The cellular mechanisms underlying glial cell swelling, a central cause of edema formation in the brain and retina, are not yet known. Here, we show that glial cells in the postischemic rat retina, ...but not in control retina, swell upon hypotonic stress. Swelling of control cells could be evoked when their K
+ channels were blocked. After transient ischemia, glial cells strongly downregulated their K
+ conductance and their prominent Kir4.1 protein expression at blood vessels and the vitreous body. In contrast, the expression of the aquaporin-4 (AQP4) (water channel) protein was only slightly altered after ischemia. Activation of D
2 dopaminergic receptors prevents the hypotonic glial cell swelling. The present results elucidate the coupling of transmembraneous water fluxes to K
+ currents in glial cells and reveal the role of altered K
+ channel expression in the development of cytotoxic edema. We propose a mechanism of postischemic glial cell swelling where a downregulation of their K
+ conductance prevents the emission of intracellularly accumulated K
+ ions, resulting in osmotically driven water fluxes from the blood into the glial cells via aquaporins. Inhibition of these water fluxes may be beneficial to prevent ischemia-evoked glial cell swelling.
To determine whether the expression of voltage-gated Ca2+ channels in human Müller glial cells changes during normal aging and in cells from patients with proliferative vitreoretinopathy (PVR).
...Müller cells were enzymatically isolated from retinas of healthy donors and from excised retinal pieces of patients with PVR, and the whole-cell, voltage-clamp technique was used to characterize the current densities of transient, low-voltage-activated calcium channels and of sustained. high-voltage-activated calcium channels, respectively. To obtain maximal currents through both channel types, Na+ ions were used as the charge carrier.
During normal aging, Müller cells developed a hypertrophy, as indicated by an increase of the cell membrane capacitance. The mean membrane capacitance of cells from aged donors (> or = 60 years old) was elevated by 25% compared with cells from younger donors. The hypertrophy was not accompanied by a changed density of low-voltage-activated currents, whereas the density of the high-voltage-activated currents was enhanced by 76%. The density of the high-voltage-activated currents increased in correlation with the increase of the cell membrane capacitance and with the age of the donors. In the case of PVR, Müller cells displayed a strong hypertrophy accompanied by a downregulation of both current types by approximately 65%.
Both normal aging and PVR cause a gliotic reactivity of human Müller cells, as indicated by their hypertrophy. The type of reactivity, however, differs between the two conditions. Normal aging is accompanied by an increased expression of voltage-gated Ca2+ channels, whereas in PVR Ca2+ channel expression is decreased.
The human major histocompatibility complex (MHC) class I loci, human leukocyte antigen (HLA)-A, -B, -C, encode highly polymorphic molecules that mediate immune recognition of infectious pathogens and ...can initiate the rapid rejection of transplanted tissue. Cloning of HLA class I alleles is complicated by polymorphism as well as interlocus homology. Here, HLA class I cDNAs are amplified by PCR using one common primer with one of three locus specific primers whose 3′ ends map to conserved, locus specific nucleotides. Using these primers, HLA-A, -B, and -C alleles were cloned from a number of cell lines and two different HLA-B alleles were cloned from a single, heterozygous cell line. The amplified products encode the entire extracellular portion of the class I molecules. An amplified HLA-A allele was cloned into an expression vector and the protein product was detected on the surface of a transfected cell. A premature termination codon was engineered into the HLA-A allele by site directed mutagenesis and the soluble protein product was detected in the culture medium of transfected cells. Therefore, these primers can be used to rapidly clone, alter, and express HLA class I molecules. This method may expedite the generation of reagents for testing the antigen specificity of antibodies, natural killer cells, or T cells.
Atherosclerosis is complicated by cardiovascular events such as myocardial infarction, stroke, or peripheral arterial occlusive disease. Inflammation and pathological neovascularization are thought ...to precipitate plaque rupture or erosion, both causes of arterial thrombosis and cardiovascular events. We tested the hypothesis that arterial inflammation and angiogenic events are increased throughout the arterial tree in vulnerable patients, ie, in patients who suffered from cardiovascular events, compared with patients who never suffered from complications of atherosclerosis.
In a postmortem study, we quantified the inflammatory infiltrate and microvascular network in the arterial wall of iliac, carotid, and renal arteries. Tissue microarray technology was adapted to investigate full-thickness arterial sectors. We compared 22 patients with symptomatic atherosclerosis with 27 patients who never had suffered from any cardiovascular event. The absolute intimal macrophage content was 2- to 4-fold higher in vulnerable patients at all 3 arterial sites analyzed (P<0.05). Patients with symptomatic atherosclerosis had a denser network of vasa vasorum than patients with asymptomatic disease (33+/-2 versus 25+/-2 adventitial microvessels per 1 mm2; P=0.008). Hyperplasia of vasa vasorum was an early and macrophage infiltration was a late sign of symptomatic atherosclerosis.
High intimal macrophage content and a hyperplastic network of vasa vasorum characterize vulnerable patients suffering from symptomatic atherosclerosis. These changes are uniformly present in different arterial beds and support the concept of symptomatic atherosclerosis as a panarterial disease.
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.
