Arthur J. Weber

Expression of glial fibrillary acidic protein and glutamine synthetase by Müller cells after optic nerve damage and intravitreal application of brain‐derived neurotrophic factor

Müller glia play an important role in maintaining retinal homeostasis, and brain‐derived neurotrophic factor (BDNF) has proven to be an effective retinal ganglion cell (RGC) neuroprotectant following optic nerve injury. The goal of these studies was to investigate the relation between optic nerve injury and Müller cell activation, and to determine the extent to which BDNF affects the injury response of Müller cells. Using immunocytochemistry and Western blot analysis , temporal changes in the expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) were examined in rats after optic nerve crush alone, or in conjunction with an intravitreal injection of BDNF (5 μg). GFAP protein levels were normal at 1 day post‐crush, but increased ∼9‐fold by day 3 and remained elevated over the 2‐week period studied. Müller cell GS expression remained stable after optic nerve crush, but the protein showed a transient shift in its cellular distribution; during the initial 24‐h period post‐crush the GS protein appeared to translocate from the cell body to the inner and outer glial processes, and particularly to the basal endfeet located in the ganglion cell layer. BDNF alone, or in combination with optic nerve crush, did not have a significant effect on the expression of either GFAP or GS compared with the normal retina, or after optic nerve crush alone, respectively. The data indicate that although BDNF is a potent neuroprotectant in the vertebrate retina, it does not appear to have a significant influence on Müller cell expression of either GS or GFAP in response to optic nerve injury. GLIA 38:115–125, 2002.

Effects of optic nerve injury, glaucoma, and neuroprotection on the survival, structure, and function of ganglion cells in the mammalian retina

Glaucoma is an optic neuropathy that originates with pressure‐induced damage to the optic nerve. This results in the retrograde degeneration of ganglion cells in the retina, and a progressive loss of vision. Over the past several years, a number of studies have described the structural and functional changes that characterize ganglion cell degeneration in the glaucomatous eye, and following optic nerve injury. In addition, a variety of different strategies for providing neuroprotection to the injured retina have been proposed. Many of these are based on the use of brain‐derived neurotrophic factor (BDNF), a particularly potent neuroprotectant in the mammalian eye and the basis of our research in this area. Of particular importance is the fact that BDNF not only promotes ganglion cell survival following damage to the optic nerve, but also helps to preserve the structural integrity of the surviving neurons, which in turn results in enhanced visual function. The studies presented here describe these attributes, and serve as the foundation for ongoing work that suggests a need to think beyond the eye in the development of future treatment strategies.

Brain-derived neurotrophic factor reduces TrkB protein and mRNA in the normal retina and following optic nerve crush in adult rats.

Brain-derived neurotrophic factor (BDNF) is a well-known retinal neuroprotectant, but its effectiveness is limited: higher doses do not yield increased cell survival, multiple applications are not additive, and long-term delivery does not reverse, ganglion cell death. These limitations might reflect either injury- or BDNF-induced retinal changes in TrkB, the high affinity tyrosine kinase receptor used by BDNF. Retinal levels of TrkB protein and mRNA were measured in rats following intravitreal application of BDNF alone, optic nerve crush alone, and both. Full-length receptor protein levels (TrkB.FL) were determined by Western blot analysis and mRNA (trkB.FL) levels were measured using RNAse protection assay (RPA). BDNF alone produced a rapid and prolonged decrease in normal retina TrkB.FL. Nerve crush also resulted in decreased TrkB.FL, but the reduction was not apparent before 2-week post-crush. BDNF applied at the time of the crush yielded reductions in TrkB.FL similar to that of BDNF alone. With respect to TrkB mRNA levels, injection of BDNF into normal eyes and optic nerve crush alone showed bell-shaped patterns of change: approximately 50% below normal at 24-h post-procedure, approximately 50% above normal at 3 days, normal at 7 days, and approximately 50% below normal at 2-week post-procedure. When BDNF and nerve crush were combined, trkB-FL levels reached 90% of normal 1-week post-crush/injection. The data suggest that the limitation of BDNF in promoting ganglion cell survival following optic nerve injury results, in part, due to drug-induced down-regulation of the full-length TrkB receptor needed to activate intracellular pathways.

