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The distribution of mitochondria within retinal glial (Müller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Müller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Müller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Müller cell processes. (3) Müller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Müller cells from the peripapillar myelinated region ('medullary rays') of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Müller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Müller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.

L-Glutamate (L-GLU) induced astrocyte swelling in a time- and concentration-dependent, as well as Na+- and Ca2+-dependent, and Cl(-)-independent manner. Swelling was prevented by MK-801, cystine, and ouabain. Since L-GLU swelling is ionically dependent, we determined the role of various ions in such swelling. Our results indicate that K+ uptake plays a major role in the mechanism of L-GLU-induced astrocyte swelling. Like swelling, K+ uptake is dependent on Ca2+ and Na+, but not on Cl-. Likewise, K+ uptake was inhibited by MK-801, cystine, and ouabain. The K+ channel blockers, Ba2+ and tetraethylammonium, partially prevented L-GLU-induced swelling. In addition to K+ channels, K+ influx may also be mediated through Na+/K+-ATPase, as its activity is increased by L-GLU uptake along with the required Na+. Taken together, the data suggest that K+ influx plays a key role in the mechanism of L-GLU-mediated astrocyte swelling.

Objective: The aim of this study was to define the neurologic risk of heart transplantation compared to coronary artery bypass grafting (CABG) and valve surgery., Design: A retrospective study., Setting: A university hospital., Patients: 8001 patients undergoing heart transplantation, CABG and valve surgery., Measures: The incidences of perioperative central nervous system (CNS) complications were compared between different procedures. Risk factors were analysed using univariate and multivariate methods., Results: The overall incidence of CNS complications was 19.8% (78/393) in heart transplantation, 3.1% (176/5734) in elective CABG, 9.5% (161/1689) in elective valve surgery, 10.3% (15/146) in emergency CABG and 51.3% (20/39) in emergency valve surgery. Most powerful predictors of CNS complications were preoperative intra-aortic counterpulsation (IABP) in the heart transplantation group, age >65 years in the CABG group and preoperative use of catecholamine in the valve group., Conclusions: CNS complications occur much more frequently after heart transplantation than elective CABG and valve surgery. The high incidence of CNS complications after emergency operations as well as preoperative catecholamine and IABP as powerful contributing factors suggest that preoperative cerebral hypoperfusion due to a compromised hemodynamic state facilitates postoperative CNS complications and this may partly explain the high incidence of CNS complications after heart transplantation.

Enzymatically isolated, noncultured Müller glial cells from human organ donor and patient eyes were studied using the whole-cell-voltage-clamp and the patch-clamp technique. The patients suffered mainly from choroidal melanomas, retinal detachment due to proliferative vitreorentinopathy, glaucomas, and perforating eye injuries. The organ donor eyes were used as a source of corneas for corneal transplantation. Use of the human retinal tissue was approved by the Ethics Committee of the School of Medicine, University of Leipzig. Most of the patient Müller cells showed a marked or complete loss of inwardly rectifying K+ currents, causing a dramatic increase in the input resistance. The zero current potential of the patient Müller cells, which is equivalent to the membrane potential, was significantly reduced (depolarized) as compared with the donor Müller cells. In contrast to the K+ current loss, the Na+ current density was significantly higher in patient Müller cells than in donor Müller cells; the number of Müller cells depicting Na+ currents increased from 33% (3 pA/pF) in donor Müller cells to 85% (about 12pA/pF) in patient Müller cells. Application of glutamate to the Müller cells generated a glutamate-transporter-mediated current, such as that seen in other species. A highly significant increase was noted for the high-affinity Na+-dependent glutamate-transporter-current density in patient Müller cells compared with donor cells. The application of gamma-aminobutyric acid (GABA) evoked, in addition to the GABA transporter currents already known from Müller cells of other mammalian species, GABA(A)-receptor mediated currents in human Müller cells. We found that GABA(A) receptors are expressed in human Müller cells, but not in other nonprimate mammals. Whether a difference exists between the GABA(A) current density in donor and patient Müller cells remains to be seen. The results concerning the disappearance of K+ currents and diminution of the membrane potential may demonstrate early glial changes that may possibly precede pathological neuronal changes, at least in retinas from eyes with choroidal melanomas. In later stages of the diseases, the glial changes may be deleterious for the neurons, because they could diminish glutamate uptake due to the depolarized membrane potential. However, increased extracellular glutamate concentration is toxic for most neurons.

Müller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the 'midperiphery' of retinae immunocytochemically labeled for vimentin, estimates of Müller cell densities per unit retinal surface area, and of neuron-to-(Müller) glia indices were performed. Müller cell densities were strikingly similar among the species studied (around 8000-11,000 mm-2) with the extremes of the horse (< or = 5000 mm-2) and the tree shrew (> or = 20,000 mm-2). By contrast, the number of neurons per Müller cell varied widely, being clustered at 6-8 (in retinae with many cones), at about 16, and at up to more than 30 (in strongly rod-dominated retinae). Isolated Müller cell volumes were estimated morphometrically, and cell surface areas were calculated from membrane capacities. Müller cells isolated from thick vascularized retinae (carnivores, rats, mice, ungulates) were longer and thinner, and had smaller volumes but higher surface-to-volume ratios than cells from thin paurangiotic (i.e. with blood vessels only near the optic disc) or avascular retinae (rabbits, guinea pigs, horses, zebras). In whole-cell voltage-clamp studies, Müller cells from all mammals studied displayed two dominant K+ conductances, inwardly rectifying currents and delayed rectifier currents. TTX-sensitive Na+ currents were recorded only in some species. Based on these data, the following hypotheses are presented, (a) neuron-to-(Müller) glia indices are determined by precursor cell proliferation rather than by metabolic demands; (b) Müller cell volumes depend on available space rather than on the number of supported neurons; and (c) it follows that, the specific metabolic activities of Müller cells must differ greatly between species, a difference that may contribute to distinct patterns of retinal vascularization.

