Expression of vascular endothelial growth factor in experimental choroidal neovascularization

Background: Although the choroidal neovascularization (CNV) is a common pathologic feature of a number of different eye diseases, its pathological mechanisms have not been fully elucidated. We investigated the expression of vascular endothelial growth factor (VEGF) in CNV using an experimental primate model. Method: CNV was induced by intense laser photocoagulation in four monkey eyes. Single eyes were enucleated at 1, 3, 7 or 14 days after photocoagulation and examined immunohistochemically for VEGF, macrophage antigen, von Willebrand factor and glial fibrillary acidic protein (GFAP). Expression of VEGF mRNA was examined byin situ hybridization. Results: One day after photocoagulation, the normal structure of the outer portion of the retina and the inner portion of the choroid was destroyed. Three days after photocoagulation, choroidal vascular endothelial cells migrated into the subretinal space through the defect in Bruch's membrane. Increased expression of VEGF was detected in the accumulating macrophages, migrating retinal pigment epithelial (RPE) cells and Muller cells. Maximal expression of VEGF was observed between 3 and 7 days after wounding, and many newly formed vessels extended into the subretinal space 7–14 days after photocoagulation. Conclusion: VEGF derived from RPE cells, macrophages and Müller cells may play a role in the formation of CNV.     

The use of tissue plasminogen activator in post-traumatic total hyphaema

Background: We wanted to evaluate whether intracameral injection of tissue plasminogen activator (tPA) is useful in managing traumatic hyphaema. Methods: Two eyes with total hyphaema after a severe penetrating injury were treated with a single intracameral injection of 25 g of tPA 5 and 14 days after the injury, respectively. Results: Most of the blood coagulum dissolved within 24 h, and in one of the two eyes the intraocular pressure decreased from 45 to 8 mmHg. The other eye was hypotonic. No re-bleeding or complications related to the use of tPA were noticed. Conclusion: The results in these two cases suggest that tPA is a useful adjunct in managing total hyphaema.     

Indirect scatter laser photocoagulation to subfoveal choroidal neovascularization in age-related macular degeneration

Background: Occult choroidal neovascularization (CNV), poorly defined on fluorescein angiography, is present in the majority of patients with exudative complications of age-related macular degeneration. For patients who present with this type of subfoveal CNV but who have useful visual acuity, no form of treatment is of proven benefit. Accordingly, a pilot randomized trial of indirect laser treatment was performed. The rationale of this treatment was to inhibit the CNV through laser-induced effects on the retinal pigment epithelium. Methods: Patients with occult subfoveal CNV without retinal pigment epithelial detachment and with visual acuity of 20/200 or better were randomized to treatment or control groups. A grid of laser burns was applied to the macula beyond the area of serous retinal detachment and of angiographically defined occult CNV Results: After an average follow-up of 38 months, there was no difference in mean final visual acuity (0.12 treated, 0.14 control) or clinical outcome between treated and untreated groups. Fluorescein angiography showed gradual enlargement in the occult CNV in 58% of eyes in both groups. A decrease in visual acuity to worse than 20/200 (54% of treated, 50% of control eyes) was associated with ingrowth of well-delineated CNV (6 treated, 7 control eyes) or progression to a fibroglial or atrophic scar (11 treated, 8 control eyes). Conclusions: No benefit was demonstrated for scatter photocoagulation of the macula in patients with age-related macular degeneration and occult subfoveal CNV with initially good visual acuity. There were, however, no complications related to treatment.     

