Anti-VEGF intravitreal injections in the era of COVID-19: responding to different levels of epidemic pressure.

PURPOSE: Following the first wave of the COVID-19 pandemic in early 2020, the easing of strict measures to reduce its spread has led to a resurgence of cases in many countries at both the national and local level. This article addresses how guidance for ophthalmologists on managing patients with retinal disease receiving intravitreal injections of anti-vascular endothelial growth factor (VEGF) during the pandemic should be adapted to the local epidemic pressure, with more or less stringent measures implemented according to the ebb and flow of the pandemic. METHODS: The Vision Academy's membership of international retinal disease experts analyzed guidance for anti-VEGF intravitreal injections during the COVID-19 pandemic and graded the recommendations according to three levels of increasing epidemic pressure. The revised recommendations were discussed, refined, and voted on by the 14-member Vision Academy Steering Committee for consensus. RESULTS: Protocols to minimize the exposure of patients and healthcare staff to COVID-19, including use of personal protective equipment, physical distancing, and hygiene measures, should be routinely implemented and intensified according to local infection rates and pressure on the hospital/clinic or healthcare system. In areas with many COVID-19-positive clusters, additional measures including pre-screening of patients, postponement of non-urgent appointments, and simplification of complex intravitreal anti-VEGF regimens should be considered. Treatment prioritization for those at greatest risk of irreversible vision loss should be implemented in areas where COVID-19 cases are increasing exponentially and healthcare resources are strained. CONCLUSION: Consistency in monitoring of local infection rates and adjustment of clinical practice accordingly will be required as we move forward through the COVID-19 era. Ophthalmologists must continue to carefully weigh the risk-benefits to minimize the exposure of patients and healthcare staff to COVID-19, ensure that patients receive sight-saving treatment, and avoid the potential long-term impact of prolonged treatment postponement.

Guidance for anti-VEGF intravitreal injections during the COVID-19 pandemic.

PURPOSE: There is an urgent need to address how to best provide ophthalmic care for patients with retinal disease receiving intravitreal injections with anti-vascular endothelial growth factor agents during the ongoing global COVID-19 pandemic. This article provides guidance for ophthalmologists on how to deliver the best possible care for patients while minimizing the risk of infection. METHODS: The Vision Academy's Steering Committee of international retinal disease experts convened to discuss key considerations for managing patients with retinal disease during the COVID-19 pandemic. After reviewing the existing literature on the issue, members put forward recommendations that were systematically refined and voted on to develop this guidance. RESULTS: The considerations focus on the implementation of steps to minimize the exposure of patients and healthcare staff to COVID-19. These include the use of personal protective equipment, adherence to scrupulous hygiene and disinfection protocols, pre-screening to identify symptomatic patients, and reducing the number of people in waiting rooms. Other important measures include triaging of patients to identify those at the greatest risk of irreversible vision loss and prioritization of treatment visits over monitoring visits where possible. In order to limit patient exposure, ophthalmologists should refrain from using treatment regimens that require frequent monitoring. CONCLUSION: Management of patients with retinal disease receiving intravitreal injections during the COVID-19 pandemic will require adjustment to regular clinical practice to minimize the risk of exposure of patients and healthcare staff, and to prioritize those with the greatest medical need. The safety of patients and healthcare staff should be of paramount importance in all decision-making.

SUSPENDING TREATMENT OF NEOVASCULAR AGE-RELATED MACULAR DEGENERATION IN CASES OF FUTILITY.

PURPOSE: To provide guidance on the management of patients with neovascular age-related macular degeneration and its subtypes who respond poorly to anti-vascular endothelial growth factor (anti-VEGF) therapy, and to identify cases where suspending anti-VEGF treatment may be warranted. METHODS: Through a literature review and the combined knowledge and clinical experience of retinal experts, the Steering Committee of the Bayer-sponsored Vision Academy developed an algorithm for determining when to suspend anti-VEGF treatment of neovascular age-related macular degeneration in cases of futility. RESULTS: Consideration of factors that may cause suboptimal response to anti-VEGF therapy, such as undertreatment or misdiagnosis of the underlying condition, and factors that may preclude continued treatment, such as injection- or drug-induced complications, is necessary for adjusting treatment protocols in patients who respond poorly to anti-VEGF. If poor response to treatment persists after switching to an alternative anti-VEGF agent and no change in response is observed after withholding treatment for a predetermined period of time ("treatment pause"), anti-VEGF treatment may be considered futile and should be suspended. CONCLUSION: This publication introduces an algorithm to guide the management of neovascular age-related macular degeneration in patients showing poor response to anti-VEGF treatment and provides expert guidance for suspending anti-VEGF treatment in cases of futility.