We compared the inward K+ currents of Müller glial cells from healthy and pathologically changed human retinas. To this purpose, the whole-cell voltage-clamp technique was performed on noncultured Müller cells acutely isolated from human retinas. Cells originated from retinas of four healthy organ donors and of 24 patients suffering from different vitreoretinal and chorioretinal diseases. Müller cells from organ donors displayed inward K+ currents in the whole-cell mode similar to those found in other species. In contrast, this pattern was clearly changed in the Müller cells from patient retinas. In whole-cell recordings many Müller cells had strongly decreased inward K+ current amplitudes or lost these currents completely. Thus, the mean input resistance of Müller cells from patients was significantly increased to 1,129 +/- 812 M omega, compared to 279 +/- 174 M omega in Müller cells from healthy organ donor retinas. Accordingly, since the membrane potential is mainly determined by the K+ inward conductance in healthy Müller cells, a large amount of Müller cells from patient retinas had a membrane potential which was significantly lower than that of Müller cells from control eyes. The mean membrane potentials were -37 +/- 24 mV and -63 +/- 25 mV for patient and donor Müller cells, respectively. The newly described membrane characteristic changes of Müller cells from patient eyes are assumed to interfere severely with normal retinal function: (1) the retinal K+ homeostasis, which is partly regulated by the Müller cell-mediated spatial buffering, should be disturbed, and (2) the diminished membrane potential should influence voltage-dependent transporter systems of the Müller cells, e.g., the Na(+)-dependent glutamate uptake.

The dependence of intracellular glutathione, an important radical scavenger, on the extracellular glutamate and cystine concentration and the velocity of the high affinity sodium/glutamate transporter was studied in freshly-isolated Müller glial cells of the guinea-pig, kept in vitro for up to 11 h. To this end the relative Müller cell glutathione levels were measured using the fluorescent dye monochlorobimane, using different concentrations of glutamate and cystine in Ringer solution. In some experiments L-buthionine-[S,R]-sulfoximine, a blocker of glutathione synthesis, or L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid, inhibitors of glutamate uptake, were added. The Müller cells maintained about 80% of the normal glutathione level when maintained in Ringer solution containing 100 microM glutamate for 11 h. When under these conditions 100 microM cystine was added, the glutathione level increased to values, which were even higher than those at the beginning of the incubation period. Addition of cystine without glutamate caused a run down of the glutathione level to about 45% of the normal level, which is comparable to the run down in pure Ringer solution. Likewise, application of L-buthionine-[S,R]-sulfoximine (5 mM) lead to a strong run down of the glutathione level even in glutamate/cystine (100 microM)-containing solution. A similar suppressing effect was observed using L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid in the presence of 100 microM cystine and glutamate. We conclude that the intracellular glutamate concentration of the Müller cells is determined by the extracellular glutamate concentration and the velocity of the sodium/glutamate uptake. Consequently, cystine uptake into Müller cells, which is performed by the cystine/glutamate antiporter, is fueled by the sodium/glutamate transporter with intracellular glutamate. Both glutamate and cystine are also substrates for glutathione synthesis. The glutathione level is logically limited by the capacity of the sodium/glutamate transporter to provide glutamate intracellularly for, first, cystine uptake and, second, direct insertion into glutathione. Accordingly, the glutathione level is reduced when the sodium/glutamate transporter is blocked. Thus, a diminution of the glutathione level should be taken into consideration when the effects of sodium/glutamate uptake failure and reduced intracellular glutamate concentrations are discussed.

The electrophysiological features of isolated baboon Müller cells was investigated using the whole-cell voltage-clamp technique. Application of depolarizing voltage steps evoked transient inward and delayed outward currents. The transient currents disappeared when extracellular Na+ was replaced by choline+ and were substantially decreased by application of tetrodotoxin (1 microM). The outward currents were strongly diminished by extracellular Ba2+ (1 mM), and the hyperpolarization-generated inward currents disappeared following application of Ba2+. The recently described gamma-aminobutyric acid A (GABAA) receptor currents were increased by flunitrazepam, nordiazepam, pentobarbital and Zn2+, as well as by the inverse agonist DMCM. These results suggest that the baboon Müller cells possess the same voltage-dependent current pattern as those from other species, e.g. humans, whereas their GABAA receptors react in an uncharacteristic manner to DMCM and Zn2+, when compared with neuronal GABAA receptors.

