites for calcium phosphate crystallization. Concurrently, the calcium ions can diffuse into the hydrogel particles and then the spontaneous precipitation of calcium phosphate may be caused by supersaturation due to the lower content of water in polymer, an effect which is likely predominant in vitro. The second event is the recruitment of phagocytic cells to clear calcium debris. Degeneration of these cells may then form nucleation sites for secondary calcification.

Fred Hollows lecture: digital screening for eye disease.

The purpose of this study was to explore progress, in the adaptation to community screening for blinding eye disease, of digital imaging devices and technology for storage and transmission. Available imaging systems were compared to gold standard clinical photography in terms of sensitivity and specificity for diagnosis of common blinding eye conditions. Since the use of expensive non-portable imaging devices is likely to be limited for widespread community screening purposes, a portable fundus camera (Nidek, Chiyoda-ku, Japan) and a prototype monocular digital indirect ophthalmoscope constructed at the Lions Eye Institute (LEI) were selected for comparative trials for the screening of optic disc cupping, glaucoma and clinical signs of diabetic retinopathy. Fifty-one eyes of 27 consecutive patients being assessed at the LEI clinic for glaucoma were dilated and photographed with a Zeiss retinal camera, and digital images were taken with the portable Nidek NM100 fundus camera (Carl Zeiss, Oberkochen, Germany) or with a prototype digital monocular indirect ophthalmoscope. Vertical cup: disc ratios (VCDR) were measured on the disc photographs by one ophthalmologist while three other clinicians were presented with compressed digital images in random order to estimate VCDR. Field trials were also carried out to demonstrate the practicality of compression, local storage and then transmission by mobile telephone ISDN lines and satellite, of optic discs and fundus images of patients with diabetes in either rural Western Australia or Surabaya, Indonesia. Kappa values of correlations of measurement of agreement between measured and estimated VCDR were 0.87, 0.45 and 0.84, respectively, for the three observers, corresponding to a specificity of 79-97% and a sensitivity of 70-95%. The portable Nidek fundus camera was also assessed for specificity and sensitivity in the diagnosis of diabetic retinopathy in comparison to standard Zeiss fundus camera photographs. Of 49 eyes in 25 consecutive patients attending the LEI clinic for assessment of diabetic retinopathy, three ophthalmologists assessed photographs and images in random order. When used for screening diabetic retinopathy, the digital images of the Nidek camera were graded as adequate quality in only 56% of eyes compared to 93% of the photographs. The kappa value of agreement in analysis of diabetic retinopathy was only 0.30. The prototype digital monocular indirect ophthalmoscope compared favourably with the Nidek camera. At 1:5 compression, images of size 36 kB transmitted from Surabaya to Perth took 29 s on the mobile telephone, while uncompressed images took 170 s. Images compressed 1:5 were transmitted in 60 s using the satellite telephone, while the uncompressed images took 240 s. Satellite transmission was more expensive but the lines were more stable than telephone connections from Indonesia. Digital imaging is becoming a powerful tool for ophthalmology in clinical records, teaching and research, and interoffice diagnostic opinions. It also has enormous potential for community screening for blinding eye diseases, such as glaucoma and diabetic retinopathy. Inexpensive portable imaging devices that are easy to use, and on which local health workers might be trained, must be developed and validated in terms of sensitivity and specificity of performance. The technology of image capture, image compression, transmission, data base storage and analysis is rapidly evolving and becoming less expensive.

A novel immunoassay for the evaluation of rod outer segment digestion in cultured retinal pigment epithelial cells.

In this work a novel enzyme-linked immunoabsorbent assay quantifying residual rod outer segments in the medium of rod outer segment-challenged retinal pigment epithelial cells is described. A retinal pigment epithelial cell line (D407) that produces low level of cathepsin D, and a primary human retinal pigment epithelial cell culture (HRPE51) that has normal cathepsin D levels, were challenged with bovine rod outer segments. At 3 days post-challenge, the amount of undigested or residual bovine rod outer segments left in the culture medium was quantified by an enzyme-linked immunoabsorbent assay. An antibody raised against bovine rod outer segments, which had been purified and labelled with nitroiodophenyl haptens, was used in the assay. The sensitivity of the immunoassay was less than 10(2) bovine rod outer segments per mL and the signal followed a linear curve, saturating around 10(6) bovine rod outer segments per mL. HRPE51 cells had no residual bovine rod outer segments present in the medium following a challenge with 10(4) bovine rod outer segments per mL. In the medium of D407 cells, residual bovine rod outer segment levels were higher at all bovine rod outer segment concentrations when compared to the residual bovine rod outer segment levels in HRPE51 cells, suggesting that D407 cells have a lower digestive capacity. These results demonstrated that the immunoassay for detecting bovine rod outer segments is a sensitive and reliable technique that can be used to quantify the amount of residual bovine rod outer segments, following bovine rod outer segment challenge of retinal pigment epithelial cells.

Telemedicine screening of diabetic retinopathy using a hand-held fundus camera.

