To evaluate the impact of temperature-controlled pars plana vitrectomy (PPV) on structural and functional outcomes in a rabbit eye model in vivo.

Ten healthy New Zealand White rabbits underwent temperature-controlled PPV in the right eye (group A), using a device specifically designed to heat the infusion fluid/air and integrated into the vitrectomy machine, and conventional PPV in the left eye (group B). Both eyes received ophthalmic examination and electroretinography (ERG) before and 1 week postoperatively. After 1-week ERG, rabbits were enucleated and then sacrificed. Histological and immunohistochemical examinations were performed on enucleated eyes and expression of glial fibrillary acidic protein (GFAP) and vimentin investigated.

Postoperatively, only group B showed significantly decreased amplitude and increased latency of a-wave at 3 cd·s/m2 (p = 0.001 and 0.005, respectively). Significant increase of b-wave latency at 0.01 cd·s/m2 was detected in both groups (p = 0.019 and 0.023, respectively). Postoperatively, amplitude of oscillatory potentials (OPs) increased significantly in group A (p = 0.023) and decreased in group B. In both groups, OPs latency significantly increased at 1-week test (P < 0.05). A greater number of eyes without structural retinal alterations was detected in group A compared to group B (6 vs 5, respectively). GFAP expression was higher in group B than group A, even if the difference was not statistically significant.

Temperature-controlled PPV resulted in more favorable functional and structural outcomes in rabbit eyes compared with conventional PPV, supporting the potential beneficial role of the intraoperative management of intraocular temperature in vitreoretinal surgery.

Whole-eye transplantation emerges as a frontier in ophthalmology, promising a transformative approach to irreversible blindness. Despite advancements, formidable challenges persist. Preservation of donor eye viability post-enucleation necessitates meticulous surgical techniques to optimize retinal integrity and ganglion cell survival. Overcoming the inhibitory milieu of the central nervous system for successful optic nerve regeneration remains elusive, prompting the exploration of neurotrophic support and immunomodulatory interventions. Immunological tolerance, paramount for graft acceptance, confronts the distinctive immunogenicity of ocular tissues, driving research into targeted immunosuppression strategies. Ethical and legal considerations underscore the necessity for stringent standards and ethical frameworks. Interdisciplinary collaboration and ongoing research endeavors are imperative to navigate these complexities. Biomaterials, stem cell therapies, and precision immunomodulation represent promising avenues in this pursuit. Ultimately, the aim of this review is to critically assess the current landscape of whole-eye transplantation, elucidating the challenges and advancements while delineating future directions for research and clinical practice. Through concerted efforts, whole-eye transplantation stands to revolutionize ophthalmic care, offering hope for restored vision and enhanced quality of life for those afflicted with blindness.

As innovations in the field of vascular composite allotransplantation (VCA) progress, whole-eye transplantation (WET) is poised to transition from non-human mammalian models to living human recipients. Present treatment options for vision loss are generally considered suboptimal, and attendant concerns ranging from aesthetics and prosthesis maintenance to social stigma may be mitigated by WET. Potential benefits to WET recipients may also include partial vision restoration, psychosocial benefits related to identity and social integration, improvements in physical comfort and function, and reduced surgical risk associated with a biologic eye compared to a prosthesis. Perioperative and postoperative risks of WET are expected to be comparable to those of facial transplantation (FT), and may be similarly mitigated by immunosuppressive protocols, adequate psychosocial support, and a thorough selection process for both the recipient and donor. To minimize the risks associated with immunosuppressive medications, the first attempts in human recipients will likely be performed in conjunction with a FT. If first-in-human attempts at combined FT-WET prove successful and the biologic eye survives, this opens the door for further advancement in the field of vision restoration by means of a viable surgical option. This analysis integrates recent innovations in WET research with the existing discourse on the ethics of surgical innovation and offers preliminary guidance to VCA programs considering undertaking WET in human recipients.

There are over 43 million individuals in the world who are blind. As retinal ganglion cells are incapable of regeneration, treatment modalities for this condition are limited. Since first incepted in 1885, whole-eye transplantation (WET) has been proposed as the ultimate cure for blindness. As the field evolves, different aspects of the surgery have been individually explored, including allograft viability, retinal survival, and optic nerve regeneration. Due to the paucity in the WET literature, we aimed to systematically review proposed WET surgical techniques to assess surgical feasibility. Additionally, we hope to identify barriers to future clinical application and potential ethical concerns that could be raised with surgery.

We conducted a systematic review of PubMed, Embase, Cochrane Library, and Scopus from inception to June 10, 2022, to identify articles pertaining to WET. Data collection included model organisms studied, surgical techniques utilized, and postoperative functional outcomes.

Our results yielded 33 articles, including 14 mammalian and 19 cold-blooded models. In studies performing microvascular anastomosis in mammals, 96% of allografts survived after surgery. With nervous coaptation, 82.9% of retinas had positive electroretinogram signals after surgery, indicating functional retinal cells after transplantation. Results on optic nerve function were inconclusive. Ocular-motor functionality was rarely addressed.

Regarding allograft survival, WET appears feasible with no complications to the recipient recorded in previous literature. Functional restoration is potentially achievable with a demonstrated positive retinal survival in live models. Nevertheless, the potential of optic nerve regeneration remains undetermined.

