Renal function and lipid metabolism are major predictors of circumpapillary retinal nerve fiber layer thickness-the LIFE-Adult Study.

BACKGROUND: Circumpapillary retinal nerve fiber layer thickness (cpRNFLT) as assessed by spectral domain optical coherence tomography (SD-OCT) is a new technique used for the detection and evaluation of glaucoma and other optic neuropathies. Before translating cpRNFLT into clinics, it is crucially important to investigate anthropometric, biochemical, and clinical parameters potentially affecting cpRNFLT in a large population-based dataset. METHODS: The population-based LIFE-Adult Study randomly selected 10,000 participants from the population registry of Leipzig, Germany. All participants underwent standardized systemic assessment of various cardiometabolic risk markers and ocular imaging, including cpRNFLT measurement using SD-OCT (Spectralis, Heidelberg Engineering). After employing strict SD-OCT quality criteria, 8952 individuals were analyzed. Multivariable linear regression analyses were used to evaluate the independent associations of various cardiometabolic risk markers with sector-specific cpRNFLT. For significant markers, the relative strength of the observed associations was compared to each other to identify the most relevant factors influencing cpRNFLT. In all analyses, the false discovery rate method for multiple comparisons was applied. RESULTS: In the entire cohort, female subjects had significantly thicker global and also sectoral cpRNFLT compared to male subjects (p < 0.05). Multivariable linear regression analyses revealed a significant and independent association between global and sectoral cpRNFLT with biomarkers of renal function and lipid profile. Thus, thinner cpRNFLT was associated with worse renal function as assessed by cystatin C and estimated glomerular filtration rate. Furthermore, an adverse lipid profile (i.e., low high-density lipoprotein (HDL) cholesterol, as well as high total, high non-HDL, high low-density lipoprotein cholesterol, and high apolipoprotein B) was independently and statistically significantly related to thicker cpRNFLT. In contrast, we do not observe a significant association between cpRNFLT and markers of inflammation, glucose homeostasis, liver function, blood pressure, or obesity in our sector-specific analysis and globally. CONCLUSIONS: Markers of renal function and lipid metabolism are predictors of sectoral cpRNFLT in a large and deeply phenotyped population-based study independently of previously established covariates. Future studies on cpRNFLT should include these biomarkers and need to investigate whether incorporation will improve the diagnosis of early eye diseases based on cpRNFLT.

Scleral cross-linking by riboflavin and blue light application in young rabbits: damage threshold and eye growth inhibition.

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Dose-dependent collagen cross-linking of rabbit scleral tissue by blue light and riboflavin treatment probed by dynamic shear rheology.

PURPOSE: To determine the visco-elastic properties of isolated rabbit scleral tissue and dose-dependent biomechanical and morphological changes after collagen cross-linking by riboflavin/blue light treatment. MATERIAL: Scleral patches from 87 adult albino rabbit eyes were examined by dynamic shear rheology. Scleral patches were treated by riboflavin and different intensities of blue light (450 nm), and the impact on the visco-elastic properties was determined by various rheological test regimes. The relative elastic modulus was calculated from non-treated and corresponding treated scleral patches, and treatments with different blue light intensities were compared. RESULTS: Shear rheology enables us to study the material properties of scleral tissue within physiological relevant parameters. Cross-linking treatment increased the viscous as well as the elastic modulus and changed the ratio of the elastic versus viscous proportion in scleral tissue. Constant riboflavin application combined with different blue light intensities from 12 mW/cm(2) up to 100 mW/cm(2) increased the relative elastic modulus of scleral tissue by factors up to 1.8. Further enhancement of the applied light intensity caused a decline of the relative elastic modulus. This might be due to destructive changes of the collagen bundle structure at larger light intensities, as observed by histological examination. CONCLUSION: Collagen cross-linking by riboflavin/blue light application increases the biomechanical stiffness of the sclera in a dose-dependent manner up to certain light intensities. Therefore, this treatment might be a suitable therapeutic approach to stabilize the biomechanical properties of scleral tissue in cases of pathological eye expansion.

Physiologic properties of Müller cells from human eyes affected with uveal melanoma.