The objective was to evaluate digital images of the retina from a handheld fundus camera (Nidek NM-100) for suitability in telemedicine screening of diabetic retinopathy. A handheld fundus camera (Nidek) and a standard fundus camera (Zeiss) were used to photograph 49 eyes from 25 consecutive patients attending our diabetic clinic. One patient had cataracts, making it impossible to get a quality image of one of the eyes (retina). The Nidek images were digitized, compressed, and stored in a Fujix DF-10M digitizer supplied with the camera. The digital images and the photographs were presented separately in a random order to three ophthalmologists. The quality of the images was ranked as good, acceptable or unacceptable for diabetic retinopathy diagnosis. The images were also evaluated for the presence of microaneurysms, blot hemorrhages, exudates, fibrous tissue, previous photocoagulation, and new vessel formation. kappa Values were computed for agreement between the photographs and digital images. Overall agreement between the photographs and digital images was poor (kappa < 0.30). On average, only 24% of the digital images were graded as being good quality and 56% as having an acceptable quality. However, 93% of the photographs were graded as good-quality images for diagnosis. The results indicate that the digital images from the handheld fundus camera may not be suitable for diagnosis of diabetic retinopathy. The images shown on the liquid crystal display (LCD) screen of the camera were of good quality. However, the images produced by the digitizer (Fujix DF-10M) attached to the camera were not as good as the images shown on the LCD screen. A better digitizing system may produce better quality images from the Nidek camera.

Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization.

Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization found in age-related macular degeneration. Normally expressed in low levels, this study investigates whether the overexpression of VEGF in the retinal pigment epithelium is sufficient to cause choroidal neovascularization in the rat retina. A recombinant adenovirus vector expressing the rat VEGF(164) cDNA (AdCMV.VEGF) was constructed and injected into the subretinal space. The development of neovascularization was followed by fluorescein angiography, which indicates microvascular hyperpermeability of existing and/or newly forming blood vessels, and histology. VEGF mRNA was found to be overexpressed by retinal pigment epithelial cells and resulted in leaky blood vessels at 10 days postinjection, which was maintained for up to 31 days postinjection. By 80 days postinjection, new blood vessels had originated from the choriocapillaris, grown through the Bruch's membrane to the subretinal space, and disrupted the retinal pigment epithelium. This ultimately led to the formation of choroidal neovascular membranes and the death of overlying photoreceptor cells. By controlling the amount of virus delivered to the subretinal space, we were able to influence the severity and extent of the resulting choroidal neovascularization. These results show that even temporary overexpression of VEGF in retinal pigment epithelial cells is sufficient to induce choroidal neovascularization in the rat eye.

The effects of induced acute hyperglycemia in the cat on the retinal capillary blood flow.

PURPOSE: To study the effect of acute hyperglycemia on the erythrocyte flow in specific retinal capillary paths. METHODS: A technique for fluorescent labeling of a known fraction of the erythrocyte moiety of systemic blood was combined with fluorescence viewing of the retinal capillary network in live cats. This technique was developed to enable visualization of the erythrocyte flow in the retinal capillary network and used to acquire video recording of the retinal capillary erythrocyte flow in normal feline eyes. The pattern of capillary erythrocyte flow under normal blood glucose levels and normal systemic blood pressure served as baseline. Acute hyperglycemia was induced by intravenous injection of glucose while monitoring the systemic blood pressure. Two subsets of functional capillary pathways previously defined as S (simple) and C (complex) were identified in the recorded data. The relationship between erythrocyte flux in random selections of these two pathways and the level of hyperglycemia and systemic blood pressure was determined. RESULTS: Induction of acute hyperglycemia led to acute elevation of the systemic blood pressure that returned to baseline levels within few minutes, while blood glucose remained high. The capillary erythrocyte flux in S paths was significantly higher than in C paths at all values of systemic blood pressure. The capillary erythrocyte flux in the S paths was directly proportional to the systemic blood pressure whereas the flux in C paths was minimally affected by acute hyperglycemia for the systemic blood pressure range between 110 and 160 mm Hg. CONCLUSIONS: The erythrocyte flux in S paths is affected by the changes of the systemic blood pressure and these paths act as 'shunt vessels' when acute elevation of the systemic blood pressure occurs. C paths maintain stable perfusion under changing conditions, most probably in an effort to minimally alter the basic metabolic needs of the retinal tissue. Hyperglycemia per se was not responsible for changes of the cell flux in these capillary paths.

Methods and limits of digital image compression of retinal images for telemedicine.

PURPOSE: To investigate image compression of digital retinal images and the effect of various levels of compression on the quality of the images. METHODS: JPEG (Joint Photographic Experts Group) and Wavelet image compression techniques were applied in five different levels to 11 eyes with subtle retinal abnormalities and to 4 normal eyes. Image quality was assessed by four different methods: calculation of the root mean square (RMS) error between the original and compressed image, determining the level of arteriole branching, identification of retinal abnormalities by experienced observers, and a subjective assessment of overall image quality. To verify the techniques used and findings, a second set of retinal images was assessed by calculation of RMS error and overall image quality. RESULTS: Plots and tabulations of the data as a function of the final image size showed that when the original image size of 1.5 MB was reduced to 29 KB using JPEG compression, there was no serious degradation in quality. The smallest Wavelet compressed images in this study (15 KB) were generally still of acceptable quality. CONCLUSIONS: For situations where digital image transmission time and costs should be minimized, Wavelet image compression to 15 KB is recommended, although there is a slight cost of computational time. Where computational time should be minimized, and to remain compatible with other imaging systems, the use of JPEG compression to 29 KB is an excellent alternative.