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Abstract

Purpose: The isolated and superfused retina from vertebrates is routinely used for neurophysiological measurements of basic retinal signal generation and transduction. Such an ex vivo isolated neural network moreover represents a useful model system for the investigation of drugs and toxins and may also be helpful to elucidate molecular mechanisms of CNS diseases. The present overview aims to bring observations to the reader’s attention, which, in part, have been made more than 50 years ago and were sparsely followed up upon in subsequent research, but may support the idea that the isolated bovine retina represents a useful system to investigate the physiology and pathophysiology of energy supply to neuronal tissue.

Material and methods: Parallel recording of ERGs and pyridine nucleotide oxidation in the isolated and superfused vertebrate retina. The review will focus on topics, which discuss the connection between retinal electrophysiology and underlying energy metabolism.

Results: Previous and present reports about (i) transretinal signaling cascades, (ii) the involvement of the pharmacoresistant Cav2.3 / R-type calcium channel in transretinal signaling and (iii) data about the retinal oxygen demands and concentration in different layers are collected, which elucidate that the retina may be used as a cerebral surrogate model in different research areas. Retinal tolerance to ischemia is several times larger than the tolerance in the remaining brain.

Conclusions: The retinal energy supply through retinal vessels is regulated and controlled by neurovascular coupling. Classical retinal recording techniques and special retinal abilities in electrical-vascular coupling will be set in perspective with novel recording techniques, currently brought into clinical application for the detection of impaired neurovascular coupling after aneurysmal subarachnoid hemorrhage in the brain. The present review elucidates that important pathophysiological aspects related to the upregulation of pharmacoresistant Cav2.3 / R-type calcium channels during SAH may be investigated in the isolated vertebrate retina.

Key words

Murine ERG, isolated vertebrate retina, energy demand under light stimuli, neurovascular coupling

Ischemic tolerance in the vertebrate retina

The oxygen consumption of the vertebrate retina on a per gram basis has been described as higher than that of the brain [1,2]. Oxygen cannot be “stored” in tissue so that a constant and adequate supply must be guaranteed to preserve function. Metabolic dysfunction regarding to impaired vascular supply is directly reflected by retinal oxygen saturation which can be detected noninvasively by dual wavelength fundus photography [3]. Normally, the oxygen saturation in retinal vessels differs along vascular segments and is higher in the macular region than retinal periphery [4]. Many retinal diseases are caused by a dysfunction of the vascular network. Interestingly, the venous oxygen saturation in diabetic retinopathy was higher in venules draining the macular surroundings than retinal periphery that reflect specific metabolic conditions [5, 6].

Since a relatively unobstructed light path to the photoreceptors is needed within the retina, the extent of vascularization within the retina itself is limited and varies in different regions of the same retina [7]. In the rat, under normal physiological conditions, intraretinal oxygen profiles show the highest oxygen tension (more than 40 mmHg) close to the deep choroid region, and the lowest oxygen tension (5-10 mmHg) within the retinal network. An intermediate elevation (15 – 20 mmHg) can be found close to the deep retinal capillary layer [8]. In highly energy consuming regions, the pO₂ may be demonstrated to be low, as it is around inner segments, or high, as it would be around ganglion cells that happen to be near a blood vessel. We believe, that additional vascularization is helpful for a better oxygen supply, as it is realized in mouse and man.

Although the vertebrate retina preserves a high metabolic rate, its tolerance to acute ischemia is several times larger than in the remaining brain [9], which may in part be related to a difference in glutamate release between retina and cerebral cortex following ischemia [10]. Furthermore, the activation of ATP-sensitive potassium channels in the retina plays an important role in the mechanism of preconditioning by enhancing the resistance of the retina against a severe ischemic insult [11]. In addition, the reprogramming of toll-like receptor 4 by brief ischemia or lipopolysaccharides (LPS) also contributes to better tolerance in retinal ischemia by microglia activation [12]. The preventive effects of LPS preconditioning revealed cell type-specificity of retinal cells. There was a complete rescue of ganglion cells, partial rescue of bipolar and photoreceptor cells or no rescue of amacrine cells, respectively. In healthy subjects, the difference in ischemia tolerance between the eye and the brain was investigated and proven by quantifying the time for visual alterations, peripheral light loss and blackout during and after self-induced retinal ischemia [13].