In the retina of most vertebrates there exists only one type of macroglia, the Müller cell. Müller cells express voltage-gated ion channels, neurotransmitter receptors and various uptake carrier systems. These properties enable the Müller cells to control the activity of retinal neurons by regulating the extracellular concentration of neuroactive substances such as K+, GABA and glutamate. We show here how electrophysiological recordings from enzymatically dissociated mammalian Müller cells can be used to study these mechanisms. Müller cells from various species have Na(+)-dependent GABA uptake carriers, but only cells from primates have additional GABA receptors that activate Cl- channels. Application of glutamate analogues causes enhanced membrane currents recorded from Müller cells in situ but not from isolated cells. We show that mammalian Müller cells have no ionotropic glutamate receptors but respond to increased K+ release from glutamate-stimulated retinal neurons. This response is involved in extracellular K+ clearance and is mediated by voltage-gated (inwardly rectifying) K+ channels which are abundantly expressed by healthy Müller cells. In various cases of human retinal pathology, currents through these channels are strongly reduced or even extinguished. Another type of voltage-gated ion channels, observed in Müller cells from many mammalian species, are Na+ channels. In Müller cells from diseased human retinae, voltage-dependent Na+ currents were significantly increased in comparison to cells from control donors. Thus, the expression of glial ion channels seems to be controlled by neuronal signals. This interaction may be involved in the pathogenesis of retinal gliosis which inevitably accompanies any degeneration of retinal neurons. In particular, Müller cell proliferation may be triggered by mechanisms requiring the activation of Ca(2+)-dependent K+ channels. Ca(2+)-dependent K+ currents are easily elicitable in Müller cells from degenerating retinae and can be blocked by 1 mM TEA (tetraethylammonium). In purified Müller cell cultures, the application of 1 mM TEA greatly reduces the proliferative activity of the cells. These data clearly show that Müller cells are altered in cases of neuronal degeneration and may be crucially involved in pathogenetic mechanisms of the retina.

Müller cells, the main macroglial cells of the retina, express several types of voltage and ligand-activated ion channels, including Na+ channels. Using the whole-cell voltage-clamp technique, we studied the expression of Na+ currents in acutely isolated, non-cultivated human Müller cells from retinas of healthy organ donors and patients suffering from different eye diseases. In both types of retinas transient Na+ currents could be recorded from Müller cells. The tetrodotoxin-resistant Na+ currents, which were not completely blocked even at a concentration of 10 microM tetrodotoxin, had a mean current density of 3.0 +/- 3.0 pA/pF (mean +/- SD, n = 10) in Müller cells from donor retinas and of 12.2 +/- 9.6 pA/pF (n = 74) in Müller cells from patient retinas. Only 33.3% of healthy but 88.4% of pathological Müller cells depicted such currents. The GNa+/GK+ ratio was very high in several Müller cells from patient retinas, such that action potential-like activity could be generated after prehyperpolarizing current injection in some of these cells. Apparently, the Na+ channels, due to their negative steady-state inactivation curve (Vh = -84.5 mV), do not influence the lowered membrane potential of the pathological cells, since they are inactivated at these voltages. Currently, we do not have an explanation for the increase in amplitude and frequency of Na+ currents in human Müller cells under pathological conditions. However, the up-regulation of Na+ channels may mirror a basic glial response to pathological conditions, since it has also been found previously in human hippocampal astrocytes from epileptic foci and in rat cortex stab wounds lined by an astrocytic scar.

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the "glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.

The effect of gamma-aminobutyric acid (GABA) application on acutely isolated, non-cultivated Muller glial cells from the baboon retina was studied using the whole-cell voltage-clamp technique. Application of GABA (0.1 mM) generated inward currents at a holding potential of -80 mV as well as an increase in current noise. The GABA-activated current had a reversal potential of 18.6 mV and was therefore supposed to be a Cl- current (ECl = 5 mV). The GABAA receptor agonist muscimol (0.1 mM) elicited an inward current and bicucullin (0.5 mM), a blocker of the GABAA receptor, diminished the GABA responses in our experiments completely. Baclofen (0.1 mM), a GABAB agonist, neither had an effect when applied under conditions where the dominant Muller cell K+ currents were unblocked, nor when the K+ currents were blocked by application of Ba2+ (1 mM). Glycine (0.1 mM) was ineffective as well. From these results we conclude that the baboon retinal Muller cells possess GABAA receptors. However, these have recently been discovered on skate Muller cells whereas GABAA receptors could not be found on Muller cells of guinea pig, pig, mouse, rat and rabbit.

A procedure was developed allowing repetitive depletion and recovery of K+ from the cell interior of Müller glial cells without patch pipette perfusion. To this end the whole-cell voltage-clamp technique using tight seal pipettes was applied to enzymatically isolated Müller cells of the guinea pig. When K+ was replaced by Cs+ and/or NMDG in the pipette solution voltage-activated outward currents could be generated similar to those found with normal K(+)-containing intracellular solution. This was the result of the distribution of K+ across the cell membrane due to the negative holding potential, i.e., K+ accumulated at the intracellular side of the cell membrane. This distribution should be favored by the low input resistance of Müller cells. K+ could also be removed from the cell interior by depolarizing voltage steps. Simultaneously, K+ influx had to be cut off by using a K(+)-free extracellular solution or blocking K+ channels using Ba2+. This procedure lead to reduction and eventually cessation of the K+ outward currents, indicating extinction of the intracellular K+ pool. The process is reversible, i.e., outward currents can be evoked again by depolarizing voltage steps after renewed application of extracellular K+ and/or removal of Ba2+. Hence, the intracellular K+ pool is filled up again. The described method was applied to demonstrate the dependence of the Müller cell Na+/glutamate transporter on the intracellular K+ concentration.