Scanning laser ophthalmoscope imaging of fluorescein-labelled blood cells

Purpose: To demonstrate the feasibility of a technique for the visualization by scanning laser ophthalmoscope (SLO) of fluores cein-labelled autologous leukocytes and platelets in retinal vessels. Method: Individual blood samples from rats and rabbits were centrifuged to isolate platelets and leukocytes, then passively labelled with fluorescein and reinjected into the same animal. An SLO was used to visualize and record cell displacement in the retinal circulation. Labelled platelets were analysed by flow cytometry. Results: By SLO, platelets appeared as a heterogeneous particle flow, and individual leukocytes appearing as brighter spots could easily be traced. Flow cytometry showed that after labelling platelets were well individualized and their size was slightly increased. Conclusion: Circulating blood cells can be visualized in retinal vessels by a simple method consisting of passive labelling of autologous platelets and leukocytes by fluorescein. No platelet toxicity was detected. This method could be applied to the study of blood cell movement in human retinal vascular diseases.Proprietary interest category: N     

Ocular findings in trigonocephaly

Background: Trigonocephaly, caused by premature closure of the metopic suture, is a rare form of craniosynostosis. The aim of this study was to assess the visual outcome in children operated on for trigonocephaly Methods: We present eight cases of children with trigonocephaly surgically corrected by the same craniofacial technique. CT with 3D reconstruction was performed in all cases. Genitori defined three types of trigonocephaly according to the severity of the deformity of the skull base only types II and III were included in this study directed at evaluation of the ocular disorder. A complete eye examination was performed on all children by the same observer, with a follow-up of 2–6 years Results: 3D-CT reconstruction of the skull base showed that the frontozygomatic region was affected by the deformation. Ocular examination showed considerable astigmatism in most children with late operation. A low degree of strabismus was observed in most children Conclusion: This study demonstrated that reconstructive surgery should be performed by the age of 6 months, given the immaturity of the visual system up to that time. Close cooperation between neurosurgeons, pediatricians and ophthalmologists is of paramount importance in order to prevent this bone deformation exerting an adverse effect on visual development. The ophthalmologist must possess a basic understanding of the various craniosynostoses.     

Static fundus perimetry using the scanning laser ophthalmoscope with an automated threshold strategy

Purpose: The purpose of this study was to develop software that allows the performance of routine static threshold perimetry using the scanning laser ophthalmoscope (SLO) and the comparison of the results with conventional computerized cupola perimetry. The original software does not allow performance of static threshold perimetry within a reasonable examination time. Methods: Static perimetry was performed in random order on 50 healthy eyes using our SLO staircase threshold perimetry technique and the Octopus 500 (program 38). We compared the relative sensitivities for each of 25 corresponding visual field locations. Results: Mean sensitivity in the SLO perimetry amounted to 32.7 dB (range 25–37 dB) while it was 28.7 dB in the Octopus. For all test locations the SLO showed higher dB values on average. The mean difference between both methods was 3.7±0.8 dB (range 1.4–5.8 dB) when the test locations at the blind spot were excluded (linear regression between the two methods: r=0.843, P<0.0001). The mean time interval between two stimulus presentations was 2.5 s with the SLO perimetry. Conclusion: With the Heidelberg software, automated static threshold perimetry using the SLO is possible within reasonably short examination times. The mean time interval between two test point presentations is about one tenth of that necessary using the original Rodenstock software. There is a systematic difference between SLO and Octopus fields of about 4 dB which was not very much influenced by the stimulus locations.Presented in part at the ARVO Meeting in Sarasota, Florida, 1–6 May 1994     

Precision of ultrasonic estimates of choroidal melanoma regression

Background: Important prognostic information may be gained from knowledge of the volume and, over time, the change in volume of intraocular tumours such as choroidal melanomas. Methods: The precision and time consumption of three different ultrasonographic methods were evaluated. Seven choroidal melanomas were analysed after placement of a ruthenium plaque and again after tumour regression had occurred. Perpendicular ultrasound B-scans were printed and analysed by overlay grid counting or outline tracing of tumour structures to calculate the volume. These two methods, using only two perpendicular scans, were compared with a micro-computer-controlled ultrasonographic three-dimensional rotation scanning system, where each tumour was manually outlined in 20 revolving scan planes. Results: The three-dimensional volume scanning method was the most precise, but also the most demanding in hardware and time consumption. Conclusion: Increased precision and less observer-dependent estimation of shrinkage rate after radiotherapy is available at the cost of sophisticated equipment.     