Parallel quantification of light-evoked signal transduction and oxygen consumption

Besides oxygen consumption, electrical responses are routinely recorded non-invasively from humans as ECG or EEG. Less well known is the recording of ERG-responses and oxygen consumption in parallel, which can be performed ex vivo in the isolated and superfused retina.

Ex vivo ERG recording and determination of pyridine nucleotide oxidation

Firstly in 1965, simultaneous respiratory and electrical responses to light stimulation were reported for the isolated and superfused frog retina, a poikilotherm organism [14]. While the isolated electrical recording from the ex vivo mammalian retina still had been successfully performed earlier [15], the use of absorption spectroscopy for measuring the energy status in parallel was indirectly introduced by Sickel [14]. The parallel recording of absorption changes for the oxidized versus reduced nicotinamide-adenine-dinucleotide had been developed by Chance and Williams [16]  and was combined with ERG recordings by Sickel’s systematic investigations in Boston and Cologne. He used frog retinas, which were isolated under dim red illumination and placed on a black plastic support separating two halves of the recording chamber. The nutrient solution perfused both halves of the chamber successively. Silver ring electrodes in both compartments served to record the ERG from the bathing fluid [14], a setup, which is still in use today in a modified form (Figure 1 and 2).

Figure 1. Recording chamber for the isolated and superfused retina [74].

1. Frontal view of the inner holder for the recording chamber. The retina is placed on a nylon mesh in the central hole of the holder (yellow circle), where it is held in place by an O-ring. The inner holder is then sealed by gaskets and glass plates and secured by the outer part of the recording chamber.  To prevent formation of air bubbles in the inner holder and achieve complete superfusion of the retina from both sides, nutrient solution is supplied from the bottom, flows to the rear part of the chamber and reaches the other side through a small opening above the central hole (blue arrows). Electrical contact is provided by concentric Ag/AgCl electrodes located in the front and rear side of the inner holder (anode and cathode) and on the retinal plane between anode and cathode (reference electrode).
2.  3D illustration of the recording chamber when completely assembled.

During the initial experiments in the 1960s, the retina was exposed to both an ultraviolet-analyzing and a visible-stimulating (and adapting) light beam. Single light flashes of one to several seconds or trains of 1-second stimuli with identical intervals of darkness were applied every 3 minutes. A 6 second light stimulus provoked a classical ERG response including a little rest of the visible negative deflecting a-wave and a prominent b-wave response. Concomitantly, the absorption of the UV beam decreased at the wave length of 350 nm (absorption maximum for NADH is at 340 nm). The reduction of absorption was even larger, when the light stimuli were given as a flickering light, which increased the energy demand. At that time, analogous absorption changes had been observed during induced physiological activity both in magnitude and direction in skeletal muscle [17]. This had been interpreted as oxidation of NADH via the redox partner from the mitochondrial electron transport chain with the resulting electron flow from the reduced NADH + H⁺ to oxygen. The interpretation that the absorption changes at 340 / 350 nm represent the redox status of NAD/NADH + H⁺ linked to metabolic reactions was supported by independent measurements of radiolabeled ¹⁴CO₂ released after feeding radiolabeled glucose. Under flickering light, the release of ¹⁴CO₂  increases, while the rate is reduced under longer lasting continuous light and increased during the consecutive dark adaptation period [18].

A normal metabolism based on a sufficient glucose (and oxygen)  supply was shown by electroretinography to be crucial for proper transretinal signaling [19]. The b-wave amplitude as a measure for transretinal signaling was dependent on a normoglycemic glucose supply of 5 mmol/l. Under glucose-free conditions, the b-wave was lost, while the a-wave, indicative for excitation generation, could still be recorded. Reperfusion of glucose reestablished the b-wave leading to a full ERG [19].

