Walid Albanna

Antagonists of ionotropic γ‐aminobutyric acid receptors impair the NiCl2‐mediated stimulation of the electroretinogram b‐wave amplitude from the isolated superfused vertebrate retina

Purpose:  NiCl2 (15 μM) stimulates the electroretinogram (ERG) b‐wave amplitude of vertebrate retina up to 1.5‐fold through its blocking of E/R‐type voltage‐gated Ca2+ channels. Assuming that such an increase is mediated by blocking the release of the inhibitory neurotransmitter γ‐aminobutyric acid (GABA) via ionotropic GABA receptors, we tested the effect of both GABA itself and GABA‐receptor antagonists such as (−)bicuculline (1.51‐fold increase) and (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA; 1.46‐fold increase) on the b‐wave amplitude.

Effect of ZnCl2 and Chelation of Zinc Ions by N,N-Diethyldithiocarbamate (DEDTC) on the ERG b-Wave Amplitude from the Isolated Superfused Vertebrate Retina

Purpose: NiCl2 (15 µM) enhances the ERG b-wave amplitude of vertebrate retina, up to 1.5-fold by blocking E/R-type voltage-gated Ca2+ channels, which is mediated by blocking the release of GABA onto ionotropic GABA-A and GABA-C receptors. In vivo, it is likely that zinc, rather than nickel ions, may be involved in the modulation of retinal signalling. Therefore, we tested the effect of both, ZnCl2 (10 to 500 µM) and DEDTC (100 to 500 µM), which chelates zinc ions for the capacity to influence the ERG b-wave amplitude. Methods: Transretinal potentials from the isolated bovine retina were recorded as electroretinograms and Ca2+ inward currents by patch-clamp recordings of stably Cav2.3 transfected HEK-293 cells, yielding an IC50 value of 5.3 µM for ZnCl2. Results: ZnCl2 (10–15 µM) increased the b-wave amplitude by 1.52-fold ± 0.12 (n = 6 retinas), which was partially reversible upon washout. The same 1.5-fold stimulation of the b-wave amplitude was reported recently for 15 µM NiCl2. The superfusion of isolated retinas by DEDTC (100 µM) caused a transient decrease of the ERG b-wave amplitude (0.75-fold ± 0.06; n = 4), suggesting that the co-secretion of Zn2+ ions may occur under scotopic conditions. Conclusion: The stimulatory effect of ZnCl2 on the ERG b-wave amplitude resembles the stimulatory effect of NiCl2 and may be mediated rather by the NiCl2-sensitive, Cav2.3 E-/R-type voltage-gated Ca2+ channels than by NiCl2-sensitive T-type channels.

Two separate Ni2+-sensitive voltage-gated Ca2+channels modulate transretinal signalling in the isolated murine retina

Purpose:  Light‐evoked responses from vertebrate retinas were recorded as an electroretinogram (ERG). The b‐wave is the most prominent component of the ERG, and in the bovine retina its NiCl2‐sensitive component was attributed to reciprocal signalling by pharmacoresistant R‐type voltage‐gated Ca2+ channels, which similar to other voltage‐dependent Ca2+ channels trigger and control neurotransmitter release. The murine retina has the great advantage that the effect of gene inactivation for Ni2+‐sensitive Ca2+ channels can be analysed to prove or disprove that any of these Ca2+ channels is involved in retinal signalling.