The effect of the glial toxin alpha-aminoadipic acid (AAA) upon the Na+/glutamate cotransporter of acutely isolated guinea pig retinal glial cells was studied using the whole-cell voltage-clamp technique. Glutamate evoked an inward current in these cells at negative holding potentials dependent on the presence of extracellular Na+ and intracellular K+. A reversal potential could not be found for the current. L-trans-Pyrrolidine-2.4-dicarboxylic acid (PDC), a blocker of Na(+)-dependent glutamate uptake, diminished the glutamate current also in our cells. Application of L-AAA also generated an inward current at negative holding potentials, without a reversal potential, being suppressed if extracellular Na+ or intracellular K+ was removed. The glutamate uptake blocker, PDC (200 microM), blocked the L-AAA (1 mM) current. Thus, L-AAA proved to be transported by the Na+/glutamate transporter of Müller cells. Hence, glutamate currents were diminished by L-AAA competitively with a Km of 499 microM at a glutamate concentration of 10 microM. The Na+/glutamate uptake was less sensitive to DL- and D-AAA block. It is suggested that the blocking effect of AAA on Na(+)-dependent glutamate uptake into glial cells might be involved in the well known glia toxicity of this compound.

In order to investigate GABA uptake into Müller glial cells electrophysiologically, we used the whole-cell voltage-clamp technique. Since Müller cells were insensitive to application of muscimol and baclofen, the expression of GABAA and GABAB receptors can be excluded. Therefore, the observed GABA current must be due to an electrogenic GABA transporter. This transporter is driven by the transmembrane Na+ gradient, as replacement of the extracellular Na+ by choline inhibits the GABA current. Currents evoked by cis-4-aminocrotonic acid, a GABAC specific agonist, are also inhibited by replacement of extracellular Na+, indicating that this compound is a substrate for the uptake. The competitive GABA uptake blockers beta-alanine and nipecotic acid are substrates of the transporter as well, and produce 42% and 65% of the currents elicited by the same GABA concentration, respectively. Affinity of the transporter for GABA is high, the Km value being 5 microM at -80 mV.

There is ample evidence that retinal radial glial (Müller) cells play a crucial role in retinal ion homeostasis. Nevertheless, data on the particular types of ion channels mediating this function are very rare and incomplete; this holds especially for mammalian Müller cells. Thus, the whole-cell variation of the patch-clamp technique was used to study voltage-dependent currents in Müller cells from adult rabbit retinae. The membrane of Müller cells was almost exclusively permeable to K+ ions, as no significant currents could be evoked in K(+)-free internal and external solutions, external Ba2+ (1 mM) reversibly blocked most membrane currents, and external Cs+ ions (5 mM) blocked all inward currents. All cells expressed inwardly rectifying channels that showed inactivation at strong hyperpolarizing voltages (> or = -120 mV), and the conductance of which varied with the square root of extracellular K+ concentration ([K+]e). Most cells responded to depolarizing voltages (> or = -30 mV) with slowly activating outward currents through delayed rectifier channels. These currents were reversibly blocked by external application of 4-aminopyridine (4-AP, 0.5 mM) or tetraethylammonium (TEA, > 20 mM). Additionally, almost all cells showed rapidly inactivating currents in response to depolarizing (> or = -60 mV) voltage steps. The currents were blocked by Ba2+ (1 mM), and their amplitude increased with the [K+]e. Obviously, these currents belonged to the A-type family of K+ channels. Some of the observed types of K+ channels may contribute to retinal K+ clearance but at least some of them may also be involved in regulation of proliferative activity of the cells.

Müller cells span through the entire retina and terminate with the formation of endfeet at the vitreous body. These endfeet are thought to be specialized for maintaining the K+ homeostasis in the retina based on the assumption that voltage signals can passively spread from the cell body to the endfeet. We employed the patch-clamp technique to study the physiological properties of these endfeet in a retinal wholemount preparation from guinea-pig or mouse. After assessing one endfoot with the patch pipette and establishing the whole cell recording configuration, a membrane area which approximately matched the size of one endfoot and proximal process could be voltage-clamped. This morphological correlation could be established by filling the cytoplasm with the fluorescent dye Lucifer Yellow via the patch-pipette. The morphological, immunocytochemical and ultrastructural inspection of the recorded cells revealed that mouse Müller cell endfeet were connected by only a thin stalk to the proximal process. In contrast, guinea-pig endfeet were connected by thick stalks. The endfoot current in the mouse was dominated by a voltage and time-independent K+ conductance. In contrast, in some of the recordings from guinea-pig, delayed and inwardly rectifying K+ currents were observed. These voltage-gated currents were more frequently observed or were facilitated when the membrane area under voltage clamp was increased, blocking the passive K+ currents by Ba2+ in both, mouse and guinea-pig. We thus assume that the voltage-gated currents were not in the endfeet membrane, but rather in the proximal process and could thus be better activated in the guinea-pig with its thicker stalk or after increasing the membrane area under voltage clamp control. Similar results were obtained in freshly isolated Müller cells; in contrast to the cells from the wholemount the voltage-gated currents were more frequently observed. These studies demonstrate that the Müller cell endfoot of the mouse with its vascularized retina is an electrically isolated unit and that voltage signals do not spread to the proximal process. Such a property would, however, be required for the redistribution of K+ via spatial buffer currents. In contrast, guinea-pig Müller glial cells with their stout morphological connection between endfoot and proximal process are better suited to fulfil this task.