Superfusion of elevated glucose concentrations (15 mmol/l) initially decreased the b-wave amplitude. When the hyperglycemic concentration was reduced to zero, the b-wave amplitude increased after a delay of about 15 minutes. In parallel, the oxidation of NADH was accelerated (recorded as a decrease of UV-absorption at 350 nm [Figure 3 in Sickel, 1968] [19]. In summary, these early investigations of the light effect on retinal energy metabolism lead to 3 conclusions [20]: (i) A depression of oxygen uptake or CO₂-output for the duration of the light exposure. (ii) Transitory increases of the energy metabolism during repetitive changes of light intensity, increasing or decreasing (flickering light). (iii) An oxygen debt accruing from prolonged quantum catch. Obviously, the retina represents a sensory system with high energy demand. Repetitive light intensity changes can be easily used to increase the energy metabolism.

Quantification of required oxygen and effects of electron transport inhibitors and uncouplers

Interestingly, under dark adaptation conditions a quantitative estimate was made for the oxygen needed during neural processing of a just saturating brief light flash, which requires approximately 300 pmoles of oxygen per mg dry weight of retina. It corresponds to an amount of energy several million-fold larger than that amount of the quanta absorbed with a single flash [20]. In 1994 it was reported that these early parallel recordings of electric field potential changes, and the absorption changes of the pyridine nucleotides may not have been taken up by the scientific readership, so that new data were measured and published carefully extending and proving the correlation between ERG-parameters and optical response at 350 nm [21].

A more detailed analysis followed and included the perfusion of electron transport inhibitors and uncouplers of the mitochondrial proton gradient aside from the effects of oxygen and glucose withdrawal [22]. Different energetic states could be demonstrated under the conditions selected. Superfusing the isolated frog retina in the dark with a medium containing sufficient oxygen (saturated) and glucose (5 mmol/l) led to the resting state 4, characterized by a low respiration rate and a low amount of adenosine diphosphate (ADP). The starved state 2 could be simulated by the withdrawal of glucose from the medium leading to an increase in overall transmission at 350 nm, which represents a slow transition of the NADH/NAD ratio towards the oxidized form [22]. Disturbances in the mitochondrial energy metabolism may be a major risk factor during neurodegeneration. The authors conclude that future research will help to elucidate the underlying mechanisms leading to retinal neurodegeneration as the age-related macular disease.

Constant and flickering light affect retinal metabolism differently. Under constant light, as opposed to darkness, retinal metabolism has been reported to be reduced (for an overview see Wang et al. 1997 [23]). The effect of light on retinal metabolism in the inner and outer retina is different. Steady and / or flicker light have the same metabolic effect on photoreceptors as long as the time averaged illumination is the same and it is only in the inner retina where flicker has a bigger effect on metabolism [24].

In contrast, flickering light stimulates glucose consumption not only in the amphibian but also in the primate retina, and increases retinal blood flow [25,26] and lactate formation [2]. Besides, catabolic vascularization and metabolism vary between species, for instance, rabbits are not good models for the inner retina of higher animals, because their metabolism is entirely glycolytic, and while the same principles apply to photoreceptor metabolism in poikilotherms and homeotherms, there are substantial quantitative differences.

The importance of retinal glucose supply was reconfirmed more recently in mice and compared within mitochondria from different mouse retina-mutants. Also in mice, similar as in frogs, inhibitors of the mitochondrial electron transport chain as well as uncouplers of the mitochondrial proton gradient changed the oxygen consumption rate confirming the high vulnerability of photoreceptors to altered energy homeostasis in mice [27].

Neurovascular coupling and the use of the vertebrate retina as a model system

As an extension of the brain, the eye contains a highly organized neuronal network which may be used for noninvasive investigation and early diagnosis of CNS disorders such as stroke [28], multiple sclerosis [29], Parkinson disease [30] and Alzheimer disease [31]. The vertebrate retina represents also a valuable model to study signaling from the neuronal network to the microvascular system, which is responsible for the energy supply via the blood circulation. The retina contains neurons, which are well organized in a network with reciprocal coupling for signal transduction at extreme different light intensities (see chapter 3.3 for more details). On the other hand, the isolated murine retina contains retinal arterioles and venules with similar features as small cerebral blood vessels with auto-regulative function. Even more so, retinal microvascular changes may reflect similar pathophysiological processes as in the cerebral microvasculature [32]. Indeed, retinal vessel analysis in humans is technically feasible in patients with subarachnoid hemorrhage and can detect fluctuations in vessel diameter and its autoregulatory characteristics [33].

