Susann Uhlmann

Activation of P2Y receptors stimulates potassium and cation currents in acutely isolated human Müller (glial) cells

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca2+‐activated K+ currents (IBK) caused by release of Ca2+ from intracellular stores was investigated in Müller glial cells of the human retina. Müller cells were enzymatically isolated from retinas of healthy donors or of patients with proliferative vitreoretinopathy, and the transmembrane ionic currents were recorded using the whole‐cell and the cell‐attached patch‐clamp techniques. The results of the screening experiments indicate that human Müller cells express, in addition to GABAA and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the IBK amplitude and, in a subpopulation of cells investigated, the appearance of a transient cation conductance at negative potentials. ATP and UTP increased dose‐dependently the IBK amplitude with half‐maximal effects at 0.33 and 0.50 μM, respectively. Since several different P2 receptor agonists increased the IBK, it is assumed that human Müller cells express a mixture of different types of P2Y receptors. In cell‐attached patches, extracellular application of ATP or UTP transiently increased the open probability of single putative BK channels. The increase of IBK and the appearance of the cation conductance in whole‐cell records were abolished when intracellular Ca2+ was buffered by a high‐EGTA pipette solution or when IP3 was included in the pipette solution. The expression of agonist‐evoked transient cation currents was found to be stronger in cells from patients as compared to cells from healthy donors. It is concluded that human Müller glial cells express P2Y receptors that, via IP3 formation, cause intracellular Ca2+ release. The increased intracellular Ca2+ concentration stimulates the activity of BK channels and may induce opening of cation channels. Both the ATP‐induced activity of BK channels and the increased expression of Ca2+‐gated cation channels may be important in respect to proliferative Müller cell gliosis. GLIA 37:139–152, 2002.

Early Activation of Inflammation-and Immune Response-Related Genes after Experimental Detachment of the Porcine Retina

PURPOSE To determine early alterations in retinal gene expression in a porcine model of rhegmatogenous retinal detachment. METHODS Local detachment was created in eyes of adult pigs by subretinal application of sodium hyaluronate. The gene expression in control tissues and retinas detached for 24 hours was analyzed with a pig genome microarray. Genes with at least three-fold expression changes were detected in the detached retina and in the attached retinal tissue surrounding the local detachment in situ. Structural alterations of the retina were examined by light and electron microscopy. RESULTS Identified were 85 genes that were upregulated and 7 that were downregulated in the detached retina. Twenty-eight genes were identified as upregulated in the nondetached retina of the surgical eyes. The genes upregulated in detached retinas were related to inflammation and immune responses (n = 52), antioxidants and metal homeostasis (n = 7), intracellular proteolysis (n = 6), and blood coagulation/fibrinolysis (n = 4). The upregulation of at least 15 interferon-stimulated genes indicates elevated interferon levels after detachment. Gene expression of blue-sensitive opsin was not detectable in the detached retinal tissue, suggesting an early reduction in phototransduction, especially in blue cones. Electron microscopy revealed an accumulation of microglial cells in the inner retinal tissue and of polymorphonuclear leukocytes in the vessels of detached and peridetached retinas. CONCLUSIONS Differentially expressed genes in the retina early after experimental detachment are mainly related to inflammation and immune responses, intracellular proteolysis, and protection against oxidative stress. A local immune and inflammatory response may represent a major causative factor for reactive changes in the retina after detachment. The inflammatory response is not restricted to the detached retina but is also observed in the nondetached retina; this response may underlie functional changes in these regions described in human subjects.

Evaluation of hepatic microcirculation by in vivo microscopy.