The effect of externally applied Ba2+ and Na+ on K+ currents was investigated by means of whole-cell patch-clamp in isolated and in situ Müller cells from guinea pig retina. Müller cells express a typical set of K+ currents, i.e. an ohmic current, an inactivating inward current (IK(IR)), a delayed rectifier (IK(DR)) and an inactivating outward current (IK(A)). Inactivation of the inward current did not occur when extracellular Na+ was replaced by choline. When administered in increasing concentrations, Ba2+ blocked these K+ currents in a typical sequence: the ohmic current and IK(A) were most sensitive, followed by IK(IR), whereas IK(DR) was not completely blocked even in 1 mM Ba2+. The differential sensitivity of Müller cell K+ currents to external Ba2+ may be a tool which can be used to improve our understanding of the Müller cell response to physiological stimulation of the retina.

Müller cells constitute the principal glia of the vertebrate retina. Unlike other types of neuroglial cells such as astrocytes and Schwann cells, Müller cells have not yet been demonstrated to express Na+ channels. Here we present first evidence of Müller cell Na+ currents from voltage-clamp studies in enzymatically isolated cells. Some cells from retinae of cats and dogs, but none from rabbit or guinea-pig retinae, revealed fast and rapidly inactivating inward currents in response to depolarizing voltage steps. The currents reversibly disappeared in Na+ free solutions or under tetrodotoxin (TTX, 1 microM). Activation and inactivation characteristics of these currents were strikingly similar to those of neurone-type Na+ channels.

In a consecutive series of 1109 patients undergoing aortic valve replacement (AVR) between January 1988 and December 1990, there were 48 patients (33 female, 15 male) over 80 years of age (mean age 83.5 years, median 82.9 years). Of those, 33 had aortic stenosis and 15 combined aortic valve disease, with additional coronary artery disease being present in 36. Isolated AVR was performed in 25 patients, and it was combined with coronary venous bypass grafting, with 1-4 (mean 1.8) peripheral anastomoses in 23. Two patients died within 30 days (early mortality 4.2%). Non-fatal complications included one hemiparesis, four transient cerebral disorders, two cases of pneumonia which led to ventilatory assistance, three rethoracotomies because of postoperative bleeding, 15 tachycardias and one transient AV block. Late results were obtained after a median follow up time of 22 months. There were eight late deaths (four cardiac related, four not related) and a low incidence of non-fatal complications (two episodes of gastrointestinal bleeding while on oral anticoagulation, one cerebral transient ischemic attack and one acute left ventricular failure). Nine patients are in NYHA Class I, 12 in Class I-II, 11 in Class II, three in Class II-III and three in Class III. Of the surviving 38 patients, four are currently living in a home for the aged or a nursing home, while all the others are living in their own homes and are able to sustain a relatively independent life-style. We conclude that in very old patients with aortic valve disease, AVR can be performed with low mortality and few non-fatal complications.

The ability of rabbit retinal Müller (glial) cells to perform phagocytosis was studied in vitro. Müller cells were feed with various kinds of latex beads either shortly after enzymatical isolation from adult retinae or in monolayer cell cultures derived from neonatal retinae and kept 14 days in vitro. Both types of Müller cell preparations showed intense phagocytosis of latex beads. Moreover, when entire retinae were isolated and exposed (sclerad side up) to latex beads in vitro for 30 min, Müller cells had picked up fluorescent beads and showed, after fixation, intense labeling in radial sections of such retinae. Effective phagocytosis by Müller cells was demonstrated 1.) by transmission electron microscopy, 2.) by bright-field light microscopy of unstained large beads (diameter 660 nm), or 3.) by fluorescence microscopy of small (diameter about 60 nm) and large latex beads labeled with rhodamine. These results suggest that both labeled and unlabeled latex beads are suitable tools to study the phagocytotic activity of retinal glial cells in vitro, thus providing information on important processes occurring in situ during ontogenesis, physiological renewal of retinal receptor cells, and pathological events. We found that movements of cells or cytoplasmic excrescences, and cell-cell interactions, play important roles in removal of foreign particles out of the fluid environment. Engulfed latex beads move through the elongated cells with velocities similar to slow axoplasmic transport.

During onset and offset of illumination, considerable changes in extracellular K+ concentration ([K+]c) occur within particular retinal layers. There are two ways in which glial cells may control [K+]c: (1) by space-independent processes, for example, by K+ uptake due to the Na(+)-K+ ATPase, and (2) by space-dependent processes, that is, by spatial buffering currents flowing through K+ channels. Rabbit retinal Müller (glial) cells were studied for expression of mechanisms supporting both kinds of processes. This review demonstrates that rabbit Müller cells have Na-K pumps whose distribution and properties are highly adapted to meet the needs of efficient K+ clearance. Furthermore, spatial buffering currents through specialized K+ channels of Müller cells greatly accelerate retinal K+ clearance during and after stimulation.

