nanite: using machine learning to assess the quality of atomic force microscopy-enabled nano-indentation data.

BACKGROUND: Atomic force microscopy (AFM) allows the mechanical characterization of single cells and live tissue by quantifying force-distance (FD) data in nano-indentation experiments. One of the main problems when dealing with biological tissue is the fact that the measured FD curves can be disturbed. These disturbances are caused, for instance, by passive cell movement, adhesive forces between the AFM probe and the cell, or insufficient attachment of the tissue to the supporting cover slide. In practice, the resulting artifacts are easily spotted by an experimenter who then manually sorts out curves before proceeding with data evaluation. However, this manual sorting step becomes increasingly cumbersome for studies that involve numerous measurements or for quantitative imaging based on FD maps. RESULTS: We introduce the Python package nanite, which automates all basic aspects of FD data analysis, including data import, tip-sample separation, base line correction, contact point retrieval, and model fitting. In addition, nanite enables the automation of the sorting step using supervised learning. This learning approach relates subjective ratings to predefined features extracted from FD curves. For ratings ranging from 0 to 10, our approach achieves a mean squared error below 1.0 rating points and a classification accuracy between good and poor curves that is above 87%. We showcase our approach by quantifying Young's moduli of the zebrafish spinal cord at different classification thresholds and by introducing data quality as a new dimension for quantitative AFM image analysis. CONCLUSION: The addition of quality-based sorting using supervised learning enables a fully automated and reproducible FD data analysis pipeline for biological samples in AFM.

Cone-to-Müller cell ratio in the mammalian retina: A survey of seven mammals with different lifestyle.

Mammalian retinal glial (Müller) cells are known to guide light through the inner retina to photoreceptors (Franze et al., 2007; Proc Natl Acad Sci U S A 104:8287-8292). It was shown that Müller cells transmit predominantly red-green and less violet-blue light (Labin et al., 2014; Nat Commun 5:4319). It is not known whether this optical function is reflected in the cone-to-Müller cell ratio. To determine this ratio in the retinas of mammals with different lifestyle, we evaluated the local densities of cones and Müller cells in the retinas of guinea pigs, rabbits, sheep, red deer, roe deer, domestic pigs, and wild boars. Retinal wholemounts were labeled with peanut agglutinin to mark cones and anti-vimentin antibodies to identify Müller cells. Wholemounts of guinea pig and rabbit retinas were also labeled with anti-S-opsin-antibodies. With the exceptions of guinea pig and pig retinas that had cone-to-Müller cell ratios of above one, the local densities of cones and Müller cells in the retinas of the species investigated were roughly equal. Because the proportion of S-cones is usually low (for example, 5.3% of all cones in the dorsal guinea pig retina expressed S-opsin), it is suggested that Müller cells are mainly coupled to M-cones. Exceptions are the ventral peripheries of guinea pig and rabbit retinas which are specialized areas with high S-cone densities. Here, up to 50% of Müller cells may be coupled to S-cones, and 40% of S-cones may be not coupled to Müller cells. Among the species investigated, the density of Müller cells in the central retina was inversely correlated with the axial length of the eyes. It is suggested that (with the exception of specialized S-cone areas) Müller cells support high acuity vision by predominant guidance of red-green light to M-opsin expressing cones.

Colloidal crystals of compliant microgel beads to study cell migration and mechanosensitivity in 3D.

Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. However, studying the effects of mechanics in in vitro 3D environments is challenging since current 3D hydrogel assays convolve mechanics with gel porosity and adhesion. Here, we present novel colloidal crystals as modular 3D scaffolds where these parameters are principally decoupled by using monodisperse, protein-coated PAAm microgel beads as building blocks, so that variable stiffness regions can be achieved within one 3D colloidal crystal. Characterization of the colloidal crystal and oxygen diffusion simulations suggested the suitability of the scaffold to support cell survival and growth. This was confirmed by live-cell imaging and fibroblast culture over a period of four days. Moreover, we demonstrate unambiguous durotactic fibroblast migration and mechanosensitive neurite outgrowth of dorsal root ganglion neurons in 3D. This modular approach of assembling 3D scaffolds from mechanically and biochemically well-defined building blocks allows the spatial patterning of stiffness decoupled from porosity and adhesion sites in principle and provides a platform to investigate mechanosensitivity in 3D environments approximating tissues in vitro.

Müller glial cells contribute to dim light vision in the spectacled caiman (Caiman crocodilus fuscus): Analysis of retinal light transmission.

