Susann Uhlmann

PURPOSE: Detachment of the neural retina from the pigment epithelium causes, in addition to photoreceptor deconstruction and neuronal cell remodeling, an activation of glial cells. It has been suggested that gliosis contributes to the impaired recovery of vision after reattachment surgery that may involve both formerly detached and nondetached retinal areas. Müller and microglial cell reactivity was monitored in a porcine model of rhegmatogenous retinal detachment, to determine whether gliosis is present in detached and nondetached retinal areas. METHODS: Local detachment was created in the eyes of adult pigs by subretinal application of hyaluronate. Retinal slices were immunostained against glial intermediate filaments and K+ and water channel proteins (aquaporin-4, Kir4.1, Kir2.1), and P2Y receptor proteins. In retinal wholemounts, adenosine 5'-triphosphate (ATP)-induced intracellular Ca2+ responses of Müller cells were recorded, and microglial and immune cells were labeled with Griffonia simplicifolia agglutinin isolectin I-B4. K+ currents were recorded from isolated Müller cells. RESULTS: At 3 and 7 days after surgery, Müller cells in detached retinas showed a pronounced gliosis, as revealed by the increased expression of the intermediate filaments glial fibrillary acidic protein and vimentin, by the decrease of Kir4.1 immunoreactivity and of the whole-cell K+ currents, and by the increased incidence of cells that showed Ca2+ responses on stimulation of purinergic (P)2 receptors by ATP. By contrast, the immunohistochemical expression of Kir2.1 and aquaporin-4 were not altered after detachment. The increase in the expression of intermediate filaments, the decrease of the whole-cell K+ currents and of the Kir4.1 immunolabeling, and the increase in the Ca2+ responsiveness of Müller cells were also observed in attached retinal areas surrounding the focal detachment. The density of microglial-immune cells at the inner surface of the retinas increased in both detached and nondetached retinal areas. The immunoreactivities for P2Y1 and P2Y2 receptor proteins apparently increased only in detached areas. CONCLUSIONS: Reactive responses of Müller and microglial cells are not restricted to detached retinal areas but are also observed in nondetached regions of the porcine retina. The gliosis in the nondetached retina may reflect, or may contribute to, neuronal degeneration that may explain the impaired recovery of vision observed in human subjects after retinal reattachment surgery.

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.

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.

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca(2+)-activated K(+) currents (I(BK)) caused by release of Ca(2+) 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 GABA(A) and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the I(BK) 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 I(BK) amplitude with half-maximal effects at 0.33 and 0.50 microM, respectively. Since several different P2 receptor agonists increased the I(BK), 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 I(BK) and the appearance of the cation conductance in whole-cell records were abolished when intracellular Ca(2+) was buffered by a high-EGTA pipette solution or when IP(3) 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 IP(3) formation, cause intracellular Ca(2+) release. The increased intracellular Ca(2+) 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 Ca(2+)-gated cation channels may be important in respect to proliferative Müller cell gliosis.