Pathophysiology of subarachnoid hemorrhage

Recent reviews have summarized the present understanding of the pathophysiology of aneurysmal subarachnoid hemorrhage (SAH) quite in detail [34,35]. In most patients, the reason for spontaneous SAH arises from cerebral aneurysm rupture [36]. The pathophysiological processes following SAH may cause severe impairment for the survivors with significant mortality and morbidity.

Within the course of SAH two phases are distinguished: within the first three days the initial pathophysiological changes are summarized as “early brain injury” (EBI). Angiographic vasospasm associated with delayed cerebral ischemia (DCI) can occur in the subsequent phase (classically day 4-14) causing severe neurological restrictions to the preinjured brain. The underlying mechanisms are multifactorial and not yet fully understood, one major mechanism includes microvascular dysfunction. Up to 70% of all arterioles are affected during microvascular spasms. They are thought to originate from exposure to blood components, particularly oxygenated hemoglobin and partially by blood degradation products such as bilirubin and its oxidation endproducts (BOXes) [35]. Aside from that, a set of typical pathophysiological mechanisms contributes to DCI including microvascular dysfunction, microthrombosis, cortical spreading depolarization and inflammation reactions. In the current understanding, they go far beyond angiographic vasospasm of the major cerebral vessels and additionally contribute to neuronal ischemia.

Many factors are known to regulate cerebral blood flow, and the precise physiological conditions, under which these molecules induce changes in the vessel diameter, are not yet fully understood. However, recent studies have shown that astrocytes are critical players in the regulation of cerebral blood vessel diameter for the purpose of adapted metabolism [37]. Astrocytes may act as sensors of neuronal activity and metabolism to coordinate the transport and delivery of nutrients and oxygen via the blood to metabolically active cells [38]. They are able to act bidirectional, causing either vasoconstriction or vasodilatation. For both vasomotor responses arachidonic acid is synthetized by the Ca²⁺ sensitive phospholipase A₂. For vasoconstriction, arachidonic acid is converted to 20-hydroxy­eicosatetraenoic acid, and for vasodilatation, it is converted to epoxyeicosa­trienoic acid or prostaglandins. Several factors determine the direction of action, including brain oxygen, lactate, adenosine as well as nitric oxide [38].

Within the neurovascular unit (neurons – astrocytes – parenchymal arterioles), the neurovascular coupling adopts the local blood flow to the current neuronal activity. Under normal conditions, parenchymal arterioles may dilate and constrict according to metabolic demand in normophysiologic control compartments in the brain. After aneurysmal subarachnoid hemorrhage (aSAH), neurovascular coupling is inverted [39]. The vasoconstriction observed after SAH is thought to be caused by elevated potassium concentrations outside ([K⁺] _o) the cells. It is likely that the increased spontaneous astrocytic Ca²⁺ oscillations activate endfoot large-conductance Ca²⁺-activated potassium channels leading to a lower increase of [K⁺]_o in an restricted perivascular space [39]. Such an inversion of neurovascular coupling has also been demonstrated in vivo [40,41].

Voltage-gated ion channels of the retina may be involved in SAH

The signaling cascade during neurovascular coupling which is evidently disturbed during angiographic vasospasm, should include up- or downregulated voltage-gated ion channels in addition to the mentioned Ca²⁺-activated K⁺ channels as well as less well investigated signaling components. Much is known about the beneficial effects of dihydropyridine L-type calcium (Ca²⁺) channel antagonists that are routinely recommended in clinical practice for the prevention and treatment of DCI [34]. When applied in a rather high local concentration, side effects on additional voltage-gated Ca²⁺ channels may be assumed, which are rather up- than downregulated in experimentally induced SAH in dogs [42]. For Ca_v2.3 (R-type) Ca²⁺ channels it is established that their expression and current density is increased in rabbit cerebral artery myocytes [43,44]. Similar results were found for two out of three T-type (transiently and low voltage-activated Ca²⁺ channels), named Ca_v3.1 and Ca_v3.3 [42]. The retina as an organotypical neuronal network expresses several voltage-gated calcium channels (Ca_v1.2, Ca_v1.3, and Ca_v1.4), and R-type calcium channel Ca_v2.3. The retina also contains T-type calcium channels, which were also considered to be involved in the mechanism of experimentally induced aSAH, too [42,45].