PURPOSE: To study physiologic characteristics of human Müller cells from healthy and pathologically altered eyes. METHODS: Human tissue was used from organ donors and from patients affected with uveal melanoma. Several melanoma eyes also showed retinal detachment. Incubation of freshly prepared slices with a commercial vital dye preferentially stained Müller cells. The Müller cell response to hypotonic stress was observed by recording the cross-sectional area of cell somata. Electrophysiologic properties were investigated in parallel in whole-cell patch-clamp experiments. RESULTS: Inward K+ currents mediated by inwardly rectifying Kir channels were significantly decreased in Müller cells from eyes with uveal melanoma compared with healthy controls. This was accompanied by a decrease of the membrane potential. Both effects were stronger in cells from eyes where the melanoma had caused a widespread retinal detachment. Application of a hypotonic solution did not affect Müller cells from healthy organ donors. By contrast, Müller cells from some melanoma eyes increased their soma size in response to hypotonic solution. This effect was aggravated in cells from eyes with widespread retinal detachment. The inflammatory mediator, arachidonic acid, could induce Müller cell swelling, whereas anti-inflammatory substances reduced the swelling. CONCLUSIONS: The experiments with human tissue confirm earlier data from animal models for retinal pathologies about typical alterations of reactive Müller cells. Hypotonic stress induced Müller cell swelling preferentially in cells from melanoma-affected eyes that displayed decreased inward current amplitudes. Widespread melanoma-associated retinal detachment potentiated the pathologic alterations of Müller cells.

Effects of intravitreal bevacizumab (Avastin) on the porcine retina.

BACKGROUND: To evaluate the effects of intravitreal bevacizumab (Avastin) on the porcine retina, with respect to structural alterations, expression of proteins involved in apoptosis (bax, caspase-3, caspase-9) and gliosis (vimentin, GFAP), expression of factors which influence the development of vascular edema (VEGF, PEDF), and of membrane channels implicated in retinal osmohomeostasis (Kir4.1, aquaporin-1, aquaporin-4). METHODS: One eye of seven adult pigs received a single intravitreal injection of bevacizumab (1.25 mg). Control eyes received buffered saline. For light and electron microscopy, the eyes were prepared 3 (one animal) and 7 days (two animals) after injection. Retinal slices were immunostained against gliosis- and apoptosis-related proteins. The gene expression was determined in the neuroretina and the retinal pigment epithelium of the remaining four animals with real-time RT-PCR 2 days after injection of bevacizumab. RESULTS: Intravitreal bevacizumab did not induce alterations in the retinal structure, neither at light microscopic nor at electron microscopic level. The photoreceptors were well-preserved; no signs of photoreceptor damage or mitochondrial swelling were observed. Bevacizumab did also not induce reactive gliosis (as indicated by the unaltered immunolocalization of the glial proteins vimentin, GFAP, and glutamine synthetase) or apoptosis (as indicated by the unaltered immunolocalization of bax, caspase-3, and caspase-9). Intravitreal bevacizumab decreased the transcriptional expression of VEGF-A, and increased the expression of Kir4.1 in the neuroretina and pigment epithelium, and of PEDF in the pigment epithelium. Bevacizumab did not alter the transcriptional expression of GFAP, bax, caspase-3, VEGF receptor-1 and -2, and aquaporin-1 and -4. CONCLUSIONS: A single intravitreal injection of bevacizumab does not result in structural changes of the porcine retina, nor in induction of gliosis or apoptosis. The bevacizumab-induced transcriptional downregulation of VEGF and upregulation of Kir4.1 might protect the retina from the development of vascular and cytotoxic edema.

Purinergic signaling involved in Müller cell function in the mammalian retina.