For many retinal diseases, including age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy (DR), the exact pathogenesis is still unclear. Moreover, the currently available therapeutic options are often unsatisfactory. Research designed to remedy this situation heavily relies on experimental animals. However, animal models often do not faithfully reproduce human disease and, currently, there is strong pressure from society to reduce animal research. Overall, this creates a need for improved disease models to understand pathologies and develop treatment options that, at the same time, require fewer or no experimental animals. Here, we review recent advances in the field of in vitro and ex vivo models for AMD, glaucoma, and DR. We highlight the difficulties associated with studies on complex diseases, in which both the initial trigger and the ensuing pathomechanisms are unclear, and then delineate which model systems are optimal for disease modelling. To this end, we present a variety of model systems, ranging from primary cell cultures, over organotypic cultures and whole eye cultures, to animal models. Specific advantages and disadvantages of such models are discussed, with a special focus on their relevance to putative in vivo disease mechanisms. In many cases, a replacement of in vivo research will mean that several different in vitro models are used in conjunction, for instance to analyze and validate causative molecular pathways. Finally, we argue that the analytical decomposition into appropriate cell and tissue model systems will allow making significant progress in our understanding of complex retinal diseases and may furthermore advance the treatment testing.

Vascularized composite allotransplantation of the eye is an appealing, novel method for reconstruction of the nonfunctioning eye. The authors' group has established the first orthotopic model for eye transplantation in the rat. With advancements in immunomodulation strategies together with new therapies in neuroregeneration, parallel development of human surgical protocols is vital for ensuring momentum toward eye transplantation in actual patients.

Cadaveric donor tissue harvest (n = 8) was performed with orbital exenteration, combined open craniotomy, and endonasal approach to ligate the ophthalmic artery with a cuff of paraclival internal carotid artery, for transection of the optic nerve at the optic chiasm and transection of cranial nerves III to VI and the superior ophthalmic vein at the cavernous sinus. Candidate recipient vessels (superficial temporal/internal maxillary/facial artery and superficial temporal/facial vein) were exposed. Vein grafts were required for all anastomoses. Donor tissue was secured in recipient orbits followed by sequential venous and arterial anastomoses and nerve coaptation. Pedicle lengths and calibers were measured. All steps were timed, photographed, video recorded, and critically analyzed after each operative session.

The technical feasibility of cadaveric donor procurement and transplantation to cadaveric recipient was established. Mean measurements included optic nerve length (39 mm) and caliber (5 mm), donor artery length (33 mm) and caliber (3 mm), and superior ophthalmic vein length (15 mm) and caliber (0.5 mm). Recipient superficial temporal, internal maxillary artery, and facial artery calibers were 0.8, 2, and 2 mm, respectively; and superior temporal and facial vein calibers were 0.8 and 2.5 mm, respectively.

This surgical protocol serves as a benchmark for optimization of technique, large-animal model development, and ultimately potentiating the possibility of vision restoration transplantation surgery.

Therapeutic, V.

Vascularized composite allotransplantation represents a potential shift in approaches to reconstruction of complex defects resulting from congenital differences as well as trauma and other acquired pathology. Given the highly specialized function of the eye and its unique anatomical components, vascularized composite allotransplantation of the eye is an appealing method for restoration, replacement, and reconstruction of the nonfunctioning eye. Herein, we describe conventional treatments for eye restoration and their shortcomings as well as recent research and events that have brought eye transplantation closer to a potential clinical reality. In this article, we outline some potential considerations in patient selection, donor facial tissue procurement, eye tissue implantation, surgical procedure, and potential for functional outcomes.

Maintenance of ocular viability is one of the major impediments to successful whole-eye transplantation. This review provides a comprehensive understanding of the current literature to help guide future studies in order to overcome this hurdle. A systematic multistage review of published literature was performed. Three specific questions were addressed: (1) Is recovery of visual function following eye transplantation greater in cold-blooded vertebrates when compared with mammals? (2) Is outer retina function following enucleation and reperfusion improved compared with enucleation alone? (3) Following optic-nerve transection, is there a correlation between retinal ganglion cell (RGC) survival and either time after transection or proximity of the transection to the globe? In a majority of the studies performed in the literature, recovery of visual function can occur after whole-eye transplantation in cold-blooded vertebrates. Following enucleation (and reperfusion), outer retinal function is maintained from 4 to 9 h. RGC survival following optic-nerve transection is inversely related to both the time since transection and the proximity of transection to the globe. Lastly, neurotrophins can increase RGC survival following optic-nerve transection. This review of the literature suggests that the use of a donor eye is feasible for whole-eye transplantation.

To survive anoxia, neural ATP levels have to be defended. Reducing electrical activity, which accounts for 50% or more of neural energy consumption, should be beneficial for anoxic survival. The retina is a hypoxia sensitive part of the central nervous system. Here, we quantify the in vivo retinal light response (electroretinogram; ERG) in three vertebrates that exhibit varying degrees of anoxia tolerance: freshwater turtle (Trachemys scripta), epaulette shark (Hemiscyllium ocellatum) and leopard frog (Rana pipiens). A virtually total suppression of ERG in anoxia, probably resulting in functional blindness, has previously been seen in the extremely anoxia-tolerant crucian carp (Carassius carassius). Surprisingly, the equally anoxia-tolerant turtle, which strongly depresses brain and whole-body metabolism during anoxia, exhibited a relatively modest anoxic reduction in ERG: the combined amplitude of turtle ERG waves was reduced by approximately 50% after 2 h. In contrast, the shark b-wave amplitude practically disappeared after 30 min of severe hypoxia, and the frog b-wave was decreased by approximately 75% after 40 min in anoxia. The specific A(1) adenosine receptor antagonist CPT significantly delayed the suppression of turtle ERG, while the hypoxic shark ERG was unaffected by the non-specific adenosine receptor antagonist aminophylline, suggesting adenosinergic involvement in turtle but not in shark.