Experimental glaucoma in the primate induced by latex microspheres

The injection of sterile latex microspheres into the anterior chamber of the eye is presented as a simple and cost effective method for inducing chronic elevation of intraocular pressure (IOP) and experimental glaucoma in primates. The microspheres produce elevated IOP primarily by restricting the outflow of aqueous humor through the trabecular meshwork located in the chamber angle. Different levels and durations of elevated IOP can be obtained by altering the frequency and number of microspheres injected. In comparison with other primate models of experimental glaucoma, the approach described here has the advantages of producing chronic elevations of IOP without the need for expensive ophthalmic equipment and personnel, surgical intervention or intraocular inflammation, and without compromising visibility of the optic disc, which is necessary for clinical assessment of the onset and progression of the disease.

Opto-

Electrocorticogram (ECoG) recordings, taken from electrodes placed on the surface of the cortex, have been successfully implemented for control of brain machine interfaces (BMIs). Optogenetics, direct optical stimulation of neurons in brain tissue genetically modified to express channelrhodopsin-2 (ChR2), enables targeting of specific types of neurons with sub-millisecond temporal precision. In this work, we developed a BMI device, called an Opto- μECoG array, which combines ECoG recording and optogenetics-based stimulation to enable multichannel, bi-directional interactions with neurons. The Opto- μECoG array comprises two sub-arrays, each containing a 4 × 4 distribution of micro-epidural transparent electrodes (~200 μm diameter) and embedded light-emitting diodes (LEDs) for optical neural stimulation on a 2.5×2.5 mm2 footprint to match the bilateral hemispherical area of the visual cortex in a rat. The transparent electrodes were fabricated with indium tin oxide (ITO). Parylene-C served as the main structural and packaging material for flexibility and biocompatibility. Optical, electrical, and thermal characteristics of the fabricated device were investigated and in vivo experiments were performed to evaluate the efficacy of the device.

\mu{\rm ECoG}

PURPOSE To determine whether application of BDNF to the eye and brain provides a greater level of neuroprotection after optic nerve injury than treatment of the eye alone. METHODS Retinal ganglion cell survival and pattern electroretinographic responses were compared in normal cat eyes and in eyes that received (1) a mild nerve crush and no treatment, (2) a single intravitreal injection of BDNF at the time of the nerve injury, or (3) intravitreal treatment combined with 1 to 2 weeks of continuous delivery of BDNF to the visual cortex, bilaterally. RESULTS Relative to no treatment, administration of BDNF to the eye alone resulted in a significant increase in ganglion cell survival at both 1 and 2 weeks after nerve crush (1 week, 79% vs. 55%; 2 weeks, 60% vs. 31%). Combined treatment of the eye and visual cortex resulted in a modest additional increase (17%) in ganglion cell survival in the 1-week eyes, a further significant increase (55%) in the 2-week eyes, and ganglion cell survival levels for both that were comparable to normal (92%-93% survival). Pattern ERG responses for all the treated eyes were comparable to normal at 1 week after injury; however, at 2 weeks, only the responses of eyes receiving the combined BDNF treatment remained so. CONCLUSIONS Although treatment of the eye alone with BDNF has a significant impact on ganglion cell survival after optic nerve injury, combined treatment of the eye and brain may represent an even more effective approach and should be considered in the development of future optic neuropathy-related neuroprotection strategies.