Adult-onset foveomacular vitelliform dystrophy and indocyanine green videoangiography

Background Adult-onset foveomacular vitelliform dystrophy (AOFVD) represents a heterogeneous group of disorders with different clinical, angiography, and histopathological features. The most common form is characterized by a yellow, round to oval subretinal macular lesion with or without central pigmented spot. Methods Eight patients affected by typical AOFVD underwent fluorescein an giography and indocyanine green videoangiography (ICGV). Results Fluorescein angiography showed a central hypofluorescent spot surrounded by an irregular hyperfluorescentring ICGV demonstrated a foveal nonfluorescent spot, visible during the entire examination, and a hyperfluorescent area surrounding the central spot, which became evident soon after the beginning of the examination. Conclusions In light of previous histopathological studies, the central nonfluorescent spot may be interpreted as a masking effect of a pigment clump, whereas the hyper-fluorescent area may represent dye pooling or staining of the subretinal pigment epithelial material.     

Silent myocardial ischemia in glaucoma and cataract patients

Background: Glaucoma and cataract are multifactorial diseases. They have been described to be associated with cardiovascular risk factors. Methods: Twentyfour-hour ECG monitoring was done in 22 normal-tension glaucoma patients, 27 open-angle glaucoma patients, 25 cataract patients, and 20 normal controls. The frequency of silent myocardial ischemia (SMI) as well as that of ventricular extrasystoles (VES) was evaluated. Results: At least one episode of significant asymptomatic ST-T segment depression occurred in 45% of the normal-tension glaucomas, in 25.9% of open-angle glaucomas, in 12% of cataract patients, and in 5% of normal controls. The frequency of VES was not significantly different among the groups. Conclusions: Glaucoma, especially normal-tension glaucoma, is significantly associated with the occurrence of episodic asymptomatic myocardial ischemias. Cataract patients, however, had only a slightly, statistically not significantly increased frequency of both SMI and VES compared with normals.     

Corneal neovascularization in rats as a model for photothrombotic therapy using bacteriochlorin a and an argon laser

Background: The presence of vessels has a negative influence on corneal transplant survival. Closure of such vessels prior to transplantation may improve the transplant results, and this might be achieved by irradiating the vessels with argon laser light after intravenous administration of a photosensitizer, e.g. bacteriochlorin a (BCA). A suture-induced corneal neovascularization model in rats was set up to test this hypothesis. Methods: Suture-induced vessels in the cornea of male Wistar rats were irradiated with argon laser light after intravenous administration of BCA. We applied irradiation of varying energy levels and duration and assessed the changes in the vessels by slit-lamp examination, fluorescein angiography and histology. Results: Suture-induced corneal vessels in the rat could be used effectively to study photothrombosis therapy. Intravenous administration of BCA prior to irradiation (=514.5 nm) of the corneal vessels led to vessel closure at lower energy levels and of longer duration than occurred with laser treatment alone. Conclusion: Suture-induced corneal neovascularization in the rat can be used as a model to study the efficacy of photothrombosis therapy. BCA can be used to enhance the rate and duration of vessel closure.     

Serum-induced collagen gel contraction

Purpose: To understand the molecular events underlying disease-related vitreous gel contraction, the effect of serum components on collagen was investigated. Methods: Bovine vitreous or dermal collagen was incubated with a mixture of transglutaminase (TG; factor XIIIa) and fibronectin (FN), and the biochemical changes of collagen were monitored by gel electrophoresis. In addition, serum-induced changes in the volume of the collagen gel were monitored. Results: Gel electrophoresis revealed a new high-molecular-weight band (M_r 240 000) presumably due to intermolecular cross-links of collagen peptides and FN. The serum components also were shown to cause a significant decrease in the volume of the collagen gel. Conclusion. Collagen gel contraction could be attributed to the collagen-FN-collagen cross-links catalyzed by TG.     