Visual acuity time in range: a novel concept to describe consistency in treatment response in diabetic macular oedema.

OBJECTIVE: To assess 'time in range' as a novel measure of treatment response in diabetic macular oedema (DMO). METHODS: This post hoc analysis of the Protocol T randomised clinical trial included 660 individuals with centre-involved DMO and best-corrected visual acuity (BCVA) letter score ≤78-≥24 (approximate Snellen equivalent 20/32-20/320). Study participants received intravitreal aflibercept 2.0 mg, repackaged (compounded) bevacizumab 1.25 mg, or ranibizumab 0.3 mg given up to every 4 weeks using defined retreatment criteria. Mean time in range was calculated using a BCVA letter score threshold of ≥69 (20/40 or better; minimum driving requirement in many regions), with sensitivity analyses using BCVA thresholds from 100 to 0 (20/10 to 20/800) in 1-letter increments. RESULTS: Time in range was defined as either the absolute or relative duration above a predefined BCVA threshold, measured in weeks or as a percentage of time, respectively. Using a BCVA letter score threshold of ≥69 (20/40 or better), the least squares mean time in range (adjusted for baseline BCVA) in Year 1 was 41.2 weeks with intravitreal aflibercept, 4.0 weeks longer (95% CI: 1.7, 6.3; p = 0.002) than bevacizumab and 3.6 weeks longer (1.3, 5.9; p = 0.004) than ranibizumab. Overall, mean time in range was numerically longer for intravitreal aflibercept for all BCVA letter score thresholds between 92 and 30 (20/20 to 20/250). In the Day 365-728 analysis, time in range was 3.9 (1.3, 6.5) and 2.4 (0.0, 4.9) weeks longer with intravitreal aflibercept vs bevacizumab and vs ranibizumab (p = 0.011 and 0.106), respectively. CONCLUSION: BCVA time in range may represent another way to describe visual outcomes and potential impact on vision-related functions over time for patients with DMO and provide a better understanding, for physicians and patients, of the consistency of treatment efficacy.

VEGFR1 signaling in retinal angiogenesis and microinflammation.

Five vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.

Clinical evaluation of pimecrolimus eye drops for treatment of canine keratoconjunctivitis sicca: a comparison with cyclosporine A.

The aim of this study was to evaluate the efficacy of pimecrolimus oil-based eye drops in alleviating the clinical signs of keratoconjunctivitis sicca (KCS) in dogs and to compare the efficacy with that of cyclosporine A (CsA) ointment. An open-label, multicenter study enrolling 44 dogs previously untreated with CsA was conducted. Dogs were randomly assigned to a treatment group and medicated twice daily for 8 weeks. After that time the mean increase (+/-SEM) in the Schirmer tear test was 9.2+/-1.6 mm/min in the pimecrolimus group and 5.8+/-1.1 mm/min in the CsA group (P=0.085). The improvement in clinical signs of inflammation in eyes treated with pimecrolimus was significantly greater than in eyes treated with CsA (P=0.02). The results show that 1% pimecrolimus oily eye drops are as safe as and more effective than CsA ointment in controlling KCS in dogs.

FTY720 sustains and restores neuronal function in the DA rat model of MOG-induced experimental autoimmune encephalomyelitis.

FTY720 (fingolimod) is an oral sphingosine 1-phosphate (S1P) receptor modulator under development for the treatment of multiple sclerosis (MS). To elucidate its effects in the central nervous system (CNS), we compared functional parameters of nerve conductance in the DA rat model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) after preventive and therapeutic treatment. We demonstrate that prophylactic therapy protected against the emergence of EAE symptoms, neuropathology, and disturbances to visual and somatosensory evoked potentials (VEP, SEP). Moreover, therapeutic treatment from day 25 to 45 markedly reversed paralysis in established EAE and normalized the electrophysiological responses, correlating with decreased demyelination in the brain and spinal cord. The effectiveness of FTY720 in this model is likely due to several contributing factors. Evidence thus far supports its role in the reduction of inflammation and preservation of blood-brain-barrier integrity. FTY720 may also act via S1P receptors in glial cells to promote endogenous repair mechanisms that complement its immunomodulatory action.