Electroretinographic Assessment of Inner Retinal Signaling in the Isolated and Superfused Murine Retina

ABSTRACT Purpose: Longer-lasting electroretinographic recordings of the isolated murine retina were initially achieved by modification of a phosphate-buffered nutrient solution originally developed for the bovine retina. During experiments with a more sensitive mouse retina, apparent model-specific limitations were addressed and improvements were analyzed for their contribution to an optimized full electroretinogram (ERG). Material and methods: Retinas were isolated from dark-adapted mice, transferred to a recording chamber and superfused with different solutions. Scotopic and photopic ERGs were recorded with white flashes every 3 minutes. The phosphate buffer (Sickel-medium) originally used was replaced by a carbonate-based system (Ames-medium), the pH of which was adjusted to 7.7–7.8. Moreover, addition of 0.1 mM BaCl2 was investigated to reduce b-wave contamination by the slow PIII component typically present in the murine ERG. Results: B-wave amplitudes were increased by the pH-shift (pH 7.4 to pH 7.7) from 22.9 ± 1.9 µV to 37.5 ± 2.5 µV. Improved b-wave responses were also achieved by adding small amounts of Ba2+ (100 µM), which selectively suppressed slow PIII components, thereby unmasking more of the true b-wave amplitude (100.0% with vs. 22.2 ± 10.7% without Ba2+). Ames medium lacking amino acids and vitamins was unable to maintain retinal signaling, as evident in a reversible decrease of the b-wave to 31.8 ± 3.9% of its amplitude in complete Ames medium. Conclusions: Our findings provide optimized conditions for ex vivo ERGs from the murine retina and suggest that careful application of Ba2+ supports reliable isolation of b-wave responses in mice. Under our recording conditions, murine retinas show reproducible ERGs for up to six hours.

Retinal Vessel Analysis (RVA) in the Context of Subarachnoid Hemorrhage - A Proof of Concept Study.

Background Timely detection of impending delayed cerebral ischemia after subarachnoid hemorrhage (SAH) is essential to improve outcome, but poses a diagnostic challenge. Retinal vessels as an embryological part of the intracranial vasculature are easily accessible for analysis and may hold the key to a new and non-invasive monitoring technique. This investigation aims to determine the feasibility of standardized retinal vessel analysis (RVA) in the context of SAH. Methods In a prospective pilot study, we performed RVA in six patients awake and cooperative with SAH in the acute phase (day 2–14) and eight patients at the time of follow-up (mean 4.6±1.7months after SAH), and included 33 age-matched healthy controls. Data was acquired using a manoeuvrable Dynamic Vessel Analyzer (Imedos Systems UG, Jena) for examination of retinal vessel dimension and neurovascular coupling. Results Image quality was satisfactory in the majority of cases (93.3%). In the acute phase after SAH, retinal arteries were significantly dilated when compared to the control group (124.2±4.3MU vs 110.9±11.4MU, p<0.01), a difference that persisted to a lesser extent in the later stage of the disease (122.7±17.2MU, p<0.05). Testing for neurovascular coupling showed a trend towards impaired primary vasodilation and secondary vasoconstriction (p = 0.08, p = 0.09 resp.) initially and partial recovery at the time of follow-up, indicating a relative improvement in a time-dependent fashion. Conclusion RVA is technically feasible in patients with SAH and can detect fluctuations in vessel diameter and autoregulation even in less severely affected patients. Preliminary data suggests potential for RVA as a new and non-invasive tool for advanced SAH monitoring, but clinical relevance and prognostic value will have to be determined in a larger cohort.

Unconjugated bilirubin modulates neuronal signaling only in wild-type mice, but not after ablation of the R-type/Cav2.3 voltage-gated calcium channel

The relationship between blood metabolites and hemoglobin degradation products (BMHDPs) formed in the cerebrospinal fluid and the development of vasospasm and delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been the focus of several previous studies, but their molecular and cellular targets remain to be elucidated.

Selected aspects of retinal signaling and energy metabolism and its perspective as a cerebral surrogate model

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. 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 Correspondence to: Walid Albanna, Department of Neurosurgery, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany, Tel: +49 241 8036706; Fax: +49 241 8082420; E-mail: walbanna@ukaachen.de. Toni Schneider, Institute of Neurophysiology, University of Cologne, RobertKoch-Str. 39, D-50931 Köln, Germany, Tel: +49-221-4786968; Fax: +49-2214786965; E-mail: toni.schneider@uni-koeln.de.