[Thrombolysis treatment and multi- disciplinary management of central retinal artery occlusion in comparison with traditional ophthalmological treatment options]. / Arteria centralis retinae elzáródás thrombolysiskezelése és multidiszciplináris ellátása a hagyományos szemészeti kezelési formákkal összehasonlítva.

Background and purpose:

The management of central retinal artery occlusion (CRAO) has long been conservative therapy with limited efficacy carried out in ophthalmology departments together with etiolo­gi­cal investigations lacking a standardised protocol. However, CRAO is analogous to ischemic central nervous system stroke and is associated with increased stroke risk, thus, systemic thrombolysis treatment and multidisciplinary management can be beneficial. Since May 2022, at Semmelweis University CRAO patients diagnosed within 4.5 hours are given intravenous thrombolysis therapy and undergo etiologic workup based on current stroke protocols. Here we report our experience with the multidisciplinary, protocol-based management of CRAO in comparison with former non-protocol based ophthalmological conservative treatment.

We reviewed CRAO patients’ data treated conservatively and with paracentesis within 6 hours at the Department of Ophthalmology between 2013 and 2022 including changes in visual acuity, neurolo­gical and cardiovascular findings compared to those in the thrombolysis project. 

Of the 78 patients receiving non-protocol care, visual improvement was seen in 37% with natural course, 47% with conservative treatment and 47% with paracentesis. Four patients had significant carotid stenosis (2 underwent endarterectomy), 1 carotid dissection, 6 cardioembolism and 1 giant cell arteritis. Of the 4 patients within 4,5 hours, 3 gave their consent to the clinical trial and were treated with thrombolysis and underwent a full etiological assessment. 
2 pa­tients had improved visual acuity, 2 pa­tients had significant carotid stenosis and underwent endarterectomy, 1 patient was started on anticoagulation for newly diagnosed atrial fibrillation.

CRAO patients presenting within 4,5 hours are rare and more patients are needed in our study to establish the efficacy of thrombolysis. However uniform protocollized evaluation helps identifying embolic sources thus, avoiding further and potentially more serious thromboembolic events.

Complex contribution of cyclophilin D to Ca2+-induced permeability transition in brain mitochondria, with relation to the bioenergetic state.

Cyclophilin D (cypD)-deficient mice exhibit resistance to focal cerebral ischemia and to necrotic but not apoptotic stimuli. To address this disparity, we investigated isolated brain and in situ neuronal and astrocytic mitochondria from cypD-deficient and wild-type mice. Isolated mitochondria were challenged by high Ca(2+), and the effects of substrates and respiratory chain inhibitors were evaluated on permeability transition pore opening by light scatter. In situ neuronal and astrocytic mitochondria were visualized by mito-DsRed2 targeting and challenged by calcimycin, and the effects of glucose, NaCN, and an uncoupler were evaluated by measuring mitochondrial volume. In isolated mitochondria, Ca(2+) caused a large cypD-dependent change in light scatter in the absence of substrates that was insensitive to Ruthenium red or Ru360. Uniporter inhibitors only partially affected the entry of free Ca(2+) in the matrix. Inhibition of complex III/IV negated the effect of substrates, but inhibition of complex I was protective. Mitochondria within neurons and astrocytes exhibited cypD-independent swelling that was dramatically hastened when NaCN and 2-deoxyglucose were present in a glucose-free medium during calcimycin treatment. In the presence of an uncoupler, cypD-deficient astrocytic mitochondria performed better than wild-type mitochondria, whereas the opposite was observed in neurons. Neuronal mitochondria were examined further during glutamate-induced delayed Ca(2+) deregulation. CypD-knock-out mitochondria exhibited an absence or a delay in the onset of mitochondrial swelling after glutamate application. Apparently, some conditions involving deenergization render cypD an important modulator of PTP in the brain. These findings could explain why absence of cypD protects against necrotic (deenergized mitochondria), but not apoptotic (energized mitochondria) stimuli.

Forward operation of adenine nucleotide translocase during F0F1-ATPase reversal: critical role of matrix substrate-level phosphorylation.

In pathological conditions, F(0)F(1)-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F(0)F(1)-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F(0)F(1)-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal K(ATP) channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.

A re-evaluation of the role of matrix acidification in uncoupler-induced Ca2+ release from mitochondria.

Massive amounts of Ca(2+) can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca(2+) efflux, the release of sequestered Ca(2+) by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca(2+)-phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca(2+) release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca(2+)-loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pH(o)) 6.8 and pH(o) 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca(2+) loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na(+) was excluded from the medium, preventing Na(+)/Ca(2+) exchange. At both pH(o) values, Delta pH was in the range 0.11-0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pH(o) 6.8 and in the presence of oligomycin, the uncoupler-induced Ca(2+) release started in the -80 to -50 mV range, whereas in the absence of oligomycin, the release occurred at approximately -15 mV. Stigmatellin induced minor Ca(2+) release only in the presence of oligomycin, starting at approximately -4 mV. At pH(o) 7.8, the uncoupler-induced Ca(2+) release started at approximately -11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca(2+) release, starting at approximately -10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca(2+) release from mitochondria.