ERG components of negative polarity in the light-adapted and in the dark-adapted inner retina are reviewed from a clinical perspective and include consideration of experimental research. Field potentials are inherently complex including summating contributions from specialized neurons as well as from glial elements. This property applies to the PERG, PhNR and to the STR. Experimental research can contribute to identifying the sites/cells of origins i.e. by determining depth profiles and by pharmacological manipulation. Intraretinal microelectrode-studies and pharmacological dissection of light-evoked responses have elucidated the origin of field potentials from the retinal pigment epithelium to the retinal ganglion cells. Thresholds for dark-adapted response components have been compared. Attenuation of the STR by anesthesia was found in cats in vivo when compared to threshold intensities used in isolated eye preparations in vitro, suggestive of depression of inner retinal activity by anesthetics. Evidence has been presented for antidromically elicited retinal responses of negative polarity that resemble the STR and summate with the light-evoked retinal response. This observation supports the notion that negative field potentials and components as recorded in the vitreous and at the cornea receive contributions from retinal ganglion cells. The weight of this contribution appears to vary among species, at least concerning the STR. The ocular negative responses from the inner retina are compared to cortical excitatory mechanisms generating negativity in the baseline of the EEG.

The mouse has become a key animal model for ocular research. This situation reflects the fact that genes implicated in human retinal disorders or in mammalian retinal function may be readily manipulated in the mouse. Visual electrophysiology provides a means to examine retinal function in mutant mice, and stimulation and recording protocols have been developed that allow the activity of many classes of retinal neurons to be examined and which take into account unique features of the mouse retina. Here, we review the mouse visual electrophysiology literature, covering techniques used to record the mouse electroretinogram and visual evoked potential, and how these have been applied to characterize the functional implications of gene mutation or manipulation in the mouse retina.

To study the effect of body temperature on the murine electroretinogram (ERG).

The corneal ERG elicited by a strobe flash from dark-adapted mice was recorded using a saline wick electrode while measuring rectal temperature continuously. The mouse was placed within a cylindrical coil of tubing through which water circulated from a temperature controlled bath. The body temperature of the mouse was changed stepwise between 30 and 37 degrees C.

ERGs of approximately normal configuration were recorded at body temperature ranging between 30 and 37 degrees C. The maximum amplitude of the a- and b-waves varied linearly with temperature. The rate of change of b-wave amplitude was about 100 microV/degree. At 30 degrees C, maximum b-wave amplitude was about 400 microV; at 37 degrees C it was about 1000 microV. A change in body temperature produced a rapid change in ERG amplitude.

The murine ERG is very sensitive to changes in temperature. In order to monitor the ERG accurately over time, continuous recording of body temperature is essential.

The effort to isolate and maintain alive in vitro an intact mammalian eye is rewarded by the full control provided over the arterial input and exclusion of systemic regulatory or compensatory mechanisms. Electrical recording of typical light-evoked field potentials from retina and optic nerve can be complemented by single-cell recording. Thus, light-induced electrical activity reflects the function of the retinal pigment epithelium, of the layers of the retina and of the ganglion cells or their axons. Retinal function in vitro is documented by electrophysiological and morphological methods revealing subtle features of retinal information processing as well as optic nerve signals that approach-at threshold stimulus intensity-the human psychophysical threshold. Such sensitivity of third-order retinal neurons is described for the first time. This well controlled in vitro preparation has been used successfully for biophysical, metabolic and pharmacological studies. Examples are provided that demonstrate the marked sensibility of the rod system to changes in glucose supply. Moreover, histochemical identification of glycogen stores revealed labeling of the second- and third-order neurons subserving the rod system, in addition to labeling of Müller (glial) cells in the cat retina. The glycogen content of the cat retina is augmented by prolonged anesthesia, largely depleted by ischemia after enucleation and enhanced by insulin. Pharmacological experiments using agonists and antagonists of putative retinal neurotransmitters are summarized and outlined using the muscarinic cholinergic agonist QNB as an example. Actions and uptake of the neuromodulator adenosine are presented in detail, including inhibitory effects on physiologically characterized ganglion cells. Neuronal effects of adenosine are distinguished from those resulting from vasodilatation and from glycogenolysis induced by the neuromodulator. To open the blood-retina barrier, a hyperosmotic challenge can be applied transiently. This process is monitored histochemically using FITC-albumin and with electrophysiological parameters. Changes in vitreo-scleral resistance and in the amplitude of the EOG-light peak appear to reflect the open/closed status of the barrier. This overview of the uses of the isolated perfused mammalian eye in retinal research concludes with a discussion of potential implications for clinically relevant topics.

To determine the cellular mechanisms involved in the hypoxia-induced alteration of the retinal pigment epithelium (RPE) potentials and the light-evoked responses of the RPE in chicks. In addition, to determine the mechanisms involved in the recovery of the RPE during the post-hypoxic period.

In vitro preparations of chick retina-RPE-choroid were studied by potassium-selective microelectrodes placed in the subretinal space. In addition, single-barrel microelectrodes were used to obtain intracellular recordings from the RPE cells. The perfusate was bubbled continuously with 95% oxygen and 5% carbon dioxide for the control condition and replaced by 95% nitrogen and 5% carbon dioxide to induce hypoxia.

Hypoxia induced a significant reduction of the trans-tissue potential which was found to result from the depolarization of the apical membrane of the RPE. This depolarization was induced by an increase of subretinal [K+]o. The c-wave was also markedly decreased or abolished during hypoxia. There were two phases of post-hypoxic recovery: an initial small increase in the trans-tissue potential resulting from a basal membrane depolarization followed by an apical membrane hyperpolarization. The trans-tissue potential and the c-wave also were supernormal in two phases during this post-hypoxic period. The c-wave amplitude was temporarily elevated (263.7 +/- 77.4% of pre-hypoxic control) because of the enhanced trans-epithelial c-wave and without a light-evoked decrease in subretinal [K+]o.