Arachidonic acid, which is thought to be involved in pathogenetic mechanisms of the central nervous system, has been shown previously to modulate neuronal ion channels and the glutamate uptake ...carrier of retinal glial (Müller) cells. We have used various configurations of the patch-clamp technique to determine the effects of arachidonic acid on the K
+ currents of freshly isolated Müller glial cells from rabbit and human. Arachidonic acid reduced the peak amplitude of the transient (A-type) outward K
+ currents in a dose-dependent and reversible manner, with a 50% reduction achieved by 4.1
μM arachidonic acid. The inward rectifier-mediated currents remained unchanged after arachidonic acid application. The amplitude of the Ca
2+-activated K
+ outward currents (K
Ca), which were blocked by 1
mM tetraethylammonium chloride and 40
nM iberiotoxin, respectively, was dose-dependently elevated by bath application of arachidonic acid. The activation curve of the K
Ca currents shifted towards more negative membrane potentials. Furthermore, arachidonic acid was found to suppress inwardly directed Na
+ currents. In cell-attached recordings with 3
mM K
+ in the bath and 130
mM K
+ in the pipette, the K
Ca channels of rabbit Müller cells displayed a linear current–voltage relation, with a mean slope conductance of 102
pS. In excised patches, the slope conductance was 220
pS (150
mM K
+
i/130
mM K
+
o). The opening probability of the K
Ca channels increased during membrane depolarization and during elevation of the free Ca
2+ concentration at the intracellular face of the membrane patches. Bath application of arachidonic acid caused a reversible increase of the single-channel opening probability, as well as an increase of the number of open channels. Arachidonic acid did not affect the single-channel conductance. Since arachidonic acid also stimulates the K
Ca channel activity in excised patches, the action of arachidonic acid is assumed to be independent of changes of the intracellular calcium concentration.
Our results demonstrate that arachidonic acid exerts specific effects on distinct types of K
+ channels in retinal glial cells. In pathological cases, elevated arachidonic acid levels may contribute to prolonged Müller cell depolarizations, and to the initiation of reactive glial cell proliferation.
For macular translocation surgery, the native attached retina has to be detached either locally or completely. Although different surgical techniques are used, there is a general search for ...supporting procedures that facilitate and accelerate the retinal detachment.
Pars plana vitrectomies were performed in pigmented rabbits. In all experimental groups, a local retinal detachment was created by infusing the test solution with a thin glass micropipette attached to a glass syringe. In control animals a standard balanced salt solution was used at room temperature, in combination with a standard vitrectomy light source. In two test groups, a calcium- and magnesium-free solution was used for the vitrectomy, under illumination by a standard light source in group I (solution at room temperature) and group II (solution heated up to body temperature). In group III the rabbits were dark-adapted for half an hour, and then, during surgery, a red filter was used in front of the light source (standard balanced salt solution at room temperature). After the rabbits were killed at the end of surgery, the adherence of the retinal pigment epithelium (RPE) to the neural retina in the detachment area was quantified microscopically, and the morphologic integrity of the detached retinal tissue was examined by light and electron microscopy. No electrophysiology was performed.
In all four groups, it was possible to detach the retina. The maximum adherence of the RPE cells to the neural retina was observed in the control group. Virtually no decrease in adherence was found in test group II (36 degrees C solution without calcium and magnesium), whereas a significant decrease was seen in both group I (calcium- and magnesium-free solution at room temperature) and group III (dark adaptation-red light technique; standard balanced salt solution at room temperature). In none of the experimental groups was any obvious damage of the retinal structure observed, even after exposure to the test solutions for 60 minutes.
Both dark adaptation (red illumination) and the use of a calcium chloride- and magnesium chloride-free solution (at room temperature) can facilitate retinal detachment in macular translocation surgery. Both techniques are proposed as a gentle support for the operation, because they protect an intact RPE cell layer and do not cause retinal damage at the ultrastructural level.
In this letter we present a hierarchical unit cell incorporating a nonlinear energy sink (NES) that functions as a low-pass amplitude filter. The hierarchical unit cell is composed of an outer and ...inner mass coupled by essentially nonlinear springs. Other than its nonlinear coupling to the outer mass, the inner mass is otherwise unconstrained and thus acts as an NES. Using numerical simulations, we show that the nonlinear unit cell passively filters incident waves as a function of their amplitude. The resonator is inactive at low amplitudes, permitting waves to pass. At high amplitudes, the NES activates and its resonant behavior reflects incident waves. Consequently, the unit cell effectively operates as an amplitude limiter for specific signal frequency and amplitude ranges. Notably, the passing of low-amplitude incident energy while bypassing the nonlinearity results in very little distortion in transmitted signals. We analytically investigate this behavior further using a harmonic balance method. The analysis supports the transmission filtering behavior as well as the activation of the nonlinear energy sink. Lastly, we design and construct the nonlinear unit cell and perform tests using incident and transmitted waveguides composed of monatomic chains. Both the nonlinear unit cell and monatomic chains are assembled using steel masses and additively-manufactured springs. We test the fabricated filter using low and high amplitude signals and observe amplitude filtering at various excitation frequencies that document strong agreement with numerical simulations and the analytical model. The ability to passively reflect high amplitude waves, and transmit low amplitude waves with minimal distortion, may inspire new devices for hearing protection, or for isolating and protecting sensitive equipment and instruments.
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