In vivo microscopy is an excellent technique for investigating the microcirculation and until recently the only one that allowed direct visualization. Rat liver has been widely studied, because microcirculatory disorders play a pivotal role in the pathogenesis of organ failure during hepatic ischemia, transplantation, hemorrhagic shock, endotoxemia, and sepsis. The state of the microcirculation is an important prognostic factor for the reestablishment of organ function after these injuries. This article introduces the most common procedures for in vivo microscopy of the rat liver, summarizes the available fluorescent dyes, and gives an overview of criteria for the expression and evaluation of microscopic findings. Particular emphasis is given to a description of the different parameters assessed by direct observation of hepatic microcirculation, such as perfusion rate, leukocyte-endothelium interactions, leukocyte velocities, and phagocytic activity. Examples of normal range values are given. This overview is intended to help those wanting to introduce this method into their research and who are embarking on intravital microscopy for the first time, and to enable them to decide which techniques are appropriate for answering special questions.

Glial cell-mediated spread of retinal degeneration during detachment: A hypothesis based upon studies in rabbits

In human subjects with peripheral retinal detachments, visual deficits are not restricted to the detached retina but are also present in the non-detached tissue. Based upon studies on a rabbit model of rhegmatogenous retinal detachment, we propose a glial cell-mediated mechanism of spread of retinal degeneration into non-detached retinal areas which may also have importance for the understanding of alterations in the human retina. Both detached and attached portions of the rabbit retina display photoreceptor cell degeneration and cystic degeneration of the innermost layers. An inverse mode of photoreceptor cell degeneration in the attached tissue suggests a disturbed support of the photoreceptor cells by Müller cells which show various indications of gliosis (increased expression of intermediate filaments, cell hypertrophy, decreased plasma membrane K(+) conductance, increased Ca(2+) responsiveness to purinergic stimulation) in both detached and attached tissues. We propose that gliotic alterations of Müller cells contribute to the degeneration of the attached retina, via disturbance of glial homeostasis mechanisms. A down-regulation of the K(+) conductance of Müller cells may prevent effective retinal K(+) and water clearance, and may favor photoreceptor cell degeneration and edema development.

Expression of CXCL8, CXCR1, and CXCR2 in neurons and glial cells of the human and rabbit retina.

PURPOSE Several eye diseases are accompanied by inflammatory processes. The authors examined the expression of the proinflammatory chemokine CXCL8 and the corresponding receptors in healthy human retinas, in cellular membranes from patients with proliferative vitreoretinopathy (PVR) or human glial cell cultures and in an animal model of PVR in rabbit eyes. METHODS The authors used immunohistochemical methods, Western blotting, RT-PCR, and real time RT-PCR to characterize the expression of CXCL8, CXCR1, and CXCR2 in human and rabbit retinas. Functionality of the receptors in cultured glial cells was tested by Ca(2+) imaging. RESULTS Immunohistochemical examinations of normal human and rabbit retinas revealed a distinct expression of CXCR1 and CXCR2 in several neuronal cell types. CXCL8 mRNA was demonstrated only by RT-PCR in normal retinas, and receptor expression was confirmed by Western blotting and RT-PCR. The presence of CXCR1 and CXCR2, but not CXCL8, was detected by immunostaining in glial fibrillary acidic protein-positive glial cells of cellular PVR membranes. Immunoreactivity for CXCL8, CXCR1, and CXCR2 was observed in virtually all cultured glial cells and in the human Müller cell line MIO-M1. Müller cells responded to the application of CXCL8 with increased cytosolic Ca(2+) concentrations. In PVR rabbit retinas, CXCR1 expression is increased in Müller cells, and CXCL8 and CXCR2 are strongly expressed in microglial cells. CONCLUSIONS Expression of CXCL8 and CXCL8 receptors in glial cells of human PVR membranes and rabbit PVR retinas suggests an involvement in glial reactivity. Furthermore, the prominent expression of CXCR1 and CXCR2 in neurons of the healthy human and rabbit retina suggests additional physiological functions.