The trans-tissue potential and the c-wave were markedly decreased during hypoxia. During the post-hypoxic period, both potential recovered with transient supernormalities in two phases. The results suggested that the hypoxic changes resulted directly from changes of the RPE membranes and indirectly from a change in the subretinal [K+]o but were not mediated by the light-evoked decrease in subretinal [K+]o.

Acute hypoxemia (low PaO2) leads to changes in oxygen consumption and electrical responses of the outer retina of cats, but inner retinal ERG components and ganglion cell responses have been shown to be quite resistant to hypoxemia. The purpose of this study was to determine whether the resistance of the inner retina depends on (1) the stimulus conditions, specifically the degree of light adaptation; and (2) the ability of the photoreceptors to increase glycolysis during hypoxemia. To address these issues, recordings of single ganglion cell action potentials and of the b-wave and scotopic threshold response (STR) of the electroretinogram (ERG) were made from the eyes of anesthetized cats during hypoxemia alone and hypoxemia plus hypoglycemia. Ganglion cells appeared to be equally resistant to hypoxemia at high and low backgrounds (3.3 to 9.7 log equivalent quanta(555 nm)-deg-2(-s)-1), and the STR, recorded with dim stimuli during dark adaptation, when photoreceptor oxygen consumption is most susceptible to hypoxemia, was unchanged until PaO2 was below 30 mm Hg. The amplitude of the b-wave was similarly resistant to hypoxemia when the animal was normoglycemic. During hypoglycemia, however, both the b-wave and the STR became more sensitive to hypoxemia, beginning to change at PaO2s as high as 50 mm Hg when blood glucose was 40-50 mg/dl. It is argued that hypoglycemia limits or prevents the increased glycolytic ATP production that would ordinarily occur when the photoreceptor oxygen supply decreases, and that increased photoreceptor glycolysis is essential in the protection of the retina against mild hypoxemia.

Studies reporting the effect of retinal cooling on the electroretinogram of mammals have, in most cases, made use of an in vitro approach where the temperature of the retina was lowered by reducing the temperature of the bathing media. The purpose of our study was to investigate, in rabbits, the effect of in vivo retinal cooling using an experimental approach never before reported in order to determine if some of the temperature-related ERG effects previously shown could have been, in part, amplified by alterations in the physiological status of the retina due to preparation for in vitro study.

In order to reduce the temperature of the retina, a 20 gauge plastic tubing was coiled around the eye from the limbus to the optic nerve head and glued to the sclera. Cold (15 degrees C) tap water entered the tubing at the limbal extremity and exited (18 degrees C) at the optic nerve extremity. Intraretinal temperature was measured to be within 1 degrees C of that of the circulating water.

Our results indicate that with progressive retinal cooling the a- and b-waves are gradually reduced to 66.9 +/- 17.3 and 90.9 +/- 10.4% of control respectively. The most dramatic temperature induced ERG modifications were observed in the oscillatory potential recordings where the mean summed OP amplitude (OP2 + OP3 + OP4) was reduced to less than 23.9 +/- 13.5% of control with OP2 being the least affected. The peak times of all the ERG components were significantly delayed with cooling.

Of all the ERG components examined, the OPs were those most severely affected by our manipulation. We believe that our results constitute further evidence in support of the concept that the OPs are more sensitive to retinal disturbance than the b-wave.

The effect of 4 weeks streptozotocin-induced diabetes on ocular vascular resistance responses to noradrenalin (NA), adrenalin (A), phenylephrine (PHE), isoproterenol (ISOP), prostaglandin F2 alpha (PGF2 alpha). 5-hydroxytryptamine (5-HT) and angiotensin II (ANG II), was determined using a newly-developed, isolated, arterially-perfused rat eye preparation, by comparing responses from control and diabetic eyes. After extensive preliminary experiments to establish optimum parameters, the ophthalmic artery of enucleated control and diabetic rat eyes was cannulated and the retinal and uveal vasculature perfused at a constant flow with Na(+)-Krebs solution after streptozotocin-induced diabetes had been established for 4 weeks. The eyes were maintained in an environment-controlled organ bath. Perfusion pressure was monitored as increasing log M concentrations of agonists were added to the perfusate. Total ocular resistance could be calculated from knowledge of flow and pressure. In control eyes, NA, A, PHE, PGF2 alpha, and 5-HT all produced dose-dependent increases in total vascular resistance, with the following order of potency: NA = A > 5-HT > PHE = PGF2 alpha at 10(-4) M. The ocular circulation was not sensitive to isoproterenol and angiotensin II. In diabetic eyes responses to NA, A, PGF2 alpha and 5-HT were altered. Diabetic responses to NA and A had lower thresholds with larger resistance increases at low concentrations. However, the rate of increase in resistance with concentration was more gradual in diabetic eyes so that at 10(-4) M control responses were larger. Diabetic resistance responses to PGF2 alpha had the same threshold as in control eyes, but were greater in magnitude with an earlier peak at 10(-4) M. In contrast diabetic resistance responses to 5-HT were reduced, peaked at a lower resistance at 10(-4) M, but had the same threshold as those in the control eye. Basal vascular resistances in control: 3.14 +/- 0.32 mmHg min microliter-1 (n = 28), and diabetic eyes: 3.44 +/- 0.19, mmHg min microliter-1 (n = 36), were not significantly different. Vasoactivity in the early diabetic eye is disturbed with the effective balance between different agonists altered in favour of catecholamines at physiological concentrations. This may be related to the early changes in blood flow and oxygen distribution already reported in the rat eye, as well as changes to autonomic function. The isolated perfused rat eye is a valuable technique for investigating such vascular reactivity in animal models of retinal disease.