In this study, we show the capability of Müller glial cells to transport light through the inverted retina of reptiles, specifically the retina of the spectacled caimans. Thus, confirming that Müller cells of lower vertebrates also improve retinal light transmission. Confocal imaging of freshly isolated retinal wholemounts, that preserved the refractive index landscape of the tissue, indicated that the retina of the spectacled caiman is adapted for vision under dim light conditions. For light transmission experiments, we used a setup with two axially aligned objectives imaging the retina from both sides to project the light onto the inner (vitreal) surface and to detect the transmitted light behind the retina at the receptor layer. Simultaneously, a confocal microscope obtained images of the Müller cells embedded within the vital tissue. Projections of light onto several representative Müller cell trunks within the inner plexiform layer, i.e. (i) trunks with a straight orientation, (ii) trunks which are formed by the inner processes and (iii) trunks which get split into inner processes, were associated with increases in the intensity of the transmitted light. Projections of light onto the periphery of the Müller cell endfeet resulted in a lower intensity of transmitted light. In this way, retinal glial (Müller) cells support dim light vision by improving the signal-to-noise ratio which increases the sensitivity to light. The field of illuminated photoreceptors mainly include rods reflecting the rod dominance of the of tissue. A subpopulation of Müller cells with downstreaming cone cells led to a high-intensity illumination of the cones, while the surrounding rods were illuminated by light of lower intensity. Therefore, Müller cells that lie in front of cones may adapt the intensity of the transmitted light to the different sensitivities of cones and rods, presumably allowing a simultaneous vision with both receptor types under dim light conditions.

Two different mechanosensitive calcium responses in Müller glial cells of the guinea pig retina: Differential dependence on purinergic receptor signaling.

Tractional forces or mechanical stimulation are known to induce calcium responses in retinal glial cells. The aim of the study was to determine the characteristics of calcium responses in Müller glial cells of the avascular guinea pig retina induced by focal mechanical stimulation. Freshly isolated retinal wholemounts were loaded with Mitotracker Deep Red (to fill Müller cells) and the calcium-sensitive dye Fluo-4/AM. The inner retinal surface was mechanically stimulated with a micropipette tip for 10 ms. Stimulation induced two different cytosolic calcium responses in Müller cells with different kinetics in dependence on the distance from the stimulation site. Müller cells near the stimulation site displayed an immediate and long-lasting calcium response with high amplitude. This response was mediated by calcium influx from the extracellular space likely triggered by activation of ATP-insensitive P2 receptors. More distant Müller cells displayed, with a delay of 2.4 s, transient calcium responses which propagated laterally in a wave-like fashion. Propagating calcium waves were induced by a calcium-independent release of ATP from Müller cells near the stimulation site, and were mediated by a release of calcium from internal stores triggered by ATP, acting in part at P2Y1 receptors. The data suggest that mechanically stimulated Müller cells of the guinea pig retina release ATP which induces a propagating calcium wave in surrounding Müller cells. Propagating calcium waves may be implicated in the spatial regulation of the neuronal activity and homeostatic glial functions, and may transmit gliosis-inducing signals across the retina. Mechanical stimulation of guinea pig Müller cells induces two calcium responses: an immediate response around the stimulation site and propagating calcium waves. Both responses are differentially mediated by activation of purinergic receptors. GLIA 2016 GLIA 2017;65:62-74.

Early evolution of radial glial cells in Bilateria.

Bilaterians usually possess a central nervous system, composed of neurons and supportive cells called glial cells. Whereas neuronal cells are highly comparable in all these animals, glial cells apparently differ, and in deuterostomes, radial glial cells are found. These particular secretory glial cells may represent the archetype of all (macro) glial cells and have not been reported from protostomes so far. This has caused controversial discussions of whether glial cells represent a homologous bilaterian characteristic or whether they (and thus, centralized nervous systems) evolved convergently in the two main clades of bilaterians. By using histology, transmission electron microscopy, immunolabelling and whole-mount in situ hybridization, we show here that protostomes also possess radial glia-like cells, which are very likely to be homologous to those of deuterostomes. Moreover, our antibody staining indicates that the secretory character of radial glial cells is maintained throughout their various evolutionary adaptations. This implies an early evolution of radial glial cells in the last common ancestor of Protostomia and Deuterostomia. Furthermore, it suggests that an intraepidermal nervous system-composed of sensory cells, neurons and radial glial cells-was probably the plesiomorphic condition in the bilaterian ancestor.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible.

