Cyclosporine A Protects Retinal Explants against Hypoxia.

The retina is a complex neurological tissue and is extremely sensitive to an insufficient supply of oxygen. Hypoxia plays a major role in several retinal diseases, and often results in the loss of cells that are essential for vision. Cyclosporine A (CsA) is a widely used immunosuppressive drug. Furthermore, treatment with CsA has neuroprotective effects in several neurologic disorders. No data are currently available on the tolerated concentration of CsA when applied to the retina. To reveal the most effective dose, retinal explants from rat eyes were exposed to different CsA concentrations (1-9 µg/mL). Immunohistochemistry with brain-specific homeobox/POU domain protein 3a (Brn3a) and TUNEL staining was performed to determine the percentage of total and apoptotic retinal ganglion cells (RGCs), as well as the responses of micro- and macroglial cells. Furthermore, optical coherence tomography (OCT) scans were performed to measure the changes in retinal thickness, and recordings with multielectrode array (MEA) were performed to evaluate spontaneous RGC spiking. To examine the neuroprotective effects, retinas were subjected to a hypoxic insult by placing them in a nitrogen-streamed hypoxic chamber prior to CsA treatment. In the biocompatibility tests, the different CsA concentrations had no negative effect on RGCs and microglia. Neuroprotective effects after a hypoxic insult on RGCs was demonstrated at a concentration of 9 µg/mL CsA. CsA counteracted the hypoxia-induced loss of RGCs, reduced the percentage of TUNEL+ RGCs, and prevented a decrease in retinal thickness. Taken together, the results of this study suggest that CsA can effectively protect RGCs from hypoxia, and the administered concentrations were well tolerated. Further in vivo studies are needed to determine whether local CsA treatment may be a suitable option for hypoxic retinal diseases.

Hypothermia protects retinal ganglion cells against hypoxia-induced cell death in a retina organ culture model.

BACKGROUND: Hypoxia contributes to retinal damage in several retinal diseases, including central retinal artery occlusion, with detrimental consequences like painless, monocular loss of vision. Currently, the treatment options are severely limited due to the short therapy window, as the neuronal cells, especially the retinal ganglion cells (RGCs), are irreversibly damaged within the first few hours. Hypothermia might be a possible treatment option or at least might increase the therapy window. METHODS: To investigate the neuroprotective effect of hypothermia after retinal hypoxia, an easy-to-use ex vivo retinal hypoxia organ culture model developed in our laboratory was used that reliably induced retinal damage on a structural, molecular and functional level. The neuroprotective effect of hypothermia after retinal hypoxia was analysed using optical coherence tomography scans, histological stainings, quantitative real-time polymerase chain reaction, western blotting and microelectrode array recordings. RESULTS: Two different hypothermic temperatures (30°C and 20°C) were evaluated, both exhibited strong neuroprotective effects. Most importantly, hypothermia increased RGC survival after retinal hypoxia. Furthermore, hypothermia counteracted the hypoxia-induced RGC death, reduced macroglia activation, attenuated retinal thinning and protected from loss of spontaneous RGC activity. CONCLUSIONS: These results indicate that already a mild reduction in temperature protects the RGCs against damage and could function as a promising therapeutic option for hypoxic diseases.

A comprehensive small interfering RNA screen identifies signaling pathways required for gephyrin clustering.