Detection of herpes simplex virus after penetrating keratoplasty by polymerase chain reaction: correlation of clinical and laboratory findings

Background: The study was carried out to investigate the possible correlation of clinical findings, histopathologic features and detection of herpes simplex virus DNA in corneal buttons obtained after penetrating keratoplasty. Methods: We examined 47 consecutive corneal buttons sent for histopathologic examination by light microscopy and using the polymerase chain reaction for the detection of HSV1 and HSV2. Twenty-one corneal buttons from eyes with bullous keratopathy served as controls. Results: The 47 cases were graded from the clinical information available as unproven, suspected and clinically proven cases of herpetic keratitis. This grading did not correlate to specific histopathologic features or to the results of HSV1 DNA testing. None of the cases were positive for HSV2 DNA. Conclusion: HSV DNA was detected in some of the cases of clinically unsuspected herpetic keratitis. This technique of demonstrating the presence or absence of HSV in the cornea after keratoplasty is more reliable than clinical data or histopathologic findings and may be important in cases of recurrent inflammatory episodes involving grafts after keratoplasty.Presented in part at the meeting Frontiers in Penetrating Keratoplasty on 22 September 1994, Heidelberg, Germany     

Inhibition of corneal allograft reaction by CTLA4-Ig

Background: Activation of T cells requires both the interaction of T-cell receptor with major histocompatibility complex on the antigen-presenting cell and costimulatory signals, for instance the B7 antigens expressed on antigen-presenting cells and the CD28 molecule expressed on T cells. A recombinant fusion protein, CTLA4-Ig, has been produced that contains the extracellular domain of human CTLA4 fused to IgGl constant region and that binds the B7 molecule with high affinity. Blocking the CD28/B7 interaction with CTLA4-Ig inhibits T cell activation in vitro and in vivo. Methods: We used CTLA4-Ig in a fully MHC-mismatched mouse keratoplasty model. The animals were divided into four groups: (1) no treatment, (2) intraperitoneal treatment with 130 g CTLA4-Ig, (3) intraperitoneal treatment with 300 g CTLA4-Ig, (4) subconjunctival treatment with 290 g CTLA4-Ig. Results: The allograft reaction occurred in untreated animals between days 12 and 16 (mean 13.5). While topical application of CTLA4-Ig seemed to shorten the graft survival (mean 11.6 days) and systemic application of 130 g had no influence (mean 14.0), only intraperitoneal injection of 300 g of CTLA4-Ig prolonged the survival of allografts (mean >20 days) (P<0.01). Conclusion: CTLA4-Ig prolonged significantly the survival of corneal allografts in a fully MHC-mismatched mouse keratoplasty model, but the small antigen load of the corneal transplant and the anterior chamber-associated immune deviation (ACAID) may have a disadvantage to induce tolerance in this model of CTLA4-Ig therapy.Presented at JERMOV 1996 in Montpellier     

Experimental microendoscopic photoablative laser goniotomy as a surgical model for the treatment of dysgenetic glaucoma

Purpose: The aim of this study was to investigate the feasibility of photoablative Er:YAG laser goniotomy under microendoscopic control in a surgical cloudy corneal model of primary infantile glaucoma. Methods: Pectinate ligaments of 12 freshly enucleated cadaver porcine eyes were treated by ab interno single-pulse (5 mJ, 200 s) Er:YAG laser (2.94 m) photoablation. Through a clear corneal incision near the limbus an ophthalmic microendoscope (18 and 20 gauge) was inserted into the anterior chamber. Internal structures were observed and photoablative laser goniotomy was conducted under video guidance. Following treatment all eyes were prepared for light and scanning electron microscopy. Results: Anterior chamber angle structures and tissue photoablation were clearly visualized on the videoscreen using ophthalmic microendoscopy. Energy settings of 5 mJ per pulse proved to be sufficient for reproducible photoablation of pectinate ligaments, accompanied by the root of the iris falling back and exposing trabecular meshwork. This was confirmed histopathologically. Scatter thermal damage was less than 30 m. Conclusion: This new therapeutic modality, which combines endoscopic visualization of the internal structures with photoablative laser goniotomy, can be effective in the management of dysgenetic glaucoma in the presence of a cloudy cornea. High reproducibility of contact laser photoablation enabled sufficient control of incision depth and was not accompanied by inadvertent tissue damage to adjacent intraocular structures.     