Endothelin/nitric oxide balance influences hepatic ischemia-reperfusion injury

During ischemia-reperfusion an imbalance between endothelin (ET) and nitric oxide (NO) can be responsible for microcirculatory disturbances. The aim of this study was to restore the ET/NO balance to reduce the ischemia-reperfusion injury. Hepatic ischemia was induced for 30 min in 56 Wistar rats. Sham operation, ischemia and treatment groups with the ET receptor antagonist (ERA) bosentan (1 mg/kg body weight i.v.) and the NO donor L-arginine (400 mg/kg body weight i.v.) were performed. For evaluation of hepatic microcirculation in vivo microscopy was carried out 30-90 min after reperfusion. Local hepatic tissue PO2, laser Doppler flow and aspartate aminotransferase/alanine aminotransferase (AST/ALT) levels were measured. Increased ET caused sinusoidal constriction after reperfusion to 76% of the sham group (p < 0.05), leading to significant decrease in perfusion rate (82%), liver tissue PO2 (6.9 mmHg) and erythrocyte flux (45.2% of sham group). Hepatocellular damage could be detected 6 h after reperfusion by AST/ALT increase (p < 0.05). Sinusoidal diameters were maintained at baseline in the ERA (98%) and NO (102%) groups (p < 0.05). Increased percentage of leukocytes sticking in sinusoids (144%) and venules (435%) was reduced by therapy to 110/253% (ERA) and 111/324% (NO), respectively (p < 0.05). Perfusion rate was increased to 93 and 94% (p < 0.05 vs ischemia). Local hepatic tissue PO2 was improved 30 min after reperfusion in the ERA (11.0 mmHg) as well as in the NO group (11.5 mmHg; p < 0.05 vs ischemia). Measurement with a laser Doppler flow meter revealed significant improved erythrocyte flux in both therapy groups (p < 0.05 vs ischemia). Also, the post-ischemic AST/ALT increase was reduced by therapy. In conclusion, ET evokes strong constriction of post-ischemic sinusoids, leading to microcirculatory disturbances. The maintenance of the ET/NO balance by blocking ET receptors, or providing an NO donor, protects liver microcirculation and reduces hepatic ischemia-reperfusion injury.

Experimental Dispase-Induced Retinopathy Causes Up-Regulation of P2Y Receptor-Mediated Calcium Responses in Müller Glial Cells

During proliferative vitreoretinopathy (PVR) Müller glial cells show an up-regulation of their responsiveness to extracellular adenosine 5′-triphosphate (ATP). In the present study, we investigated if such a glial cell response is also a feature for other retinopathies besides PVR. To this aim, the proteolytic enzyme, dispase (0.1 U), was injected into the vitreous of rabbit eyes. After 3 weeks, a distinct retinopathy had developed which showed no signs of PVR. The retinopathy was characterized by strong alterations of the retinal vasculature in the medullary rays, by photoreceptor degeneration, retinal atrophy, and activation of microglial cells. Müller cells became reactive, as indicated by up-regulation of glial fibrillary acidic protein immunoreactivity and by hypertrophy involving subretinal fibrosis. Müller cell reactivity was also evidenced electrophysiologically by a down-regulation of their inwardly rectifying potassium currents and by an up-regulation of their responsiveness to extracellular ATP. Significantly more Müller cells from dispase-treated eyes showed ATP-evoked calcium (83%) and current responses (69%) when compared with cells from control eyes (13 and 9%, respectively). The results indicate that increased responsiveness to extracellular ATP may be a more general feature of Müller cell gliosis, and is also observed in retinopathies besides PVR.

Upregulation of purinergic P2Y receptor-mediated calcium responses in glial cells during experimental detachment of the rabbit retina

To investigate injury-induced alterations of purinergic P2Y receptor-mediated calcium responses in glial (Müller) cells of the rabbit retina, neural retinae were experimentally detached from the pigment epithelium. The ATP-evoked calcium responses were recorded in the endfeet of glial cells at the vitread surface of retinal wholemounts. In control retinae, approximately 7% of the glial cells investigated showed ATP-evoked calcium responses. Within 24 h of detachment, significantly more retinal glial cells (42%) showed calcium responses, and glial ATP responsiveness increased further in retinae which were detached for 48 (44%) or for 72 h (64%). The results indicate that in the detached retina, glial cells upregulate their responsiveness to extracellular ATP within 24 h of injury. Thus, P2Y receptor-mediated signalling may be involved in the early steps of glial response to retinal injury.