The postsynaptic scaffold protein gephyrin is clustered at inhibitory synapses and serves for the stabilization of GABA(A) receptors. Here, a comprehensive kinome-wide siRNA screen in a human HeLa cell-based model for gephyrin clustering was used to identify candidate protein kinases implicated in the stabilization of gephyrin clusters. As a result, 12 hits were identified including FGFR1 (FGF receptor 1), TrkB, and TrkC as well as components of the MAPK and mammalian target of rapamycin (mTOR) pathways. For confirmation, the impact of these hits on gephyrin clustering was analyzed in rat primary hippocampal neurons. We found that brain-derived neurotrophic factor (BDNF) acts on gephyrin clustering through MAPK signaling, and this process may be controlled by the MAPK signaling antagonist sprouty2. BDNF signaling through phosphatidylinositol 3-kinase (PI3K)-Akt also activates mTOR and represses GSK3ß, which was previously shown to reduce gephyrin clustering. Gephyrin is associated with inactive mTOR and becomes released upon BDNF-dependent mTOR activation. In primary neurons, a reduction in the number of gephyrin clusters due to manipulation of the BDNF-mTOR signaling is associated with reduced GABA(A) receptor clustering, suggesting functional impairment of GABA signaling. Accordingly, application of the mTOR antagonist rapamycin leads to disinhibition of neuronal networks as measured on microelectrode arrays. In conclusion, we provide evidence that BDNF regulates gephyrin clustering via MAPK as well as PI3K-Akt-mTOR signaling.

An optoelectronic neural interface approach for precise superposition of optical and electrical stimulation in flexible array structures.

Optical stimulation of genetically modified nerve cells has become one of the state-of-the-art methods in neuroscience. This so-called optogenetic approach allows cell-type specific activation in comparison to more generalized electrical stimulation. Combinations of both stimulation modalities would be desirable to investigate effects in detail and specify differences. This work presents the design of a miniaturized optoelectronic device that allows optical and electrical activation at the same spot. Indium tin oxide (ITO), which is transparent to visible light, has been chosen as electrode material. Light emitting diodes were assembled on a polyimide substrate with integrated interconnection lines, directly behind the electrodes to compare optical with electrical stimulation. The optical transparency of the ITO-polyimide layer stack was investigated and showed sufficient transmission in the required wavelength range. ITO electrodes with diameters up to 1000 µm were electrochemically characterized using electrical impedance spectroscopy (EIS). Several diameters did show comparable results to platinum, a commonly used electrode material. Fully assembled devices were used in combination an ex vivo setting with genetically modified retina to demonstrate the functionality of this approach. Retinal ganglion cells were excited by both, optical and electrical stimulation at the same spot and signals were recorded via standard microelectrode arrays (MEA) as reference. The simultaneous stimulation and recording of directly evoked action potentials indicates a similar mode of action of the two stimulation modalities. Further engineering work is needed to transfer the presented and proven concept into devices for chronic implantation, might it be in animal or first-in-human studies.

A flexible protruding microelectrode array for neural interfacing in bioelectronic medicine.

Recording neural signals from delicate autonomic nerves is a challenging task that requires the development of a low-invasive neural interface with highly selective, micrometer-sized electrodes. This paper reports on the development of a three-dimensional (3D) protruding thin-film microelectrode array (MEA), which is intended to be used for recording low-amplitude neural signals from pelvic nervous structures by penetrating the nerves transversely to reduce the distance to the axons. Cylindrical gold pillars (Ø 20 or 50 µm, ~60 µm height) were fabricated on a micromachined polyimide substrate in an electroplating process. Their sidewalls were insulated with parylene C, and their tips were optionally modified by wet etching and/or the application of a titanium nitride (TiN) coating. The microelectrodes modified by these combined techniques exhibited low impedances (~7 kΩ at 1 kHz for Ø 50 µm microelectrode with the exposed surface area of ~5000 µm²) and low intrinsic noise levels. Their functionalities were evaluated in an ex vivo pilot study with mouse retinae, in which spontaneous neuronal spikes were recorded with amplitudes of up to 66 µV. This novel process strategy for fabricating flexible, 3D neural interfaces with low-impedance microelectrodes has the potential to selectively record neural signals from not only delicate structures such as retinal cells but also autonomic nerves with improved signal quality to study neural circuits and develop stimulation strategies in bioelectronic medicine, e.g., for the control of vital digestive functions.

Electrode-size dependent thresholds in subretinal neuroprosthetic stimulation.