Carteolol, a nonselective beta-antagonist, was administered intra-arterially in perfused cat eyes. Carteolol increased both photopic and scotopic electroretinogram b-wave amplitude dose-dependently and reversibly, but carteolol failed to induce significant changes in the flow rate of perfusate. This study suggests that carteolol may increase selectively the retinal perfusion flow rate, though it did not reflect the total perfusion flow, or carteolol may have an interaction with retinal beta-adrenergic receptors related to the origin of the b-wave. These ideas are supported by carteolol's intrinsic sympathomimetic activity and effects on endothelium of vessels.

Various concentrations of three dopamine antagonists (fluphenazine, haloperidol and sulpiride) with different affinities to the receptor subtypes were applied in order to test their effect on responses from outer (isolated PIII-component of the ERG), middle (b-wave) and inner (optic nerve response, ONR) retinal layers, recorded from dark-adapted, isolated, arterially-perfused cat eyes. In the range of concentrations tested (e.g. from 4 nmol ml-1 to 16 mumols ml-1 for haloperidol) none of the drugs added to the perfusion medium had any effect on either slow or fast PIII-amplitude or on the temporal characteristics of the response. All dopamine antagonists increased the rod b-wave with a comparable molar efficacy. The rod b-wave latency and implicit time showed no drug-induced changes. In the rod ONR the D-1 antagonist fluphenazine increased the fast transient on-component while it simultaneously strongly decreased the off-component. IN contrast, concentrations of the D-2 antagonists sulpiride that had a comparable effect on the ONRs fast transient on-component influenced neither the slow transient on-component nor the off-component. These findings indicate that the D-1 and D-1 receptors play different roles in the transmission of rod signals at the border of middle and inner retina.

The process of light adaptation in cat horizontal cells was studied by means of intracellular recordings in the optically intact, in situ eye. Response vs intensity profiles were measured using increment- as well as decrement-flashes of "white light" on steady backgrounds. The effects of background illumination in the mesopic and photopic range on both purely rod-driven horizontal cells and mixed rod and cone input horizontal cells were investigated. Increasing the background illumination for mixed-input horizontal cells strongly reduced the contribution from the rod system. The rod aftereffect in the responses to high intensity flashes is totally suppressed at higher background levels. Light adaptation resulted in a sustained hyperpolarization without a substantial effect on the total response range of the response vs intensity curve. At higher background intensities more of the response range is made up of depolarizing responses to decrements of light. Increasing the background illumination also shifted the operating curve to higher intensities. Increment threshold functions, measured with a 3.9 deg diameter test spot on a large background (8.8 deg diameter) showed a linear relation between log threshold intensity and log background intensity with a slope of, on average, 0.64. The response vs intensity curve for the rod horizontal cell typically spanned a narrower intensity range and was displaced toward lower intensities as compared to that for the mixed input horizontal cells. Background illumination greatly reduced the total response range for rod horizontal cells. Increment threshold curves for rod horizontal cells clearly indicated loss in sensitivity due to response compression.

Full-field electroretinograms (ERGs) were recorded from isolated cat eyes perfused through the ophthalmociliary artery with the cGMP-PDE inhibitor, 3-isobutylmethylxanthine (IBMX). Under dark-adapted conditions perfusion with IBMX resulted in reduced ERG b-wave amplitudes at low stimulus luminances and supernormal b-wave amplitudes at high stimulus luminances with reduced b-wave sensitivity; b-wave implicit times were more delayed at low than at high stimulus luminances. Presentation of a steady white background or high calcium fully reversed the supernormal amplitudes and partially reversed the delayed implicit times produced by IBMX. Rod ERG b-wave sensitivity, reduced with IBMX alone, was partially reversed with calcium but further reduced with background light. Perfusion with the cation channel blocker, L-cis-diltiazem, also reversed the supernormal amplitudes produced by IBMX but had no effect on implicit times or b-wave sensitivity. Possible mechanisms of action of these antagonists and clinical implications of these findings are considered.

We studied the effects of single doses of different dopamine agonists and antagonists on the electroretinogram of a group of healthy volunteers. The results demonstrated significant b-wave amplitude changes after drug administration, suggesting that electroretinograms can be employed to evaluate the effects on retinal dopaminergic activity induced by psychotropic drugs and that the study of the electroretinographic effects of psychopharmacologic agents can provide new insights into the relationship between retinal dopaminergic mechanisms and the electroretinogram b-wave origin.

The spatial properties of horizontal cells in the cat retina have been studied by means of intracellular recordings in the optically intact, in situ, eye. The spread of potentials in the horizontal cell layer and the spatial summation properties have been investigated using "white light" stimuli in several different configurations. Area-response curves were measured with flashing spots carefully centered on the receptive field. The lateral spread of potentials was studied using long, narrow slits of light and circular spots of different sizes at different positions in the receptive field. Two-dimensional receptive field profiles showed that the receptive field structure was, both on a large scale and at higher resolutions, relatively homogeneous and isotropic. The size and shape of the receptive fields have been characterized by applying a simplified version of the model proposed by Naka and Rushton (1967) of electrical coupling in the horizontal cells layer in fish. Results show that the model describes the variation of response amplitude reasonably well both as a function of spot size and spot or slit position. However, deviations were found for area-response curves measured at higher light intensities and for receptive field profiles measured with relatively small spots. Furthermore, the estimated length constants resulting from the different experiments were not in agreement. Different model parameters were needed in order to describe the spatial summation properties and the lateral spread of potentials respectively. It is concluded that passive electrical spread and linear summation of potentials cannot account for the observed spatial properties.