Epidemiological and Clinical Baseline Characteristics as Predictive Biomarkers of Response to Anti-VEGF Treatment in Patients with Neovascular AMD.

Purpose. To review the current literature investigating patient response to antivascular endothelial growth factor-A (VEGF) therapy in the treatment of neovascular age-related macular degeneration (nAMD) and to identify baseline characteristics that might predict response. Method. A literature search of the PubMed database was performed, using the keywords: AMD, anti-VEGF, biomarker, optical coherence tomography, treatment outcome, and predictor. The search was limited to articles published from 2006 to date. Exclusion criteria included phase 1 trials, case reports, studies focusing on indications other than nAMD, and oncology. Results. A total of 1467 articles were identified, of which 845 were excluded. Of the 622 remaining references, 47 met all the search criteria and were included in this review. Conclusion. Several baseline characteristics correlated with anti-VEGF treatment response, including best-corrected visual acuity, age, lesion size, and retinal thickness. The majority of factors were associated with disease duration, suggesting that longer disease duration before treatment results in worse treatment outcomes. This highlights the need for early treatment for patients with nAMD to gain optimal treatment outcomes. Many of the identified baseline characteristics are interconnected and cannot be evaluated in isolation; therefore multivariate analyses will be required to determine any specific relationship with treatment response.

Effect of somatostatin analogues on chemically induced ischaemia in the rat retina.

This study investigated the neuroprotective effect of somatostatin, cortistatin and agonists at somatostatin(2) (sst(2)) receptors in retinal explants subjected to chemical ischaemia. Eyecups of female Sprague-Dawley rats (250-300 g) were immersed in PBS buffer or PBS containing iodoacetic acid (IAA; 0.5, 5, 50, 100 mM) and sodium cyanide (NaCN; 2.5, 25, 250, 500 mM) (chemical ischaemia solution) for 15, 30, 45, 60, 120 min (pilot study). Subsequently, eyecups were incubated with (1) PBS, (2) chemical ischaemia solution (5 mM IAA/25 mM NaCN) or (3) somatostatin, cortistatin, BIM23014 or MK678 (0.1, 1, 10 microM) together with the chemical ischaemia solution for 60 min, followed by a second 60-min incubation in PBS (control and ischaemia groups) or ligands in PBS (neuroprotection groups). The eyecups were subsequently fixed and sectioned for immunohistochemistry. Treatment of the eyecups with IAA/NaCN (5/25 mM) for 60 min abolished choline acetyltransferase (ChAT), tyrosine hydroxylase and brain nitric oxide synthase immunoreactivity in the inner nuclear, inner plexiform and ganglion cell layers. It also abolished protein kinase C immunoreactivity in rod bipolar cells and terminals, but did not damage ganglion cells labelled for microtubule-associated protein-1. TUNEL staining provided evidence of cell death in the ischaemic retina. Cortistatin, BIM23014 and MK678 attenuated the retinal damage caused by the chemical ischaemia in a concentration dependent manner. The ligands afforded approximately 58, 76 and 49% neuroprotection, respectively, of the ChAT immunoreactive cells. These results demonstrate that somatostatin analogues can protect the retina from ischaemic damage. The chemical ischaemia model is presently employed for the elucidation of the mechanisms involved in the neuroprotection.

Genetic predictive biomarkers of anti-VEGF treatment response in patients with neovascular age-related macular degeneration.