Hypoosmotic and glutamate-induced swelling of bipolar cells in the rat retina: comparison with swelling of Müller glial cells.

Regulation of cellular volume is of great importance to avoid changes in neuronal excitability resulting from a decrease in the extracellular space volume. We compared the volume regulation of retinal glial (Müller) and neuronal (bipolar) cells under hypoosmotic and glutamate-stimulated conditions. Freshly isolated slices of the rat retina were superfused with a hypoosmotic solution (60% osmolarity; 4 min) or with a glutamate (1 mM)-containing isoosmotic solution (15 min), and the size changes of Müller and bipolar cell somata were recorded. Bipolar cell somata, but not Müller cell somata, swelled under hypoosmotic conditions and in the presence of glutamate. The hypoosmotic swelling of bipolar cell somata might be mediated by sodium flux into the cells, because it was not observed under extracellular sodium-free conditions, and was induced by activation of metabotropic glutamate receptors and sodium-dependent glutamate transporters. The glutamate-induced swelling of bipolar cell somata was mediated by sodium chloride flux into the cells induced by activation of NMDA- and non-NMDA glutamate receptors, glutamate transporters, and voltage-gated sodium channels. The glutamate-induced swelling of bipolar cell somata was abrogated by adenosine and γ-aminobutyric acid, but not by vascular endothelial growth factor and ATP. The data may suggest that Müller cells, in contrast to bipolar cells, possess endogenous mechanisms which tightly regulate the cellular volume in response to hypoosmolarity and prolonged glutamate exposure. Inhibitory retinal transmission may regulate the volume of bipolar cells, likely by inhibition of the excitatory action of glutamate.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression.

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP(-/-)Vim(-/-) mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration.

Transcriptional regulation of aquaporin-3 in human retinal pigment epithelial cells.

The expression of aquaporin (AQP) water channels may influence the development of retinal edema. We investigated the transcriptional regulation of AQP3 in cultured human retinal pigment epithelial (RPE) cells. As shown by RT-PCR and immunocytochemistry, cultured RPE cells express AQP3 mRNA and protein. The AQP3 mRNA level in RPE cells was elevated under the following conditions: chemical hypoxia induced by CoCl(2), hyperosmolarity induced by 100 mM NaCl, and upon stimulation of the cultures with PDGF, arachidonic acid, prostaglandin E(2), and blood serum, respectively. Chemical hypoxia increased AQP3 gene expression through MEK/ERK and JNK activation. The hyperosmolarity-, PDGF-, and serum-induced upregulation of AQP3 was prevented by inhibition of the phospholipase A(2), but not by inhibition of the cyclooxygenase. Triamcinolone acetonide prevented the upregulation of AQP3 induced by arachidonic acid and prostaglandin E(2), but not by the other factors tested. It is concluded that AQP3 is transcriptionally activated in RPE cells by various pathogenic factors involved in the development of retinal edema in situ. Activation of phospholipase A(2) is a critical factor which induces AQP3 in RPE cells.

Müller glial cell-provided cellular light guidance through the vital guinea-pig retina.

In vertebrate eyes, images are projected onto an inverted retina where light passes all retinal layers on its way to the photoreceptor cells. Light scattering within this tissue should impair vision. We show that radial glial (Müller) cells in the living retina minimize intraretinal light scatter and conserve the diameter of a beam that hits a single Müller cell endfoot. Thus, light arrives at individual photoreceptors with high intensity. This leads to an optimized signal/noise ratio, which increases visual sensitivity and contrast. Moreover, we show that the ratio between Müller cells and cones-responsible for acute vision-is roughly 1. This suggests that high spatiotemporal resolution may be achieved by each cone receiving its part of the image via its individual Müller cell-light guide.

The human Müller cell line MIO-M1 expresses opsins.