A method is introduced for the quantification of specific compliance and the elasticity of small pieces of living retinal tissue. These pieces are fixed at their margins by means of tissue glue, and loaded with a small iron spherule the bending force of which can be gradually enhanced by the action of an electromagnet. Retinal bending caused by such calibrated forces is measured by a horizontal light microscope, and used for estimations of specific compliance and elasticity of the tissue. Three different particular regions of the rabbit retina--periphery, visual streak, and (prospective) medullary rays--were tested at several post-natal developmental stages. From very early stages on (day 2 p.p.) up to adulthood the peripheral retina was found to be significantly more tensile than the two other central regions. This can be shown to depend greatly on the thickness of the tissue which is lower in the retinal periphery. During early post-natal development, all retinal regions except the (prospective) medullary rays become thinner. The tensility of the tissue increases, with the exception of the medullary rays which reduce their compliance strongly. In the adult retina, however, the tensility of all retinal regions is reduced as compared with the neonatal tissue. This seems to be caused by a constant gradual increase of the elasticity of the retina during development which, in turn, may be caused by several developmental parameters, e.g. the formation of synapses, the outgrowth of glial side branches ensheathing neighbouring neuronal cells, or a reduction in extracellular clefts. It is proposed that these differences in tensility between different retinal regions, may be the cause for differential retinal expansion driven by the intraocular pressure. Thus, simple mechanical features of the tissue may contribute to the formation of important topographic specializations of the retina, e.g. the visual streak as the site of highest visual acuity.

To examine if the choice of volatile agents influences cardiac outcome in coronary artery surgery, 1178 patients undergoing elective coronary artery bypass grafting without additional operations received enflurane (608) or isoflurane (570) as their primary anesthetics. The inspired concentration of volatile agent (administered with 50% nitrous oxide) was adjusted depending on the level of blood pressure at the discretion of the anesthesiologist. In addition to the volatile agent assigned, each patient received small doses of fentanyl at induction and before sternotomy (total 0.006-0.008 mg/kg). The groups did not differ in preoperative and surgical characteristics except for a more frequent history of renal dysfunction in patients given isoflurane. The rates of postoperative myocardial infarction, administration of positive inotropic agents at the time of weaning from cardiopulmonary bypass, and in-hospital deaths in the enflurane and isoflurane groups were 1.8% and 4.0% (P less than 0.05), 4.9% and 8.1% (P less than 0.05%), and 0.3% and 2.1% (P less than 0.01), respectively. Although the mechanism of the adverse effects of isoflurane could not be clarified in this study, these results demonstrate that the use of isoflurane could be inappropriate in patients undergoing coronary artery bypass grafting.

Retinae of early postnatal rabbits were enzymatically dissociated and explanted in a culture system. The prospective myelinated region was discarded in order to avoid the presence of astrocytic or mesenchymal cells. After about 14 days in vitro (DIV), outgrowing glial (Müller) cells formed what light optically appeared to be confluent monolayers but by electron microscopy was shown to consist of flat epithelioid cells which overlapped considerably by extension of cytoplasmic tongues. Applying the freeze-fracture technique, apposed membranes of these cells were demonstrated to express infrequently but consistently both gap and tight junctions. This kind of junctions has never been observed on the membrane of rabbit Müller cells in situ. In comparison with Müller cell membranes in situ, the density of intramembrane particles was considerably reduced. Orthogonal arrays of particles which are characteristic elements of Müller cells in situ were not detected. Our results suggest that in homogeneous cell culture, Müller cells form some kind of epithelium-like specialized intercellular junctions. This situation resembles that of closely related glial cell types which form homogeneous layers in situ as e.g. retinal pigment epithelium cells expressing tight junctions, and marginal astrocytes being coupled by extensive gap junctions.

The freeze-fracture technique was applied to the retina of early postnatal and adult rabbits to investigate the distribution and density of orthogonal arrays of intramembrane particles (OAP) within the vitread endfoot membranes. In adult animals, two distinct types of endfoot membranes were observed within the central myelinated retina but not in the retinal periphery. One type of endfoot membranes contained low density of individual 'background' particles, and a more or less stripe-like pattern of OAP; this type was found only within the myelinated centre and is concluded to represent membranes of retinal astrocytes. The other type of endfoot membranes was rather tightly packed with individual 'background' particles, and contained OAP which formed rows only at the margins of footplates; this kind of membrane was found throughout the whole retina, and represents Müller cell endfeet. The density of OAP in both types of endfoot membranes was higher within the central myelinated retina than in the Müller cell endfeet of the retinal periphery. In early postnatal retinae, a discrimination between the two types of endfeet was impossible. At the day of birth, only very few OAP were observed, and the majority of footplates were free of OAP. Within the next 10 days, both density and size of OAP were found to increase but fail to reach adult levels. Quantitative data are presented with the hope of providing a basis for future correlation with functional maturation of rabbit retinal glia.

From 1973 through December 1980, 41 children with total anomalous pulmonary venous connection (TAPVC) were seen in our hospital, and 35 underwent surgery. The supracardiac type of TAPVC was found in 19 cases (46%), the intracardiac type in 6 infants (15%), the infracardiac type in 15 cases (37%), and the mixed type in one child (2%). Clinical and cardiac catheterization data demonstrated that children with the infracardiac type of TAPVC were referred very early in life and had the highest pulmonary artery pressures. Operation was performed in 35 cases, 15 of supracardiac, 6 of cardiac, 13 of infracardiac, and 1 of mixed type of TAPVC. Total in-hospital mortality was 39%, and was 28% in the operated children. The initial operative mortality of 71% in the years 1973 to 1975 has decreased to 18% in the last 5 years. Twelve of the surviving infants underwent re-catheterization showing excellent results. Only in one case, with additional aplasia of the left lung, did half-systemic pulmonary artery pressure persist. Corrective surgery offers the only chance of survival in most children with TAPVC. The operative risk can be reduced by increased surgical experience supported by optimal medical preparation of the patient. After adequate surgical correction long-term results appear to be excellent.