Anti-vascular endothelial growth factor (anti-VEGF) therapies for neovascular age-related macular degeneration (nAMD) have proven efficacy at a study-population level, although individual patient responses vary, with most of the patients responding well to anti-VEGF therapies, while a few respond poorly. The pathogenesis of AMD is known to have a genetic component, but it is unclear if any particular genotype can predict response to anti-VEGF therapy. With the advent of less expensive genotyping technology, there have been numerous studies within this area. Here we analyze potential biomarker candidates identified that could be used in a clinical setting to predict response to anti-VEGF treatment of nAMD. We analyze single nucleotide polymorphisms (SNPs) identified from 39 publications. The SNPs that appeared to be of most importance fell into two main groups: those previously associated with AMD pathogenesis and those within the signaling pathway targeted by anti-VEGF therapies. A number of small studies found evidence supporting an association between anti-VEGF treatment response and two SNPs, CFH rs1061170 and VEGFA rs699947, but results from randomized controlled trials found no such association. It is possible that, in the future, the cumulative effect of several high-risk SNPs may prove useful in a clinical setting and that other genetic biomarkers may emerge.

Functional damage to inner and outer retinal cells in experimental glaucoma.

PURPOSE: To investigate the cellular sources underlying the functional damage observed by multifocal electroretinography (mfERG) responses of glaucomatous eyes of monkeys. METHODS: First- and second-order (K1 and K2, respectively) mfERG responses of three normal and three experimentally induced glaucomatous eyes of cynomolgus monkeys were measured at two different levels of luminance. Retinal contributors to the responses were isolated by intravitreal injections of pharmacological agents that suppress specific retinal cells. gamma-Aminobutyric acid (GABA) and glycine were administered to block inner retinal function, followed by 2-amino-4-phosphonobutyric acid (APB), to block ON-bipolar cells. RESULTS: An inner retinal component removed by GABA and glycine was found in both the normal and glaucomatous eyes. However, it was attenuated in the latter, correlating with changes observed in the baseline K1 responses. Delays in the latency of outer retinal components were found in the responses of the glaucomatous eyes. K2 responses were dominated by an inner retinal contribution and were diminished in the responses of glaucomatous eyes. The outer retina responded to increased luminance with a shorter implicit time. A distinct wave part of the inner retinal component responded to increased luminance with increased amplitudes. CONCLUSIONS: The integration of the retinal sources forming the mfERG response was compared between normal and glaucomatous monkey eyes. Both inner and outer retinal functions were aberrant in the responses of the glaucomatous eyes, with the attenuation of the inner retinal function more conspicuous. Nevertheless, glaucomatous eyes retained certain inner retinal activity, despite the advanced stage of disease. K2 responses were more sensitive to glaucomatous changes than were K1 responses.

The effect of contrast and luminance on mfERG responses in a monkey model of glaucoma.

PURPOSE: To evaluate the effect of contrast and luminance attenuation on the multifocal electroretinogram (mfERG) responses of normal and glaucomatous eyes of cynomolgus monkeys. METHODS: Nine adult male cynomolgus monkeys with unilateral experimentally induced glaucoma were used. Hypertension-induced damage was confirmed by tomography of the optic disc. mfERGs were recorded with five different stimulus contrasts and/or luminance settings. The first-order and the first slice of second-order responses were analyzed. RESULTS: Waveforms of normal and glaucomatous eyes differed in shape and amplitude. Second-order responses contributed to first-order responses of the signals in the normal eyes, but made a negligible contribution to the signals in the glaucomatous eyes. Contrast and luminance attenuation affected both first- and second-order responses. The differences between signals in normal and glaucomatous eyes were sufficiently large for an unsupervised clustering algorithm to achieve accurate segregation. CONCLUSIONS: The observations in this study indicate that outer and inner retinal generators participate in first-order mfERG responses and that both inner and outer retinal contributors respond to contrast and luminance changes in stimulus. The hypertension-induced changes in the mfERG furthermore suggest damage to both inner and outer retina.

Functional and structural analysis of the visual system in the rhesus monkey model of optic nerve head ischemia.