The purpose of this study was to examine if there is an ERG component (alpha-wave, b-wave, oscillatory potentials) which can be used to account for the intraretinal processing time. To address this issue, ERGs and LGN unit responses were recorded simultaneously from anesthetized and paralyzed rabbits. A gradual decrease in the intensity of the light stimulus yields a progressive increase in the latency of the LGN unit response. A similar, highly correlated (r = 0.91 +/- 013), latency shift was also noted for one oscillatory potential (OP2). In comparison, correlation coefficients of r = 0.63 +/- 0.27 and r = 0.70 +/- 0.29 were obtained for the alpha- and b-wave respectively. Furthermore, in 77.6% of the cells examined, OP2 preceded LGN unit activity while the b-wave preceded LGN activity in only 17.8% of the cases. Our results suggest that, of all the retinal potentials considered, the peak time of OP2 better reflects the primary visual processes and the intraretinal time taken for visual processing.

We have attempted to determine in this study whether the arterial administration of vincristine produces in cat the functional defects associated with hereditary and vincristine-induced night blindness in man. Using the isolated perfused cat eye, it has been possible to mimic some of the essential features of human night blindness, namely, retention of normal rhodopsin chemistry and normal photoreceptor activity, with marked suppression of the ERG b-wave. In addition, we find that vincristine produces an early, rapid fall in the c-wave, a potential that arises largely in the pigment epithelium. Ultrastructurally, it appears that many classes of retinal neuron are affected by the drug, but the principal changes in cytoarchitecture are seen in the photoreceptors. Except for the outer segments, paracrystalline deposits were found in all parts of the visual cell. The disruption of the normal microtubular organization of these cells suggests that the drug interferes with the functional integrity of the transport system by which synaptic activity is maintained. By reducing the efficacy of communication between visual cells and their second-order neurons, the electrical responses of post-synaptic elements is degraded. The route by which vinca alkaloids reach the neural retina is still uncertain, but our preliminary studies using HRP indicate that the relatively high concentration of vincristine used in this study may be responsible for compromising the blood-retinal barrier.

Arterial perfusion of the isolated cat eye with a perfusate augmented with a 20% (wt/vol) emulsion of the oxygen carrying-fluorocarbon, FC43, produces a much larger b-wave in the electroretinogram than use of the same perfusate lacking FC43. The c-wave is either unchanged or slightly reduced by use of this solution. Halving the oxygen content of the FC43-augmented perfusate also reversibly reduces the b-wave. The use of perfusates with and without FC43 but containing similar amounts of oxygen produces b-waves of similar amplitude. The large b-wave recorded during use of a well-oxygenated, FC43-augmented perfusate suggests that the use of perfusates containing less oxygen produces retinal hypoxia. The b-wave recorded in vivo is considerably smaller than that recorded from the isolated eye during perfusion with a well-oxygenated, FC43-augmented perfusate, but the waveforms are very similar. In particular, the ratios of the b-wave to a-wave are almost identical. The larger ERG recorded in vitro is a result of increasing the shunt resistance to ground upon enucleation. Histological examination of retinas perfused for two hours with the FC43-augmented and standard perfusate reveal no clear signs of tissue hypoxia in either retina. However, even two hours of complete ischemia produces limited inner retinal deterioration and virtually no outer retinal damage, suggesting that the histological state of the retina may not be a reliable indicator of its physiological state. Despite the improved electrophysiological state of the retina produced by increasing its oxygen supply, the survival time of the isolated eye was not increased.

The effects of the beta-adrenergic agonist nylidrin and the beta 2-adrenergic agonist clenbuterol on electroretinogram and optic nerve response were studied in the isolated and arterially perfused, light-adapted cat eye. Two cone mechanisms, short wavelength-sensitive and long wavelength-sensitive, were functionally separated by means of intense yellow adaptation. A reversible increase in b-wave amplitude in response to nylidrin or clenbuterol was observed for the cone systems. Both drugs also caused a reversible alteration in configuration of the optic nerve response, mainly a depression of the late components related in time to the changes in the electroretinogram. These observations suggest that beta-adrenergic mechanisms are involved in cone systems. The greater increase in b-wave amplitude on 558-nm stimulation and preliminary evidence for greater increase in sensitivity observed in the V-log I function compared with 439 nm further suggest that the short and long wavelength cone systems are affected differently by beta-adrenergic agonists.

Exclusion of extraocular variables facilitates neuropharmacological retinal experiments. Isolated tissue or organs such as arterially perfused mammalian eyes provide this advantage. Electrophysiology comprising field potentials (ERG and optic nerve response) and recordings from single cells is used to assess various levels of retinal information processing. Several directions of research using the perfused cat eye are indicated. Pharmacological studies are presented in a description of an approach to identify adrenergic mechanisms in the cat retina. Studies on effects of changes in physico-chemical parameters and on drug actions reveal a number of parallels to problems relevant in clinical eye research.

Amplitude and implicit-times of responses representing different retinal layers (PIII, b-wave and optic-nerve response) were measured in electrophysiological recordings from isolated, arterially perfused cat eyes. The amplitude of these potentials was found to saturate at lower stimulus irradiances than the implicit-time in dark-adapted eyes. Light adaptation had a strong effect on the amplitudes, whereas the implicit-times were altered only slightly. Similar results were obtained in double-flash experiments. The injection of phosphodiesterase inhibiting drugs had different effects on amplitude, latency and implicit-time.