A case of transient unilateral twelfth nerve Paresis after puncture of the internal jugular vein is reported. The anatomy of the puncture site makes a connection possible.

A transfusion reaction was observed with blood warmed by a microwave blood warmer. Analysis of the blood remaining in the bag excluded the possibility of incompatibility but revealed hemolysis and clot formation which was attributed to overheating by the blood warmer.

Radial glial (Müller) cells of the rabbit retina were studied by various techniques including Golgi impregnation, scanning electron microscopy, horseradish peroxidase application, and staining of enzymatically isolated cells. This combination of methods produced detailed information on the specialized morphology of the Müller cells within the different topographical regions of the retina, and of the Müller cell processes within the various retinal layers. As a general rule, the retinal periphery contains short thick Müller cells with big endfeet, whereas the thick central retina is occupied by long slender cells with small endfeet. Independent of their location within the retina, Müller cell processes were found to be adapted to the structure of the surrounding retinal layers. Within the outer and inner nuclear layers, Müller cell processes (and somata) extend thin cytoplasmic "bubbles" ensheathing the neuronal somata, as do the "velate" astrocytes in the brain. In the plexiform layers, Müller cells extend many fine side branches between the neuropil, comparable to the protoplasmic astrocytes of the brain. In the thick myelinated nerve fibre layer of the central retina the Müller cell processes are rather smooth, similar to those of fibrous astrocytes. It is concluded that the neuronal microenvironment determines the morphology of a given glial process, or even of a part of a glial process running through a specialized neuronal compartment.

In cardiosurgical patients the haemodynamic effects of dobutamine 2.5 microgram/kg . min and 5 microgram/kg . min dobutamine were investigated during neuroleptanalgesia, intra- and immediately postoperatively. Intraoperative measurements were performed in 8 coronary surgical patients each after sternotomy and pericardiotomy, but before the aortocoronary venous bypass operation. The following haemodynamic parameters increased significantly: cardiac index (2.5 microgram/kg . min: 2.6 leads to 2.1 1/min . m2; 6 microgram/kg . min: 1.5 leads to 2.24 1/min . m2), heart rate (80 leads to 91 min-1; 86 leads to 107 min-1), stroke index (16%, 27%), mean arterial pressure (70 leads to 90 mm Hg; 70 leads to 93 mm Hg), mean pulmonary arterial pressure (8%; 14%), LV dp/dtmax (72%; 121%) and calculated myocardial oxygen consumption Eg (35%; 52%). Changes in right (PRA) and left ventricular filling pressure (PLVED), in total systemic resistance and total pulmonary vascular resistance were not significant. Postoperative measurement immediately after open heart operations (ASD-correction n = 5, aortocoronary venous bypass (n = 3) in neuroleptanalgesia too, showed the same haemodynamic results as intraoperatively before correction of coronary stenosis. Only a few premature ventricular beats were observed in 3 patients and there were no changes in S-T segments during dobutamine infusion. In another group of 15 patients selective vascular responses to an infusion of 10 microgram/kg . min dobutamine were examined during steady state cardiopulmonary bypass excluding heart and lungs from the circulation. No relevant direct influence on the arteriolar resistance vessels and the venous capacitance vessels were found. In a dose range of 2.5--5.0 microgram/kg . min dobutamine proved to be a potent inotropic agent causing almost no peripheral and relatively little positive chronotropic effects. But the increase in heart rate was more pronounced than in other clinical investigations in conscious patients, which might be due to an attenuation of vagal reflex by anaesthesia. The results indicate, that dobutamine may be a valuable drug in the treatment of intra- and postoperative low output syndromes especially in patients with coronary heart disease.

The influence of dihydroergotamine on volume content and compliance of the extrathoracic capacitance vessels during extracorporeal circulation was investigated. Compared with an untreated control group the compliance decreased (0.44 ml/mm Hg/kg b. wt.) and 490 ml of blood was mobilized and shifted from the circulation into the blood reservoir of the machine. Therefore DHE counteracts the effects of venous pooling.

Rabbit retinal Müller (glial) cells were isolated by means of papain and mechanical dissociation. In a special perfusion chamber, the cells were penetrated with a recording microelectrode. Membrane potential changes were recorded in response to extracellular application of both high-K+ solutions and of ouabain, and that during perfusion with normal and Na+-free solutions, respectively. In other Müller cell preparations, Na+,K+-adenosine triphosphatase (ATPase) activity was measured using a radiochemical method, and its Na+ dependence was determined. All results strongly suggest that the Müller cell's Na+,K+ pump can be activated in the presence of extremely low amounts of Na+. This provides additional evidence for significant differences between the glial and the neuronal enzyme.

Radial glial (Müller) cells were isolated from postnatal rabbit retinae by enzymatic dissociation in papain-containing solution, air-dried, and submitted to Pappenheim's panoptic stain. Morphometric data of these cells were evaluated by light microscopy. During postnatal development, the cells become substantially thicker and shorter, their nuclei lose the rod shape and move more toward scleral layers, and the nucleus-cytoplasm volume relation decreases. Whereas the cell volume increases from birth on, substantial outgrowth of fine side branches within the plexiform layers fails to occur before electrical activity is established there, i.e. after postnatal day 9. A model is proposed relating the growth of sheath-bearing glial processes to local protein synthesis stimulated by external K+ accumulation due to neuronal activity. Early myelinated nerve fibers are suggested to bear mechanical resistance to growing radial glial processes thus causing a splitting of these processes when they enter developing nerve fiber layers.