PURPOSE: A redistribution of neurochemicals has been identified in the visual cortex of monkeys with laser-induced glaucoma. Examined were functional, structural, and neurochemical changes to the retina, optic nerve, and central visual system in a nonhuman primate model of optic nerve head (ONH) ischemia caused by sustained unilateral administration of endothelin (ET)-1 to the optic nerve. METHOD: ET-1 or sham control solution was delivered by osmotic minipump to the retrolaminar region of one optic nerve of rhesus monkeys (Macaca mulatta) for 1.5 years. ONH topography and blood flow velocity were serially studied with scanning laser tomography and laser Doppler flowmetry, respectively. Retinal and cortical electrophysiologic measurements from pattern-derived stimuli were obtained quarterly. Immunohistochemistry was used to identify the distribution of calbindin (CB) and c-Fos labeled neurons in the visual cortex areas V1 and V2, and lateral geniculate nucleus (LGN). Retinal ganglion cell counts and optic nerve axon density were determined by light microscopy. RESULTS: No significant changes in retinal and ONH morphology, ONH blood flow velocity, and retinal and cortical pattern-derived functional activity were detected. Measurement of CB-positive cell density in V1 and V2 showed a significant decrease in CB labeling to the contralateral side of the ET-1-treated eye (P < 0.04). CB-positive cells were present in the magnocellular layers of the LGN with no differences noticed between the ET-1- and sham-treated eyes. c-Fos-labeled neurons were found in striate area V1 and extrastriateV2 of both groups. No c-Fos labeling was observed in the LGN. CONCLUSIONS: Administering ET-1 to the orbital optic nerve alters neuronal metabolic activity in the visual cortex in rhesus monkeys. Metabolic activity reductions in the visual cortex precede the ability to detect functional and structural alterations in the retina, ONH, and visual cortex in this animal model.

Hypoxia activates matrix metalloproteinase expression and the VEGF system in monkey choroid-retinal endothelial cells: Involvement of cytosolic phospholipase A2 activity.

PURPOSE: To determine whether the gene expression of matrix metalloproteinases (MMPs) as well as that of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and its receptors change in response to hypoxic exposure in a primate choroid-retinal endothelial cell line, and furthermore, whether cytosolic phospholipase A2 (cPLA2) plays a role in this process. METHODS: Rhesus macaque choroid-retinal endothelial (RF/6A) cells were incubated under hypoxic conditions for 1, 2, 4, or 8 h prior to RNA extraction. In some experiments cells were pretreated with the cPLA2 inhibitor AACOCF3 (10 microM) for 30 min prior to hypoxia. Changes in gene expression were determined by RT-PCR and quantified by real-time PCR for urokinase plasminogen activator (uPA), collagenase-1 (MMP-1), membrane type-1 metalloproteinase (MT1-MMP), gelatinases A and B (MMP-2, MMP-9), tissue inhibitor-2 (TIMP-2), VEGF and its receptors, Flt-1 (VEGFR-1), KDR (VEGFR-2), and neuropilin-1 (NP-1). MMP-2 secreted by the cells was evaluated by zymography. VEGF release was measured by ELISA. In tube-formation studies, endothelial cells (EC) were seeded into collagen gel, exposed to hypoxia for 4 h, then incubated under normoxic conditions for 72 h. RESULTS: Hypoxia triggered a three fold increase in the gene expression of MT1-MMP, MMP-2, and TIMP-2, and a ten fold increase in MMP-2 levels. Moreover it also induced tube formation in EC. Expression of uPA, MMP-1, and MMP-9 mRNA was not detected. Pretreatment with AACOCF3 abolished hypoxia-induced tube formation and MT1-MMP, MMP-2, and TIMP-2 transcription. Furthermore, hypoxia produced a significant, sustained increase in the gene expression and release of VEGF-165, the only VEGF-A isoform detected in these cells. AACOCF3 reduced the hypoxia-induced VEGF release at 8 h of hypoxia. VEGF receptors KDR and NP-1 were constitutively expressed in EC and up-regulated under hypoxic conditions. CONCLUSIONS: In monkey choroid-retinal EC, hypoxia selectively induces MMP-2 activity. This induction is preceded by MT1-MMP, MMP-2, and TIMP-2 mRNA expression, as well as that of the VEGF-165 isoform and its receptors KDR and NP1. These increases possibly result from hypoxia-induced activation of cPLA2 and subsequent release of arachidonic acid and its conversion to prostaglandins. These molecular changes in EC could, in part, contribute to the angiogenic response that occurs in the development of ischemic retinopathies and choroidal neovascularization.

Partial antagonism of endothelin 1-induced vasoconstriction in the human choroid by topical unoprostone isopropyl.