PURPOSE: To determine whether the human Müller cell line Moorfields/Institute of Ophthalmology-Müller 1 (MIO-M1) expresses opsins. METHODS: The gene expression of opsins was determined by reverse-transcription PCR (RT-PCR). The presence of opsin proteins was determined by western blotting and immunocytochemistry. The light sensitivity of the cells was examined with imaging experiments using the calcium-sensitive dye Fluo-4. RESULTS: MIO-M1 cells express glial (glutamine synthase [GLUL], vimentin [VIM], glial fibrillary acidic protein [GFAP], cellular retinaldehyde-binding protein [RLBP1], glial high-affinity glutamate transporter [SLCA1], aquaporin-4 [AQP4], inwardly rectifying potassium channel Kir4.1 [Kir4.1]), neuronal (Thy-1 cell surface antigen [THY1], heavy neurofilament polypeptide [NEFH], microtubule-associated protein 2 [MAP2], neurogenic differentiation 1 [NEUROD1], neuronal nuclei [NEUN]), and neural progenitor markers (Nestin [NES], paired-type homeobox transcription factor [PAX6], neurogenic locus notch homolog 1 [NOTCH1]). The cells contain mRNA for the following opsins: blue opsin (OPN1SW), rhodopsin (OPN2), panopsin (OPN3), melanopsin (OPN4), neuropsin (OPN5), and peropsin (RRH), as well as for the transducins (guanine nucleotide binding protein [GNAZ], alpha transducing activity polypeptide 1 [GNAT1], alpha transducing activity polypeptide 2 [GNAT2]). The presence of blue opsin and melanopsin was confirmed with immunocytochemistry and western blotting. The immunoreactivity and mRNA of red-green opsin were found in some but not all cultures, while the immunoreactivity for rhodopsin was absent in all cultures investigated. Repetitive stimulation with 480 nm light evoked slow and fast transient calcium responses in the majority of cells investigated, while irradiation with 600 nm light was ineffective. CONCLUSIONS: The human Müller cell line MIO-M1 expresses opsins. This suggests immortalized Müller cells could be used as a cellular source to produce human opsins for their potential application as therapeutic agents in patients with retinitis pigmentosa.

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.

Dim light vision--morphological and functional adaptations of the eye of the mormyrid fish, Gnathonemus petersii.

The African weakly electric fish Gnathonemus petersii is well known for its electrosensory capabilities. These animals can detect and distinguish objects through active electrolocation in complete darkness. Because of their nocturnal lifestyle, a low contribution of vision for orientation and object detection has been expected. However, as we show in this review, the retina of G. petersii is highly specialized with hundreds of rods and tens of cones grouped together in bundles in a complex way, ensheathed by a tapetum lucidum. The structure of the bundles goes beyond what would be expected if only photon catch was supposed to be increased. During daytime, the structure of these "macro-receptors" changes dramatically depending on retinomotor movements. During the day, the rods and cones are located in different compartments of the bundle, separated by a narrow canal in the form of a "bottle neck". Investigations on cell structure and neurochemistry in the retina indicate a general organization that is simpler in terms of bipolar and ganglion cell diversity than in tetrachromatic species such as goldfish, yet similar in terms of neurochemical differentiation of amacrine cells. In both respects, the inner retina of the elephantnose fish bears the greatest similarity to catfish and some deep-sea fish retinae. Neuronal circuits and bundle structure give hints of possible adaptations for contrast and/or movement detection. Behavioral experiments suggest that, in contrast to the vision specialists Lepomis gibbosus, pattern detection of G. petersii is not affected by higher spatial frequencies. A pattern of low spatial frequencies, however, was equally well detected by G. petersii and L. gibbosus. Optomotor response experiments indicate that motion vision is important for Gnathonemus, narrowing down the search for the functional specialization of the Gnathonemus retina and providing a starting point for work on multisensory integration in these fish.

Three-dimensional correlative single-cell imaging utilizing fluorescence and refractive index tomography.

Cells alter the path of light, a fact that leads to well-known aberrations in single cell or tissue imaging. Optical diffraction tomography (ODT) measures the biophysical property that causes these aberrations, the refractive index (RI). ODT is complementary to fluorescence imaging and does not require any markers. The present study introduces RI and fluorescence tomography with optofluidic rotation (RAFTOR) of suspended cells, facilitating the segmentation of the 3D-correlated RI and fluorescence data for a quantitative interpretation of the nuclear RI. The technique is validated with cell phantoms and used to confirm a lower nuclear RI for HL60 cells. Furthermore, the nuclear inversion of adult mouse photoreceptor cells is observed in the RI distribution. The applications shown confirm predictions of previous studies and illustrate the potential of RAFTOR to improve our understanding of cells and tissues.

A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy.

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

Glutamate-evoked alterations of glial and neuronal cell morphology in the guinea pig retina.