Müller (glial) cells from the rabbit retina were isolated by means of papain and mechanical dissociation. Their Na+,K+-adenosine triphosphatase (ATPase) activity was measured using a radiochemical method, and its K+ dependence was determined. In contrast to that of photoreceptors (data from the literature), the Na+,K+-ATPase of Müller cells could be shown to increase its activity greatly when the [K+] was enhanced up to 10 mM. The functional implications of this behaviour for the K+ clearance in the retina are discussed.

Two cases of cardiac asystole and one case of severe bradycardia were seen following a single injection of suxamethonium in a series of 46 adult patients in whom anaesthesia was induced with fentanyl and etomidate. It is suggested that the vagomimetic effects of fentanyl and, possibly also of etomidate, may contribute to the enhancement of the bradycardic effects of suxamethonium.

The haemodynamic effects of the new selective beta 1-receptor-agonist prenalterol were investigated in two groups each of 8 patients. Dosages of 500 microgram/5 min and 100 microgram/1 min were given after direct closure of atrial septal defect under neuroleptanalgesia. Prenalterol 500 microgram/5 min increased cardiac index (37.5%), heart rate (19%), stroke index (16.2%), mean arterial pressure (26.5%) and dp/dtmax (84.5%) significantly. Total systemic resistance, total pulmonary vascular resistance and mean pulmonary pressure remained constant. Filling pressures of both ventricles decreased slightly but significantly. Maximal hemodynamic changes after infusion of 100 microgram/1 min prenalterol were half as great as with 500 microgram/5 min and occurred 3 min earlier. The haemodynamic action of prenalterol was rather long lasting. One hour after cessation of the drug-infusion there were still detectable haemodynamic effects. In contrast to investigations in awake patients there was a greater increase in heart rate, which is referred to influences on the vagal reflex caused by anaesthesia.

The effect of 0.015 mg/kg KHE i.v. on the circulatory system was investigated in 10 patients after closure of an atrial septal defect under neuroleptanalgesia. The arterial pressure increased due to an increase of total systemic resistance. The filling pressures of the right and the left ventricle increased while the stroke index remained unchanged. The maximal rate of rise of left ventricular pressure did not change despite the marked changes of pre- and afterload. The data support the drug mechanism of a volume shift from the peripheral to the central circulation. The possible reasons for the missing stroke index increase are discussed.

To define the role of vecuronium in the occurrence of bradyarrhythmia, haemodynamic changes after the induction of anaesthesia were studied in 96 patients undergoing coronary artery bypass grafting. Patients were assigned to one of six groups according to different combinations of induction agents (etomidate 0.3 mg kg-1 or thiopentone 3 mg kg-1, with fentanyl 0.003 mg kg-1; etomidate 0.4-0.5 mg kg-1 or thiopentone 4-6 mg kg-1, without fentanyl) and neuromuscular blocking drugs (vecuronium 0.112 mg kg-1, pancuronium 0.112 mg kg-1 or suxamethonium 1 mg kg-1). Anaesthesia was maintained with enflurane and nitrous oxide in oxygen. After initial diverse changes, heart rate decreased in all groups. Thirty minutes after intubation, the reduction of heart rate showed statistically significant differences between the different combinations of drugs: fentanyl-etomidate-vecuronium (group I) (the largest reduction) greater than etomidate-vecuronium (II) = fentanyl-thiopentone-vecuronium (IV) greater than thiopentone-vecuronium (V) = fentanyl-thiopentone-suxamethonium (VI) = fentanyl-etomidate-pancuronium (III). Five patients in group I, two in group IV and one each in groups II and V had a heart rate slower than 45 beat min-1, whereas a similar value was never seen in groups III and VI. These results indicate that vecuronium has a bradycardic effect. This effect is more pronounced in association with etomidate than in association with thiopentone, and is augmented by the addition of fentanyl.

Retinal glial (Müller) cells were grown from explants of early postnatal rabbit retinae. The resulting monolayers of flat cells were exposed to control media (containing 5.85 mM K+), and to media with enhanced K+ concentrations (10 and 20 mM) or arginine-vasopressin (AVP, 20 micrograms/ml) or epithelial growth factor (EGF, 10 ng/ml). Autoradiographically, protein synthesis was quantified as L-[3H]-lysine incorporation, and DNA synthesis as [3H]-thymidine incorporation. Furthermore, the activity of Na+,K(+)-ATPase was measured radiochemically. Short exposure to either moderately enhanced K+ concentrations (10 mM) or to AVP, stimulated L-[3H]-lysine incorporation into the cells. Long-lasting exposure to either high K+ concentrations (20 mM) or to EGF stimulated [3H]-uptake. The Na+,K(+)-ATPase activity of cell cultures increased with increasing K+ concentration of the media. It is suggested that release of K+ by active neuronal compartments stimulates local protein synthesis of glial cells, resulting in the formation of glial sheaths with active K+ uptake capacity. Strong K+ release may even induce glial proliferation.