BACKGROUND: There is increasing evidence that reduced ocular blood flow plays a role in the pathogenesis of glaucoma. In patients with normal-tension glaucoma, ocular blood flow abnormalities may be associated with dysfunction of the endothelin 1 (ET-1) regulation system. OBJECTIVE: To test the hypothesis that unoprostone, a topical docosanoid, may affect ET-1--induced vasoconstriction in the human choroid. METHODS: In a placebo-controlled, randomized, double-masked, 2-way crossover design, ET-1 (2.5 ng/kg per minute for 150 minutes) was administered intravenously to 24 healthy individuals. Thirty minutes after the start of ET-1 infusion, 1 drop of unoprostone or placebo was instilled into the right eye. After another 30 minutes, 2 drops of unoprostone or placebo was topically administered. This procedure was continued and the dose was increased further until 4 drops of unoprostone or placebo was reached. Subfoveal and pulsatile choroidal blood flow were assessed using laser Doppler flowmetry and laser interferometric measurement of fundus pulsation amplitude, respectively. RESULTS: Administration of exogenous ET-1 decreased choroidal blood flow (mean +/- SEM, 17% +/- 2%; P<.001) and fundus pulsation amplitude (mean +/- SEM, 19% +/- 2%; P<.001). This effect was significantly blunted when topical unoprostone was coadministered (mean +/- SEM decrease in choroidal blood flow, 7% +/- 2%; P =.04 vs. placebo; mean +/- SEM decrease in fundus pulsation amplitude, 12% +/- 2%; P<.001 vs. placebo). CONCLUSION: There is a functional antagonism between ET-1 and topical unoprostone in the choroidal vasculature. CLINICAL RELEVANCE: Our findings of a functional antagonism between ET-1 and topical unoprostone in the choroidal vasculature may be important in vascular eye diseases associated with increased ET-1.

3H-Noradrenaline release from mouse iris-ciliary body: role of presynaptic muscarinic heteroreceptors.

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris-ciliary bodies. Tissue preparations were preincubated with (3)H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris-ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked alpha(2)-autoinhibition as indicated by the release-enhancing effect of the alpha(2)-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free (3)H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, alpha(2)-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of (3)H-noradrenaline release. Secondly, muscarinic inhibition of (3)H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 muM and rauwolscine 1 muM throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC(50) of 0.33 muM and maximal inhibition of 51%. The concentration-response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M(2) subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris-ciliary bodies are predominantly M(2). Finally, inhibition of (3)H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of (3)H-noradrenaline from mouse iris-ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris-ciliary body are predominantly M(2). Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.

Multifocal electroretinographical changes in monkeys with experimental ocular hypertension: a longitudinal study.

PURPOSE: To study the time course of changes in the multifocal electroretinograms (mfERG) in monkeys with experimental ocular hypertension (OHT). METHODS: The mfERGs were recorded in 12 eyes out of 6 monkeys. Two baseline measurements were used to quantify the reproducibility, the inter-ocular and the inter-individual variability of the ERG signals. Thereafter, the trabeculum of one eye of each animal was laser-coagulated in one to three sessions to induce OHT. ERG measurements were repeated regularly in a period of 18 months and the changes in ERG waveforms were quantified. RESULTS: All animals displayed OHT (between 20 and 50 mmHg) in the laser-coagulated eyes. An ERG change was defined as the sum of differences during the first 90 ms between the laser-coagulated eye and the same eye before laser coagulation and between the laser-coagulated eye and the non-treated fellow eye. Three animals displayed significant changes for nearly all retinal areas and all stimulus conditions. The three remaining animals displayed significant changes only in one comparison, indicating very mild changes. The data indicate that a high stimulus contrast is more sensitive to detect changes, probably because of a better signal-to-noise ratio. Moreover, the comparisons with the fellow eye are more sensitive to detect changes than comparisons with the measurements before laser-coagulation. CONCLUSIONS: OHT does not always lead to ERG changes. Comparisons with fellow eyes using high contrast stimuli are more sensitive to detect changes related to OHT.

Effects of intravitreally and intraperitoneally injected atropine on two types of experimental myopia in chicken.