Important Role for Endothelins in Acute Hepatic Ischemia/Reperfusion Injury

The aim of this study was to explore the complex role of endothelins (ETs) in hepatic ischemia-reperfusion injury and to minimize this type of injury by nonselective ET receptor blockade. In an in vivo rat model, hepatic ischemia was induced for 30 min. The rats were divided into three groups: (1) sham operated, (2) untreated ischemic, and (3) group treated with the nonselective ET receptor antagonist bosentan (1 mg/kg body weight iv). Blockage of the ET system during ischemia-reperfusion was assessed by: (1) in vivo microscopic analysis, (2) measurement of local tissue PO2, (3) laser Doppler flowmetry, (4) transaminases, and (5) tumor necrosis factor (TNF)- serum levels. During liver ischemia, anoxia (mean liver pO2 decreased from 14.7 to 1.5 mm Hg) and TNF- (levels rose from 0 pg/mL to 145.3 pg/mL at the end ofischemia) were associated with the release of ETs. Immunoreactive ET-1 (ir-ET-1) plasma levels (basal levels: 12.1+/-1.8 pg/mL) went up by 2.6-fold (32.1+/-6.8 pg/mL) after 15 min and by 11.7-fold (142.1+/-32.6 pg/mL) after 120 min of reperfusion. Increased plasma levels of ir-ET-1 were associated with sinusoidal constriction to 77.6+/-7.1% of basal diameters. This constriction led to significant decreases in perfusion rate (77+/-3%), local tissue PO2 (6.9+/-2.7 mm Hg), and erythrocyte flux (61.7+/-13.8% of basal values). Hepatocellular damage, evaluated via the serum level of aspartate aminotransferase (AST, increase to 393.5+/-68.3 U/L, preoperative 23.9+/-2.0 U/L) was detectable 6 h after reperfusion (p < .05). Administration of bosentan before 30 min of ischemia significantly reduced ischemia-reperfusion injury and was associated with an increase of ir-ET-1 levels to 110.8+/-12.0 pg/mL and 94.1+/-25.0 pg/mL after 15 and 120 min of reperfusion. Sinusoidal diameters were maintained at nearly 100% in the treatment group instead of 77%, while perfusion rate (88+/-2%) and tissue PO2 (12.1+/-1.0 mm Hg) rose significantly in contrast with the nontreatment group (p < .05). Hepatocellular damage was reduced (AST levels after 6 h of reperfusion 244.0+/-34.4 U/L, p <.05), and leukocyte sticking and rolling were diminished (p < .01). In the treatment group, bosentan values of 5.6+/-0.7 and 2.9+/-0.4 ng/mL after 15 and 120 min of reperfusion were measured. In conclusion the release of endothelins is combined with microcirculatory disturbances and local hypoxia, thereby causing liver damage. By protecting the liver microcirculation, ET receptor blockade of both receptors at a low dose increased blood and oxygen supply to the liver and reduced hepatocellular injury. These results constitute the bases for further studies and transfer into clinical practice.The aim of this study was to explore the complex role of endothelins (ETs) in hepatic ischemia-reperfusion injury and to minimize this type of injury by nonselective ET receptor blockade. In an in vivo rat model, hepatic ischemia was induced for 30 min. The rats were divided into three groups: (1) sham operated, (2) untreated ischemic, and (3) group treated with the nonselective ET receptor antagonist bosentan (1 mg/kg body weight iv). Blockage of the ET system during ischemia-reperfusion was assessed by: (1) in vivo microscopic analysis, (2) measurement of local tissue pO2

Endothelin receptors in the detached retina of the pig.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ET(A) and ET(B), in control and locally detached retinas of the pig. Immunoreactivity for ET(A) was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ET(B) was expressed by the innermost retinal layers, by ganglion cell somata, and by Müller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ET(A) may suggest a hypoperfusion of the retina after detachment.