In the experiments presented here we have used the isolated perfused eye technique to investigate the distribution of ERG potentials on the sclera over local retinal lesions induced by xenon arc photocoagulation. Three lesioned dog eyes were examined. In two cases the lesion was allowed 28 days to stabilize, whilst in the third the experiment was performed two hours after treatment. Immediately after enucleation, the eyes were placed in a perfusion system capable of maintaining retinal function for many hours. The scleral ERG profile was then measured on the scleral surface over both treated and untreated areas of retina in the same eye. It was found that the ERG amplitudes were consistently lower over the treated areas of retina when compared to the untreated side of the same eye. More surprising was the discovery that the ERG distribution on the untreated side was significantly different to that observed in control eyes. In particular it was found that the limbal areas of sclera on the untreated side now had significant ERG amplitudes present, whereas the control eyes had close to zero amplitude in this region. It is concluded that the induced retinal lesions caused a widespread redistribution of ERG potentials around the entire surface of the globe. The potential applications of these results in the assessment of local retinal function is discussed. A schematic model is presented for the distribution of ERG potentials on the surface of control and lesioned eyes.

The effect of various concentrations of the dopamine antagonist fluphenazine on ocular field potentials, recorded under scotopic conditions from isolated, arterially perfused cat eyes, was studied. Responses from outer (isolated PIII-component of the electroretinogram, ERG), middle (b-wave), and inner (optic nerve response, ONR) retinal layers were separated. Neither the fast or slow PIII-amplitude nor the temporal characteristics of the response were influenced by any of the drug concentrations tested. In contrast, fluphenazine reversibly increased the rod b-wave amplitude over a large range of concentrations. Only very high drug concentrations led to an irreversible loss of the b-wave. In the ONR the initial transient on-response increased during drug injection, whereas the sustained on-response and off-response decreased. In summary, the dopamine antagonist fluphenazine affects mainly the signal processing of the rod pathway in the inner retinal layers, while responses from outer retinal layers are not influenced. On- and off-responses of the ONR are affected differently.

Buphenine (nylidrin), a beta-adrenergic agonist, is used therapeutically for its vasodilating effect on the peripheral circulation and possibly on the cerebral circulation as well. In spite of its ophthalmic usage in degenerative retinal disease and glaucoma, buphenine's ocular effects and their mechanisms are not sufficiently established. Consequently, we studied the action of 4.5-120 microM buphenine in isolated, arterially perfused cat eyes, and then used light-evoked electrical signals, perfusion flow rates and the diameters of the retinal vessels as parameters for the drug's effect. Our findings showed that buphenine induced a marked, dose-related, reversible increase in the amplitude of the electroretinogram (ERG) b-wave, a decrease in the c-wave, but no significant changes in the standing potential and light peak of the DC-ERG. The compound action potential of the optic nerve revealed dose-dependent and reversible changes in configuration. However, the flow of perfusate was not affected by the drug, and the diameter of retinal vessels did not change significantly. Our studies suggest that the interaction of buphenine with retinal adrenergic receptors is not related to the vasculature present but to elements involved in information processing. It is likely that these receptors are linked to neurons, since the beta-agonist affected the ERG b-waves as well as the compound action potential of the optic nerve.

Full-field electroretinograms (ERGs) were recorded from isolated cat eyes perfused through the ophthalmociliary artery with isobutylmethylxanthine (IBMX), an inhibitor of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase. Low doses of IBMX (0.1-0.3 mM) produced decreased rod ERG amplitudes at low stimulus luminances and increased rod ERG amplitudes at high stimulus luminances. A high dose of IBMX (1.0 mM) initially produced the same effect as the low doses and then led to decreased rod ERG amplitudes at all stimulus luminances. Perfusion with IBMX also resulted in elevations in the semi-saturation luminance (sigma), delayed rod a-wave latencies, delayed rod a-wave and b-wave implicit times, and reduced rod a-wave slopes. Eyes perfused with IBMX (1.0 mM) were also found to have elevated levels of retinal cyclic GMP. These effects of IBMX on the rod ERG are considered in the context of previously described ERGs in selected cases of human retinal degeneration.

The isolated arterially perfused eye preparation has become a valuable tool in the investigation of ocular function. Normal retinal function can be maintained for several hours with the measurement of the gross electroretinogram (ERG) serving as a useful monitor of the electrophysiological condition of the preparation. This paper deals with the manner in which the amplitude of the ERG is affected by the electrode recording locations and describes the characteristic distribution of potentials on the surface of the isolated arterially perfused dog eye. The ERG is recorded between a fixed corneal contact lens electrode and a movable electrode on the uppermost surface of the sclera. The scleral electrode is moved in small increments parallel to the optic axis and lowered onto the uppermost surface of the sclera on a line from the corneal limbus to the optic nerve. The resulting ERG profile is characterised by minimal b-wave amplitude at the corneal limbus and little growth until a point 5 or 6 mm from the corneal limbus is reached, followed by a region of rapidly increasing amplitude up to a maximum at the point where the optic nerve exits from the globe. There follows a region of large but stable signal amplitude along the optic nerve. The ERG profile is largely unaffected by a reorientation of the globe about the optic axis, demonstrating that the potential distribution on the surface of the globe may be described in terms of points equidistant from the limbus being isopotential.(ABSTRACT TRUNCATED AT 250 WORDS)

The off-response of dark adapted cat ganglion cells shows a tripartite response-intensity function in the optic nerve response (ONR) as well as in extracellular recordings of single cells. While responses increase when stimuli of low or high intensities are increased, the rod driven off-response shows a strong decrease (dip) for intermediate intensities before the cone part of the function starts to rise. In contrast, on-responses increase monotonically or stay at a maximum. The dip in the response-intensity function of the off-response has a constant shape with test lights of increasing as well as of decreasing irradiance. The action spectrum of the descending part of the function peaks at 500 nm, indicating that a rod driven mechanism is responsible for the response reduction reflected by the dip. Changing the stimulus diameter from 24 deg to a 1 deg test field centred on a ganglion cell's receptive field has minimal effect on the response reduction. This points to a temporal rather than a spatial mechanism being responsible for the dip.