Hemodynamic parameter estimation from ocular fluorescein angiograms

Background: A method is proposed for parameterizing choroidal blood flow from fluorescein angiograms. Methods: After digitizing and aligning the angiographic sequence, the intensity build-up curves of fluorescence are analysed per pixel (approx. 10 m in fundo). Two models are compared. A one-compartment model predicts an exponential build-up curve, from which the following parameters are estimated: maximum fluorescence, dye appearance time and local perfusion rate (reciprocal of the time constant of the exponential). To account for the contribution of the systemic circulation to the shape of the build-up curve, a two-compartment model is used which predicts a bi-exponential curve. Results: Introduction of the second (systemic) compartment resulted in a significant improvement of fit in 37 of 48 patients studied. The rate constants of the systemic compartment found were mainly in the range of 0.30–1.00 s^–1. Conclusion: For the individual patient, the local perfusion rates may vary strongly, with lower perfusion rates possibly being of prognostic value for ocular diseases such as glaucoma or diabetic retinopathy.     

Course of the optic nerve fibers through the lamina cibrosa in human eyes

Background: This study investigated whether regional variations in the course of the optic nerve fibers through the lamina cribrosa may be one of the reasons why the local susceptibility for glaucomatous optic nerve fiber loss differs among the various regions of the optic disc. Methods: The study included 34 human eyes enucleated because of a malignant melanoma of the peripheral choroid without involvement of the anterior chamber angle or the optic nerve. Anterior-posterior sections through the pupil and the optic disc were histomorphometrically evaluated. In the central region and the peripheral part of the optic disc, we measured the thickness of the lamina cribrosa and the length of the lamina cribrosa channels through which the nerve fibers pass. Results: In the peripheral parts of the disc, compared with its central region, the lamina cribrosa was significantly thicker (P<0.0001, Wilcoxon test), the lamina cribrosa channels with the nerve fibers passing through were significantly longer (P<0.0001), and the ratio of length of the fiber channels to the thickness of the lamina cibrosa was significantly higher (P=0.0001). Conclusion: The lamina cribrosa is thicker and the course of the optic nerve fibers through the lamina cribrosa is more curvilinear in the disc pheriphery than in the disc center. These variations in the anatomy of the lamina cribrosa may be one of several factors influencing the regional susceptibility for glaucomatous optic nerve fiber loss within the optic nerve head.     

How often do patients need visual field tests?

Background: This study was undertaken to determine whether the interval between visual field tests affects the ability to detect progres sive glaucomatous field loss. Methods: One hundred and nineteen retinal locations which were deteriorating significantly by 1 dB/year (untreated normal tension glaucoma patients: 6 eyes) were studied. Analysis was repeated using thinned visual field tests: one test per year instead of the complete three per year over a period of 4 years. Results: The thinned tests identified only 45.4% of the deteriorating points over the 4-year period. Furthermore, there was a mean delay of 1.10 years in detection (P<0.01). Conclusions: Less frequent visual field testing detects fewer progressing locations and detects them later.Presented in part at the Association for Research in Vision and Ophthalmology Annual Meeting, May 1995 and at the American Academy of Ophthalmology Meeting, October 1995.The authors have no proprietary interest in any of the materials used in this study.     