Atropine, a non-selective muscarinic receptor antagonist, is currently the most potent agent used to prevent myopia in animal models and children. However, the ocular target tissues are not well defined. To learn more about the effect of atropine on experimental myopia, atropine was applied both intravitreally and systemically (intraperitoneally) to chickens wearing either negative lenses or light diffusers. Furthermore, the effect of ipsilateral intravitreal atropine on myopia development in the saline-treated fellow eye was studied. Monocular intravitreal injections of atropine were performed daily for a period of 4 successive days, starting at day 8 post-hatching. Fellow eyes received saline injections. Chicks were fitted with -7D lenses, either over the atropine-injected eyes only (unilateral "lens-induced myopia (LIM)"), or over both eyes (bilateral LIM). Other groups of chicks were fitted with translucent diffusers over the atropine-injected eyes (unilateral "form deprivation myopia (FDM)"). Finally, atropine was intraperitoneally injected for 4 days in chicks that wore monocularly -7D lenses. Refractive errors (RE) were measured with infrared photoretinoscopy and axial length (AL) with A-scan ultrasonography. Atropine prevented development of myopia in both unilateral LIM and FDM in a dose-dependent fashion. Fifty percent inhibition of myopia was observed at a dose of 25 microg (unilateral LIM) or 90 microg atropine (bilateral LIM) and complete inhibition at 750 microg; in unilateral FDM, 50% inhibition occurred at 2.5 microg and almost 100% inhibition at 250 microg. Interestingly, at the highest dose of atropine (2500 microg), the treated eyes became even more hyperopic compared to the saline-injected contralateral eyes with normal visual experience. In the bilateral LIM model, atropine suppressed development of myopia in both the treated and the saline-injected control eye. However, about 8.3 times higher doses were necessary to achieve comparable contralateral suppression. Since this ratio is lower than the vitreous volume to blood volume ratio (about 1:23 in young chicks), it seems unlikely that systemic dilution of the intravitreally injected drug can fully account for the contralateral suppression. Intraperitoneal injection inhibited myopia development only at the highest dose (2500 microg) but, strikingly, this inhibition was still less when the same dose was provided through the vitreous of the fellow eye. Both eyes seem to be coupled by a yet unknown, perhaps neuronal pathway. Estimations of the scleral concentrations of atropine after intravitreal injection are compatible with the assumption that the suppression of myopia by atropine occurs by direct inhibition of scleral chondrocytes.

Blood flow velocity response of the ophthalmic artery and anterior optic nerve head capillaries to carbogen gas in the rhesus monkey model of optic nerve head ischemia.

PURPOSE: To determine the effect on blood flow velocity of the ophthalmic artery and anterior superficial optic nerve head (ONH) capillaries by changing inhaled gas from 100% oxygen to carbogen (95% oxygen, 5% CO(2)) in rhesus monkeys receiving chronic unilateral orbital endothelin-1 administration. METHODS: The right eye of six young male rhesus monkeys (Macaca mulatta) received endothelin-1 (ET-1) by osmotic minipumps to the perineural optic nerve (0.3 microg/day) for 8 months. Three additional monkeys (control group) received the ET-1 vehicle (Sham) solution to the right optic nerve for the same period of time. The left eye served as a nontreated control in both groups. The blood flow velocities of the anterior ONH capillaries and ophthalmic artery were assessed in both eyes using confocal laser scanning flowmetry (CSLF) and color Doppler imaging (CDI), respectively. RESULTS: A slight increase in the CDI blood flow velocities and a small decrease in the resistive index of the ophthalmic artery, and increased flow of the ONH capillaries in rhesus monkeys were detected when inhaled gas was changed from 100% oxygen to carbogen. The difference in CSLF blood flow in the nasal ONH between the endothelin-1 (ET-1) treated right eye and the normal left eye of the same individual monkeys was significantly greater than the difference in blood flow between the Sham-treated right eye and the normal left eye in control animals under the conditions of carbogen and oxygen inhalation. CONCLUSION: Carbogen inhalation slightly influences the microcirculation of the globe and ONH in rhesus monkeys. These data suggest that low dose ET-1 administration has a subtle vasorelaxing effect in the ONH microcirculation in this animal model of ONH ischemia.