A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT.

A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.

Identification of protease-activated receptor-4 (PAR-4) in puromycin-purified brain capillary endothelial cells cultured on Matrigel.

An improved method is described for culturing primary rat brain capillary endothelial cells (RBCEC) on glass, covered by Matrigel. The procedure using Matrigel yields spindle-shaped endothelial cells exhibiting close cell-cell appositions seen on electron microscopic sections. These cells permanently express tight junction proteins ZO-1, claudin-5 and the adherent junction protein beta-catenin, as revealed by immunofluorescence. Furthermore, glass coverslips covered with Matrigel provide a stable and low-background fluorescent base for microfluorimetric calcium measurements. By this method, hereby we show that the PAR-4 agonist peptide induces transient [Ca2+]i changes with different kinetics compared to that due to activation of the PAR-1 receptor. This indicates that RBCE cells grown on Matrigel express PAR-4 receptors.

Selective cardiac plasma-membrane K(ATP) channel inhibition is defibrillatory and improves survival during acute myocardial ischemia and reperfusion.

ATP-dependent potassium channels (K(ATP)) have been implicated in cardioprotection both during myocardial ischemia and reperfusion. We compared the effect of a non-selective K(ATP) inhibitor glibenclamide, a selective mitochondrial K(ATP) inhibitor 5-hydroxy-decanoate (5-HD) and a selective sarcolemmal K(ATP) blocker HMR 1883, on survival and incidence of arrhythmias during myocardial ischemia in conscious, and during ischemia-reperfusion in pentobarbitone anesthetized rats. Glibenclamide (5 mg/kg i.p.) or HMR 1883 (3 mg/kg i.v.) reduced ischemia-induced irreversible ventricular fibrillation and improved survival during myocardial ischemia (64% and 61% vs. 23% in controls, respectively). 5-HD (5 mg/kg i.v.) did not influence survival and the incidence of ventricular arrhythmias. The incidence of reperfusion-induced arrhythmias was reduced by both glibenclamide and HMR 1883 (3 or 10 mg/kg) resulting in improved survival during reperfusion (81%, 82% and 96% vs. 24% in controls, respectively) in anesthetized rats. 5-HD did not reduce the incidence of lethal reperfusion arrhythmias. Glibenclamide and HMR 1883 prolonged (89+/-4.6 and 89+/-4.9 ms vs. 60+/-2.4 ms in controls), while 5-HD did not change the QT interval. In conclusion, inhibition of sarcolemmal K(ATP) reduces the incidence of lethal ventricular arrhythmias and improves survival both during acute myocardial ischemia and reperfusion in rats. This beneficial effect correlates with the prolongation of repolarization. Inhibition of mitochondrial K(ATP) does not improve survival or reduce the occurrence of ischemia and/or reperfusion-induced arrhythmias and does not prolong the QT interval. The present results also suggest that the antiarrhythmic effect of K(ATP) inhibitors is not influenced by pentobarbitone anesthesia.

Preconditioning effects of levosimendan in a rabbit cardiac ischemia-reperfusion model.

The preconditioning effects of levosimendan were investigated on ischemia-reperfusion induced morphological and functional cardiac damage. Langendorff-perfused rabbit hearts were reserved as controls or subjected either to global myocardial ischemic preconditioning or to perfusion with levosimendan (0.1 micromol/l) for two 5-minute cycles. After a washout period, all hearts were then subjected to 30 minutes of global ischemia and 120 minutes of drug-free reperfusion. Intraventricular pressure and coronary flow were measured, and infarct size determined after nitroblue-tetrazolium staining on completion of the experiments. Levosimendan pretreatment resulted in a significantly smaller elevation from the preischemic level in left ventricular end-diastolic pressure during reperfusion (37 +/- 17 mm Hg) compared with controls (56 +/- 14 mm Hg) and ischemia-preconditioned hearts (53 +/- 34 mm Hg). The left ventricular developed pressure-representing the functional recovery of the heart after ischemia-that was significantly improved by levosimendan pretreatment (38 +/- 6% vs 16 +/- 5% in controls, P < 0.05). In addition, contractility and relaxability parameters (+dP/dt and -dP/dt, respectively) were better preserved in the levosimendan hearts. The volume of infarcted myocardium after global ischemia-reperfusion was significantly (P < 0.05) decreased by both ischemic preconditioning (38 +/- 2%) or levosimendan pretreatment (45 +/- 2%) versus controls (52 +/- 2%). The results of this study suggest that levosimendan pretreatment is capable of decreasing infarct size in an ischemia-reperfusion model and improving recovery of cardiac function following ex vivo global ischemia.

Distinct modulation of angiotensin II-induced early left ventricular hypertrophic gene programming by dietary fat type.

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.