Purines (in particular, ATP and adenosine) act as neuro- and gliotransmitters in the sensory retina where they are involved in bidirectional neuron-glia signaling. This review summarizes the present knowledge about the expression and functional importance of P1 (adenosine) and P2 (nucleotide) receptors in Müller glial cells of the mammalian retina. Mammalian Müller cells express various subtypes of adenosine receptors and metabotropic P2Y receptors. Human Müller cells also express ionotropic P2X(7) receptors. Müller cells release ATP upon activation of metabotropic glutamate receptors and/or osmotic membrane stretching. The osmotic mechanism is abrogated under conditions associated with ischemia-hypoxia and inflammation, resulting in swelling of the Müller cells when the extracellular milieu is hypoosmotic. However, exogenous glutamate, which induces the release of ATP and adenosine, and thus activates P2Y(1) and A(1) adenosine receptors, respectively, prevents such osmotic swelling under pathological conditions, suggesting unimpaired receptor-induced release of ATP. In addition to the inhibition of swelling, which is implicated in regulating the volume of the extracellular space, purinergic signaling is involved in mediating neurovascular coupling. Furthermore, purinergic signals stimulate the proliferation of retinal precursor cells and Müller cells. In normal retinal information processing, Müller cells regulate the synaptic activity by the release of ATP and adenosine. In retinopathies, abrogation of the osmotic release of ATP, and the upregulation of ecto-apyrase (NTPDase1), may have neuroprotective effects by preventing the overactivation of neuronal P2X receptors that are implicated in apoptotic cell death. Pharmacological modulation of purinergic receptors of Müller cells may have clinical importance, e.g., for the clearance of retinal edema and for the inhibition of dysregulated cell proliferation in proliferative retinopathies.

Cooperative phagocytes: resident microglia and bone marrow immigrants remove dead photoreceptors in retinal lesions.

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

Glutaminyl cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer's disease-like pathology.

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Abeta peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimer's disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimer's disease and correlated with the appearance of pE-modified Abeta. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Abeta(3(pE)-42) burden in two different transgenic mouse models of Alzheimer's disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Abeta(x-40/42), diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Abeta(3(pE)-42) acts as a seed for Abeta aggregation by self-aggregation and co-aggregation with Abeta(1-40/42). Therefore, Abeta(3(pE)-40/42) peptides seem to represent Abeta forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Abeta by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimer's disease and provides implications for other amyloidoses, such as familial Danish dementia.

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.

The activation of IL-8 receptors in cultured guinea pig Müller glial cells is modified by signals from retinal pigment epithelium.

Interleukin 8 (IL-8, CXCL8) is a pro-inflammatory chemokine which attracts neutrophils to sites of inflammation via an activation of the G-protein-coupled receptors, CXCR1 and CXCR2. However, both IL-8 and IL-8 receptors are widely expressed in various tissues and cell types, and have been suggested to be involved in other functions such as angiogenesis, tumor growth, or brain pathology. We examined the expression of IL-8 and IL-8 receptors in highly enriched primary cultures of guinea pig Muller glial cells. Immunoreactivity for CXCL8, CXCR1 and CXCR2 was observed in all cultured Muller cells. The expression of CXCL8 was confirmed by PCR, and the secretion of the CXCL8 protein from Muller cells was revealed by ELISA. Western blots showed prominent bands at approximately 40 kDa by using antibodies specific for human CXCR1 and CXCR2, and the expression of a putative CXCR2 receptor in Muller cells was confirmed by PCR. Furthermore, cultured Muller cells responded to application of recombinant human IL-8 with an increase of the cytosolic Ca(2+) concentration. If supernatants of cultured human retinal pigment epithelium (RPE) cells were applied to the Muller cell cultures, no obvious changes were observed in the CXCL8, CXCR1 and CXCR2 expression but (i) Muller cell proliferation was stimulated, and (ii) there was an increased number of CXCL8-responsive Muller cells and the amplitudes of the evoked calcium responses were enhanced. It is concluded that Muller glial cells may participate in the inflammatory response(s) of the retina during ocular diseases, and that this contribution may be modified by interactions with RPE cells.

Glycogen phosphorylase isozyme pattern in mammalian retinal Müller (glial) cells and in astrocytes of retina and optic nerve.

Müller cells, the radially oriented dominant macroglial cells of the retina, are known to contain abundant glycogen as well as the key enzyme for its degradation, glycogen phosphorylase (GP), but the expressed isozyme pattern is unknown. To elucidate the isoform expression pattern, specific antisera directed against the brain (BB) and muscle (MM) isoforms of GP were applied to retinal sections, isolated Müller cells, and sections of the optic nerve. We show that Müller cells of rat, rabbit, guinea pig, and mouse retina exclusively express the BB isoform. Astrocytes of rat and rabbit optic nerve, as well as retina express only the BB isoform. In contrast, astrocytes in the brain and spinal cord as well as the epithelial cells of the pars caeca and of the ciliary body express both the BB and MM isoform. This result may indicate some differences in the role of glycogen in retinal macroglia and brain astrocytes, reflecting a local specialization of macroglia in the retina proper.