Array: A Hybrid Neural Interface With Transparent

PURPOSE To examine whether brain-derived neurotrophic factor (BDNF), a potent neuroprotectant in the mammalian retina, also plays a role in preserving the dendritic integrity of the surviving ganglion cells after optic nerve injury. METHODS Single ganglion cells from cats that underwent unilateral optic nerve crush and received no treatment or nerve crush combined with intravitreous treatment of the affected eye with BDNF were labeled intracellularly, reconstructed using confocal microscopy, and compared quantitatively. RESULTS Optic nerve injury produced a significant decrease in the soma, dendritic field size, and dendritic complexity of alpha cells. beta Cells also displayed a significant decrease in soma size, but their dendritic fields were not affected as severely as those of alpha cells. Intravitreous treatment of the eye with BDNF at the time of injury preserved the normal somal and dendritic morphologies of both alpha and beta cells. CONCLUSIONS BDNF, in addition to promoting ganglion cell survival, plays an important role in preserving the somal and dendritic morphologies of the surviving ganglion cells, a necessary precursor to maintaining normal visual function. Ganglion cells, however, are not created equal with respect to their responses to nerve injury or to treatment of the eye with BDNF. Thus, development of effective treatment strategies for preserving ganglion cell function in optic nerve-related diseases mandates a clearer understanding of the cellular response characteristics of the specific neurons involved.

\mu{\rm ECoG}

PURPOSE To characterize a canine model of autosomal recessive RP due to a PDE6A gene mutation. METHODS Affected and breed- and age-matched control puppies were studied by electroretinography (ERG), light and electron microscopy, immunohistochemistry, and assay for retinal PDE6 levels and enzymatic activity. RESULTS The mutant puppies failed to develop normal rod-mediated ERG responses and had reduced light-adapted a-wave amplitudes from an early age. The residual ERG waveforms originated primarily from cone-driven responses. Development of photoreceptor outer segments stopped, and rod cells were lost by apoptosis. Immunohistochemistry demonstrated a marked reduction in rod opsin immunostaining outer segments and relative preservation of cones early in the disease process. With exception of rod bipolar cells, which appeared to be reduced in number relatively early in the disease process, other inner retinal cells were preserved in the early stages of the disease, although there was marked and early activation of Müller glia. Western blot analysis showed that the PDE6A mutation not only resulted in a lack of PDE6A protein but the affected retinas also lacked the other PDE6 subunits, suggesting expression of PDE6A is essential for normal expression of PDE6B and PDE6G. Affected retinas lacked PDE6 enzymatic activity. CONCLUSIONS This represents the first characterization of a PDE6A model of autosomal recessive retinitis pigmentosa, and the PDE6A mutant dog shows promise as a large animal model for investigation of therapies to rescue mutant rod photoreceptors and to preserve cone photoreceptors in the face of a rapid loss of rod cells.

Electrode Array and Integrated LEDs for Optogenetics

This paper presents a wireless-enabled, flexible optrode array with multichannel micro light-emitting diodes (μ-LEDs) for bi-directional wireless neural interface. The array integrates wirelessly addressable μ-LED chips with a slanted polymer optrode array for precise light delivery and neural recording at multiple cortical layers simultaneously. A droplet backside exposure (DBE) method was developed to monolithically fabricate varying-length optrodes on a single polymer platform. In vivo tests in rat brains demonstrated that the μ-LEDs were inductively powered and controlled using a wireless switched-capacitor stimulator (SCS), and light-induced neural activity was recorded with the optrode array concurrently.

Combined Application of BDNF to the Eye and Brain Enhances Ganglion Cell Survival and Function in the Cat after Optic Nerve Injury

The recent development of optogenetics has created an increased demand for advancing engineering tools for optical modulation of neural circuitry. This paper details the design, fabrication, integration, and packaging procedures of a wirelessly-powered, light emitting diode (LED) coupled optrode neural interface for optogenetic studies. The LED-coupled optrode array employs microscale LED (μLED) chips and polymer-based microwaveguides to deliver light into multi-level cortical networks, coupled with microelectrodes to record spontaneous changes in neural activity. An integrated, implantable, switched-capacitor based stimulator (SCS) system provides high instantaneous power to the μLEDs through an inductive link to emit sufficient light and evoke neural activities. The presented system is mechanically flexible, biocompatible, miniaturized, and lightweight, suitable for chronic implantation in small freely behaving animals. The design of this system is scalable and its manufacturing is cost effective through batch fabrication using microelectromechanical systems (MEMS) technology. It can be adopted by other groups and customized for specific needs of individual experiments.