Neuronal activity is accompanied by transmembranous ion fluxes that cause cell volume changes. In whole mounts of the guinea pig retina, application of glutamate resulted in fast swelling of neuronal cell bodies in the ganglion cell layer (GCL) and the inner nuclear layer (INL) (by approximately 40%) and a concomitant decrease of the thickness of glial cell processes in the inner plexiform layer (IPL) (by approximately 40%) that was accompanied by an elongation of the glial cells, by a thickening of the whole retinal tissue, and by a shrinkage of the extracellular space (by approximately 18%). The half-maximal effect of glutamate was observed at approximately 250 mum, after approximately 4 min. The swelling was caused predominantly by AMPA-kainate receptor-mediated influx of Na+ into retinal neurons. Similar but transient morphological alterations were induced by high K+ and dopamine, which caused release of endogenous glutamate and subsequent activation of AMPA-kainate receptors. Apparently, retinal glutamatergic transmission is accompanied by neuronal cell swelling that causes compensatory morphological alterations of glial cells. The effect of dopamine was elicitable only during light adaptation but not in the dark, and glutamate and high K+ induced strong ereffects in the dark than in the light. This suggests that not only the endogenous release of dopamine but also the responsiveness of glutamatergic neurons to dopamine is regulated by light-dark adaptation. Similar morphological alterations (neuronal swelling and decreased glial process thickness) were observed in whole mounts isolated immediately after experimental retinal ischemia, suggesting an involvement of AMPA-kainate receptor activation in putative neurotoxic cell swelling in the postischemic retina.

Ischemia-reperfusion causes exudative detachment of the rabbit retina.

PURPOSE: To characterize the activation of macroglial (Müller) and microglial cells, as well as neuronal cell degeneration, during ischemia-reperfusion in rabbit retina and to test the possible effect of triamcinolone acetonide on gliosis. METHODS: Transient retinal ischemia was produced by increasing intraocular pressure for 60 minutes. Triamcinolone (8 mg) was intravitreally applied immediately after the cessation of ischemia. At 3 and 8 days after reperfusion, the K+ currents of acutely isolated Müller cells were recorded, and the Ca2+ responses of Müller cells on stimulation of P2Y receptors were recorded fluorometrically in retinal wholemounts. Microglial/immune cells in the nerve fiber layer of retinal wholemounts were labeled with isolectin. To evaluate neuronal and Müller cell loss, the numbers of cells were counted in retinal slices. RESULTS: Transient ischemia caused exudative detachment of the central retina that was characterized by disruption of the pigment epithelial monolayer, the presence of scattered pigment epithelial and immune cells in the expanded subretinal space, and retinal folds. A significant loss of photoreceptor cells was observed at 8 days after reperfusion. At 3 and 8 days after reperfusion, Müller cell gliosis was apparent, as indicated by cellular hypertrophy, downregulation of K+ channel expression, and an increased number of cells that displayed P2Y receptor-mediated Ca2+ responses. The number of microglial/immune cells increased strongly after reperfusion. Intravitreal triamcinolone did not affect the parameters of Müller cell gliosis but decreased the number of microglial/immune cells. CONCLUSIONS: Ischemia-reperfusion of the rabbit retina causes exudative retinal detachment that is characterized by a loss of photoreceptor cells, whereas the inner retina remains largely preserved. Micro- and macroglial cells are activated early during reperfusion, even before dropout of the photoreceptor cells. Intravitreal triamcinolone may decrease the degree of microglial/immune cell activation.

Microglia mechanics: immune activation alters traction forces and durotaxis.

Microglial cells are key players in the primary immune response of the central nervous system. They are highly active and motile cells that chemically and mechanically interact with their environment. While the impact of chemical signaling on microglia function has been studied in much detail, the current understanding of mechanical signaling is very limited. When cultured on compliant substrates, primary microglial cells adapted their spread area, morphology, and actin cytoskeleton to the stiffness of their environment. Traction force microscopy revealed that forces exerted by microglia increase with substrate stiffness until reaching a plateau at a shear modulus of ~5 kPa. When cultured on substrates incorporating stiffness gradients, microglia preferentially migrated toward stiffer regions, a process termed durotaxis. Lipopolysaccharide-induced immune-activation of microglia led to changes in traction forces, increased migration velocities and an amplification of durotaxis. We finally developed a mathematical model connecting traction forces with the durotactic behavior of migrating microglial cells. Our results demonstrate that microglia are susceptible to mechanical signals, which could be important during central nervous system development and pathologies. Stiffness gradients in tissue surrounding neural implants such as electrodes, for example, could mechanically attract microglial cells, thus facilitating foreign body reactions detrimental to electrode functioning.

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.