Early glial cell reactivity in experimental retinal detachment: effect of suramin.

PURPOSE: In a rabbit model of retinal detachment, early Müller glial cell reactivity was monitored-specifically, changes in membrane features-to determine whether these changes involve an upregulation of purinergic P2 receptor-mediated responses and whether all or some of these alterations could be blocked by suramin or pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS). In addition, the immune cell reactivity (microglial cells and blood-derived immune cells) was monitored. METHODS: A local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Three, 24, 48, and 72 hours after surgery, Müller cells were acutely isolated, and patch-clamp records of the whole-cell potassium currents were made. The presence of P2 receptor-mediated responses was determined by measuring extracellular adenosine triphosphate (ATP)-induced membrane current increases, and by recording of ATP-induced calcium responses at the vitreal surface of retinal wholemounts. The density of isolectin B(4)-labeled immune cells was determined in the nerve fiber layer of retinal wholemounts. RESULTS: Within 24 hours of detachment, Müller cell reactivity was evident. The cells downregulated the density of their inwardly rectifying potassium currents to 60% and 47% of the control value at 48 hours and 72 hours of detachment, respectively. This downregulation was accompanied by an enhanced incidence of cells which showed calcium and current responses after ATP application (control: 14%; 24 hours of detachment: 42%; 72 hours of detachment: 80%). Müller cell hypertrophy was apparent at 48 and 72 hours of detachment. Application of suramin during surgery inhibited the downregulation of potassium currents, but not the elevated responsiveness to extracellular ATP; PPADS had no effect. Suramin also inhibited the inflammatory response that was induced by the surgical procedure and that was apparent by the increased number of immune cells. CONCLUSIONS: Reactive responses of Müller cells occur within 24 hours of detachment. Suramin inhibits several (but not all) reactive glial alterations and therefore may represent one candidate for further investigations in the search for drugs that limit detrimental effects of immune cell activation and Müller cell gliosis during retinal detachment.

Upregulation of extracellular ATP-induced Müller cell responses in a dispase model of proliferative vitreoretinopathy.

PURPOSE: To test whether in an animal model of proliferative vitreoretinopathy (PVR) the Müller glial cells displayed an upregulation of purinergic P2 receptor-mediated responses. METHODS: PVR was induced by intravitreal injection of the proteolytic enzyme, dispase, in the eyes of adult rabbits. The developing PVR was examined ophthalmoscopically. After 3 weeks, small retinal pieces were wholemounted and used for calcium imaging, freshly dissociated Müller cells were subjected to calcium imaging, and patch-clamp recordings were made. The presence of P2 receptor-mediated Ca(2+) responses was determined both directly--that is, fluorometrically--and indirectly, by electrophysiological recording of Ca(2+)-activated K(+) currents. RESULTS: According to earlier observations in another model of retinal detachment and PVR, the reactive Müller cells displayed hypertrophy, downregulation of inwardly rectifying K(+) currents, and depolarization of the resting membrane potential, all dependent on the severity of the PVR. Further, significant PVR-induced increase was observed in the number of Müller cells responding to adenosine 5'-triphosphate (ATP), with a transient elevation of their [Ca(2+)](i). If isolated Müller cells were exposed to ATP, 13% of the control cells, but 29% (moderate PVR) or 53% (massive PVR) of the reactive cells, showed fluorometric Ca(2+) increases. An increase of Ca(2+)-activated K(+) currents was measured in 11% of the control cells, but in 83% (moderate PVR) and 90% (massive PVR) of the reactive cells. Confocal images of retinal wholemounts revealed similar results. Because similar responses were elicited by uridine triphosphate (UTP), the dominant involvement of metabotropic (P2Y type) purinergic receptors is suggested. CONCLUSIONS: An upregulation of purinergic receptors is part of the reactive changes of Müller cells during PVR. It is suggested that ATP-evoked Ca(2+) responses may support the proliferation of Müller cells during PVR.