Indocyanine green angiography and age-related serous pigment epithelial detachment

Background: Fundus fluorescein angiography has shown that pigment epithelial detachment in age-related macular degeneration is often associated with choroidal neovascularisation (CNV). Indocyanine green angiography (ICG-A) provides a better visualisation of choroidal circulation and of CNV than fluorescein angiography (FA). Methods: We studied the ICG angiograms of 58 eyes presenting age-related pigment epithelial detachment, either with signs of occult CNV (48 eyes) or without signs of CNV (10 eyes) on FA. In selected cases the neovascular complex defined on the ICG angiogram was photocoagulated. Results: ICGA-revealed hyperfluorescence interpreted as CNV in 46 of 48 eyes with fluorescein angiographic signs of occult choroidal neovascularisation. The neovascular complex seen on the ICG angiogram was well delineated in 29 eyes and ill defined in 17 eyes. ICG-A revealed CNV in 2 of 10 eyes without signs of CNV on FA. In these two cases the neovascular complex was ill defined. Photocoagulation in selected cases resulted in stabilisation or even improvement of visual acuity and flattening of the pigment epithelial detachment in 9 of 18 cases. Conclusion: ICG-A may offer a better definition of the neovascular complex associated with pigment epithelial detachment in age-related macular disease and be helpful in guiding laser treatment. In some cases FA still outlines more clearly the lesions to be treated. FA and ICG-A should thus be used concurrently to determine treatment strategy.     

Increased release of tenascin in tear fluid after photorefractive keratectomy

Background: Extracellular matrix protein tenascin (TN) is expressed in the anterior stroma during corneal wound healing. In this study we analysed TN release in tear fluid after photorefractive keratectomy (PRK). Methods: Tear fluid TN concentrations of ten PRK patients were measured with an immunoassay. Tear fluids were collected preoperatively and 1, 2 and 7 days after PRK. The tear fluid collection time and the volume of tears collected were registered. Because tear fluid flow was greatly increased postoperatively, tear fluid flow-corrected release (TN flux) was calculated. Results: The tear fluid flow was 4.50±0.94 l/min (mean±SEM) preoperatively, 55.48±16.70 l/min (P<0.01) on the 1st, 33.91±7.91 l/min (P<0.01) on the 2nd, and 13.79±5.49 l/min (P>0.05) on the 7th postoperative day. The preoperative TN concentration was 0.85±0.20 g/ml. On the 1st postoperative day it decreased to 0.37±0.17 g/ml (P>0.05), most likely due to the dilution effect caused by hypersecretion after PRK. The TN concentration was 0.67±0.12 g/ml (P>0.05) on the 2nd and 0.78±0.15 g/ml (P>0.05) on the 7th postoperative day. The preoperative TN flux was 5.23±1.88 ng/min. On the 1st and 2nd postoperative days the TN flux was 14.40±4.99 ng/min (P<0.05) and 22.66±6.I2 ng/min (P<0.05), respectively. On the 7th postoperative day a tendency towards decreased flux (14.00±6.02 ng/min, P>0.05) was observed. Conclusion: Although there is a minor decrease in TN concentration after PRK due to increased tear fluid flow, a significant increase in TN flux was observed. Complete reepithelialization of the ablated area was observed in all eyes at the follow-up visit on postoperative day 7.     

Decorin and suramin inhibit ocular fibroblast collagen production

Background: The process of ocular wound healing with respect to glaucomatous filtering procedures is of current interest. Delaying this response in patients could possibly lead to more favorable surgical results. So far, only highly toxic antimetabolites have come into frequent clinical use. The possible efficacy of other groups of substances such as growth factor inhibitors has not yet been examined in vitro. Methods: We exposed Tenon's capsule fibroblasts in tissue culture to various concentrations of decorin and suramin. The dose responses of type I and type III collagen to these inhibitors were measured using an ELISA-type dot blot assay. Total cellular protein production was assayed by measuring the incorporation of tritiated leucine. Results: At a concentration of 10 g/ml, suramin reduced the collagen production by more than 80%. Decorin, at a concentration of 100 g/ml, reduced type I collagen production by about 50% while type III collagen was reduced by 80%. At these concentrations, the total cellular protein production was not inhibited. Conclusions: Both suramin and decorin, which specifically inhibit the action of growth factors on target cells, reduce the production of collagen synthesis by Tenon's capsule fibroblasts. This is a specific effect, because total protein production is not influenced. This sets these substances apart from antimetabolites. Decorin and suramin may have clinical relevance in that they appear to interfere with ocular wound healing more specifically than the substances so far frequently used.