OBJECTIVE: Retinal prostheses have shown promising results in restoring some visual perception to blind patients but successful identification of objects of different size remains a challenge. Here we investigated electrode-size specific stimulation thresholds and their variability for subretinal electrical stimulation. Our findings indicate the range of charge densities required to achieve identification of small objects and the object-size-specific scaling of stimulation threshold. APPROACH: Using biphasic voltage-limited current stimuli provided by a light-sensitive microchip, we determined threshold charge densities for stimulation with variable electrode sizes. The stimulated activation of the retinal network was identified by recording the spiking of retinal ganglion cells in photoreceptor-degenerated mouse rd10 retinas. Stimulation thresholds were determined for cells with saturating stimulus response relationships (SRRs) but not for cells characterized by monotonically increasing or decreasing SRRs. MAIN RESULTS: Stimulation thresholds estimated in rd10 retinas ranged between 100-900 µC cm-2 for stimulation with small electrodes (30 µm diameter). Threshold charge density decreased with increasing electrode size and plateaued at 20 µC cm-2 for an electrode diameter larger than 300 µm. This trend of decreasing threshold down to a plateau value was confirmed in wild-type mouse retina suggesting an underlying physiological source. SIGNIFICANCE: Our results suggest the following guidelines for retinal prosthetics employing biphasic current pulses. The encoding of small objects may be achieved through the activation of a confined set of different retinal ganglion cells, with individual stimulation thresholds spanning a wide range of charge densities. The encoding of increasing object sizes may be achieved by decreasing stimulation charge density.

Retinal charge sensitivity and spatial discrimination obtainable by subretinal implants: key lessons learned from isolated chicken retina.

In order to obtain functional parameters relevant to the designing of a subretinal implant, we carried out electrical stimulation experiments with isolated chicken retina. The median threshold for network activation with planar disc electrodes (diameter 10 microm) was 0.5 nC (625 microC cm(-2)) for anodal voltage impulses and 1.6 nC (2 mC cm(-2)) for cathodal impulses. Above threshold, the number of spikes evoked by a single voltage impulse increased up to saturation within a range of injected charge from 0.1 nC to 1 nC for anodal impulses and from 1 nC to 10 nC for cathodal impulses. Using needle electrodes with a tip diameter of 1 microm, we determined the electrical point spread function (EPSF) for subretinal stimulation. It had a half width in the range of 100 microm, which corresponds to a visual angle of 21' and to a visual acuity of 20/417 in the human eye. It is reasonable to conclude that with subretinal implants the minimum separable will be of the same dimension.

Neuroprotective effect of transretinal electrical stimulation on neurons in the inner nuclear layer of the degenerated retina.

Electrical stimulation has been shown to have neuroprotective effects on ganglion cells and photoreceptors in axotomized and dystrophic retinas from Royal College of Surgeons (RCS) rats. This study determined whether electrical stimulation also has a neuroprotective effect on cells in the inner nuclear layer (INL) of retinas. We cultivated retinas from adult RCS rats on microelectrode arrays and stimulated them continuously with 20 Hz for up to 5 days. Afterwards, we subjected them to quantitative immunohistochemical analysis. Using TUNEL assay we found that transretinal electrical stimulation (TRES) with charge densities within the range of 100-500 microC/cm2 reduced apoptosis of neurons in the INL of degenerated retinas from RCS -/- rats by 20% after 1 day of continuous stimulation. Antibody staining (OX-42, ED1) revealed a reduced activation of migroglial cells in RCS -/- and congenic control (RCS +/+) rat retinas by up to 50% after 1 day of stimulation. The effect of electrical stimulation on apoptosis and reduced activation of microglial cells was closely correlated with the strength and duration of the stimulation. The neuroprotective effect of TRES on neuronal cells in the INL of degenerated RCS rat retinas supports the idea that electrical stimulation may be a therapeutic option to delay the progression of retinal degeneration in patients suffering from retinitis pigmentosa.