The light peak (LP) is a slow increase in the standing potential of the eye and has been attributed to a depolarization of the basal membrane of the retinal pigment epithelium (RPE). We tested effects of different external calcium ion concentrations ([Ca++]0) on the LP in the arterially perfused cat eye. An increase in Ca++ activity by 3.2 mM depressed the LP by 85-90% of its amplitude under control conditions. In contrast the other components of the light response (c-wave, fast oscillation, and second c-wave) did not change. The small increase in [Ca++]0 had insignificant effects on the amplitude-intensity plots of the a- and b-waves of the electroretinogram. Elevated [Ca++]0 had its effect only during onset of light and not after the LP was once initiated. The effects were reversible. Since there is a very high [Ca++] concentration in the RPE cells and the pigment granulas may release Ca++ during illumination (Hess 1975), our results suggest that this ion plays a role during the initiation of the LP.

Changing flow rate (F) in arterially perfused cat eyes concomitantly alters the supply of O2 and greatly affects electrical signals: increasing F increases the standing potential (SP) and decreasing F induces the opposite. Corresponding effects on c-wave are more variable. To separate changes in supply of O2 from changes in F we performed two types of experiments: (1) changing supply of O2 while keeping F constant; this was done by switching to a perfusate of different PO2: and (2) changing F while keeping the supply of O2 constant; this was done by switching to hypoxic or hyperoxic perfusates and adjusting F to keep the b-wave amplitude constant. Results (1): Decrease in PO2 left the SP essentially unaffected, but increased the c-wave. Increase in PO2 tended to increase the SP and to decrease the c-wave. Results (2): Isolated decrease in F did not affect the SP consistently and left the c-wave unchanged. Isolated increase in F often increased the SP, but also left the c-wave unchanged. Covariation between SP and amplitude to the c-wave was absent during hyperoxia.

Electroretinographic (ERG) and extracellular potassium activity measurements were carried out in superfused eyecup preparations of several amphibians. Light-evoked changes in extracellular K+ activity were characterized on the bases of depth profile analysis and latency measurements and through the application of pharmacological agents that have selective actions on the retinal network. Three different extracellular potassium modulations evoked at light onset were identified and characterized according to their phenomenological and pharmacological properties. These modulations include two separable sources of light-evoked increases in extracellular K+: (a) a proximal source that is largely post-bipolar in origin, and (b) a distal source that is primarily or exclusively of depolarizing bipolar cell origin. The pharmacological properties of the distal extracellular potassium increase closely parallel those of the b-wave. A distal light-evoked decrease in extracellular potassium appears to be associated with the slow PIII potential, based on a combination of simultaneous intracellular Müller cell recordings and extracellular ERG and potassium activity measurements before and during pharmacological isolation of the photoreceptor responses. The extracellular potassium activity increases are discussed with respect to the Müller cell theory of b-wave generation.

The arterially perfused eyecup of the Eastern gray squirrel, Sciurus carolinensis, has been developed for the study of mammalian retinal neurons by the technique of intracellular recording. Particular emphasis is placed in this report on the development of a convenient perfusion chamber. The choice of this animal and the reason for choosing the arterially perfused open eyecup are also discussed. Intracellular recordings were made from all major types of neurons in the squirrel retina. Data are presented here from ganglion cells and bipolar cells.

The major goal of this investigation was to describe and characterize response components of graded retinal responses in man and cat as a function of check size of the spatial stimulus. The arterially perfused cat eye enabled us to record the pattern reversal electroretinogram (PERG) and simultaneously summed activity of retinal ganglion cells by recording a compound action potential of the optic nerve (ONR). Checkerboard pattern reversal stimuli were presented at a low temporal frequency (recording 2 reversals in each trace), while a number of parameters such as check size, modulation depth and field size have been varied in order to elucidate similarities in response characteristics between the PERG and ONR. The striking similarity area dependence of the PERG and ONR points to ganglion cell activity as well as summation within the latter's receptive field center, reflected in the PERG. A fractionation was obtained of the PERG into two separate components generated by specific changes in spatial contrast and by luminance, respectively. The spatial response component of the PERG recorded in man from the enucleated cat eye closely resembled and consisted of a negative deflection. This component is preceded by a positive deflection sensitive to luminance changes and prominent upon peripheral retinal stimulation. It is anticipated that mild to moderate abnormalities of the ganglion cell activity will be first reflected in the negative contrast component upon central retinal stimulation.

Hypercapnia has been shown to depress the ERG b-wave and also the light peak in vivo. To test if acidosis induced by elevation pCO2 affects the retina differently from that induced by HCl we used bicarbonate buffered and HEPES buffered perfusates, respectively, in isolated perfused feline eyes. Any decrease in pH from 7.4 to 7.1-7.0 depressed the b-wave and enhanced the flow rate of a perfusate. The light peak, in contrast, was depressed only be largely elevated pCO2. Preretinal measurements of pH in the perfused eye confirmed retinal acidosis. All effects were reversible.