Transretinal signaling of neuronal networks

In the past, the model of the isolated and superfused vertebrate retina was frequently used for the screening of drug and other toxin effects on the sensitive phototransduction and transretinal signaling [20,46-48]. Frog, bovine and murine retinas have proven to represent a valuable neuronal network including transretinal signaling via L-type, T-type and R-type voltage-gated Ca²⁺ channels. They constitute different parts of the ERG. The major L-type voltage-gated Ca²⁺ channel at the photoreceptor synapses is Ca­_v1.4 in both retinal rods and cones [49]. T-type currents have been recorded in retinal bipolar cells [50]. The function of the Ca_v2.3/R-type Ca²⁺ channel is related to reciprocal inhibition of bipolar cell signaling (Figure 2).

Figure 2.  Schematic representation of signal transduction through rod bipolar cells.

 Light stimulation hyperpolarizes rod photoreceptors, leading to decreased glutamate release and depolarization of rod bipolar cells. Rod bipolar cells excite amacrine cells, which synapse on to ON or OFF bipolar cells of the cone pathway (not shown). In addition, amacrine cells are involved in reciprocal inhibitory pathways, which modulate rod bipolar cell depolarization via GABAergic projections. Although the exact circuits remain to be elucidated, there is evidence that Ca_v2.3 channels may be involved in GABAergic reciprocal inhibition. [M⁺] = non-selective cations.

The sensitivity towards bipolar cations was used to unravel its function during transretinal signaling. Transient effects of micromolar / sub­millimolar Ni²⁺ concentrations had earlier been recorded for the frog retina [51]. Low concentrations (10 and 30 µM) augmented the b-wave amplitude while higher concentrations (100 µM) inhibited the b-wave amplitude reversibly. Starting from this observation, our group repeated the experiments for the bovine [52] and the murine retina [53]. Isolated murine retinas from control mice and from Ca_v2.3-deficient (as well as from Cav3.2/T-type-deficient) channels were compared for their response to 15 µM NiCl₂. It was concluded that Ca_v2.3 voltage-gated Ca²⁺ channels contribute to the reciprocal inhibitory control during transretinal signaling. The cell type in which Ca_v2.3 should be expressed, was assumed but not proven to be a subtype of amacrine cells. However, it is not excluded that other neurons or even glial cells may harbor the Ca_v2.3/R-type channel.

In the vascularized mammalian retina, spreading depolarization can also be demonstrated in vivo [54], which implies that voltage- (and ligand-)gated ion channels are involved in the surface spreading and must be altered in their activity in the retina as well. As the so called “pharmacoresistent” Ca_v2.3/R-type Ca²⁺ channel is expressed in the retina [55,56] and was found to be sensitive towards micromolar dihydropyridine concentrations [57,58], the isolated retina should be selected for investigating the molecular mechanisms of electric signaling during early and late stages of SAH, a disease, which is known to be worsened in outcome by a disturbed, maybe even by an inversed neurovascular coupling during which the vessels are constricted instead of being relaxed. For the isolated murine retina, the effect of blood degradation products on transretinal signaling can be investigated, and different murine genotypes can be used for the investigations to test for those targets which may be influenced by blood degradation products.

2021 Copyright OAT. All rights reserv

Conclusion and perspectives

The retina highlights a sensory system with enormous energy demand. As an embryologic part of the brain, the retina represents an organized neuronal network for early diagnosis of CNS diseases. We believe that the retina still deserves more observation through non-invasive technical tools in the future.

Acknowledgments

We would like to specially thank Mrs. Renate Clemens for her dedication and hard work.

Funding

This research program is supported by the START-Program of the Faculty of Medicine, RWTH Aachen, Germany.

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