Aqueous Angiography in Normal Canine Eyes.

Purpose: To conduct aqueous angiography (AA) using a clinically applicable technique in normal dogs and to compare findings to intravenous scleral angiography (SA). Methods: We examined 10 canine cadaver eyes and 12 eyes from live normal dogs. A gravity-fed trocar system delivered 2% sodium fluorescein and 0.25% indocyanine green (ICG) intracamerally (IC) in cadaver eyes. In vivo AA was subsequently performed in one eye of each of the 12 dogs via IC bolus of ICG under sedation. The same 12 dogs received SA via intravenous ICG (mean ± SD) 10.7 ± 3.3 days later. Identical scleral sectors were imaged using a Spectralis confocal scanning laser ophthalmoscope. Results: The gravity-fed trocar system permitted visualization of the conventional aqueous humor outflow (CAHO) pathways in cadaver eyes, but not in vivo. Fluorescence was observed superonasally in four of the 10 cadaver eyes within 24.0 ± 3.6 seconds. A single IC bolus of ICG showed CAHO pathways in vivo, demonstrating sectoral outflow patterns in the superotemporal sclera in 10 of the 12 eyes within 35.0 ± 4.3 seconds; four of the 12 eyes exhibited pulsatile aqueous movement. SA exhibited fluorescence patterns comparable to AA with weak pulsatile aqueous humor outflow. Conclusions: Angiography (AA or SA) in dogs permits visualization of the CAHO pathway and its vascular components in vivo. AA may be a more useful modality to assess aqueous humor outflow. Translational Relevance: Intracameral AA has potential utility for evaluating CAHO in vivo in dogs, an important animal model species.

Flexible, diamond-based microelectrodes fabricated using the diamond growth side for neural sensing.

Diamond possesses many favorable properties for biochemical sensors, including biocompatibility, chemical inertness, resistance to biofouling, an extremely wide potential window, and low double-layer capacitance. The hardness of diamond, however, has hindered its applications in neural implants due to the mechanical property mismatch between diamond and soft nervous tissues. Here, we present a flexible, diamond-based microelectrode probe consisting of multichannel boron-doped polycrystalline diamond (BDD) microelectrodes on a soft Parylene C substrate. We developed and optimized a wafer-scale fabrication approach that allows the use of the growth side of the BDD thin film as the sensing surface. Compared to the nucleation surface, the BDD growth side exhibited a rougher morphology, a higher sp 3 content, a wider water potential window, and a lower background current. The dopamine (DA) sensing capability of the BDD growth surface electrodes was validated in a 1.0 mM DA solution, which shows better sensitivity and stability than the BDD nucleation surface electrodes. The results of these comparative studies suggest that using the BDD growth surface for making implantable microelectrodes has significant advantages in terms of the sensitivity, selectivity, and stability of a neural implant. Furthermore, we validated the functionality of the BDD growth side electrodes for neural recordings both in vitro and in vivo. The biocompatibility of the microcrystalline diamond film was also assessed in vitro using rat cortical neuron cultures.

Wireless, passive strain sensor in a doughnut-shaped contact lens for continuous non-invasive self-monitoring of intraocular pressure.

After cataract, glaucoma is the second leading cause of blindness worldwide and real-time monitoring of intraocular pressure (IOP) is of great demand. We present a wireless, passive sensor sitting inside a customized, planar and circular doughnut-shaped contact lens capable of continuous monitoring of the change in the curvature of cornea caused by IOP fluctuations. The sensor consists of a constant capacitor and a variable inductor in the form of a stretchable, closed-loop, serpentine wire that serves as both the sensor and the antenna. Results show a pressure responsivity of 523 kHz per 1% axial strain on a pressurized polydimethylsiloxane membrane and 35.1 kHz per 1 mmHg change in the IOP of a canine eye. The sensor is tested for stability and shows unvaried characteristics after repeated cycles and parasitic movements. Predictable influences of temperature and humidity on the sensor response are also verified experimentally, which can be canceled out using real-time calibration with temperature and humidity sensors to integrate with a reader device. The design reported here has numerous advantages, such as design simplicity, component reliability, high responsivity, and low cost, thereby opening up potential opportunities for the translation of this non-invasive, continuous IOP monitoring technique into clinical applications.

A wireless, smartphone controlled, battery powered, head mounted light delivery system for optogenetic stimulation.

This paper reports the design, fabrication and characterization of a head-mounted, flexible, and ultralight optogenetic system that enables wireless delivery of light into the brains of awake and freely behaving animals. The project is focused on miniaturized design, light weight (2.7g), small volume, low cost (< 25 USD) and simple fabrication. The chip, the substrate material, the battery, and the micro light emitting diode (µLED) are commercially available. The device implementation consists of one step photolithography, soldering, and packaging along with Arduino programming. In vivo study is carried out where the battery-powered µLED stimulates the visual cortex of a rat with parameters that can be controlled wirelessly via a smart-phone user interface application. The efficacy of optical stimulation is validated using c-Fos as a report of light-evoked neuronal activity.

An implantable, miniaturized SU-8 optical probe for optogenetics-based deep brain stimulation.

This paper reports a method of making optical probes for optogenetics-based deep brain optical stimulation using SU-8, which effectively increases light coupling efficiency, has excellent mechanical stiffness, and reduces fabrication complexity. By mounting microscale LEDs (µLEDs) at the tip of a SU-8 probe and directly inserting the light source into deep brain regions, attenuation caused by light transmission in wave-guided structures such as optical fibers or optrodes can be minimized. Compared to silicon neural probes, SU-8 is more biocompatible and flexible, which can reduce brain damage. Parylene-C encapsulation can potentially improve the long-term biocompatibility and reliability of the device for chronic implantation. The functionality has been proven by clearly light-induced neural activity.

BDNF treatment and extended recovery from optic nerve trauma in the cat.

PURPOSE: We examined the treatment period necessary to restore retinal and visual stability following trauma to the optic nerve. METHODS: Cats received unilateral optic nerve crush and no treatment (NT), treatment of the injured eye with brain-derived neurotrophic factor (BDNF), or treatment of the injured eye combined with treatment of visual cortex for 2 or 4 weeks. After 1-, 2-, 4-, or 6-week survival periods, pattern electroretinograms (PERGs) were obtained and retinal ganglion cell (RGC) survival determined. RESULTS: In the peripheral retina, RGC survival for NT, eye only, and eye + cortex animals was 55%, 78%, and 92%, respectively, at 1 week, and 31%, 60%, and 93%, respectively, at 2 weeks. PERGs showed a similar pattern of improvement. After 4 weeks, RGC survival was 7%, 29%, and 53% in each group, with PERGs in the dual-treated animals similar to the 1- to 2-week animals. For area centralis (AC), the NT, eye only, and eye + cortex animals showed 47%, 78%, and 82% survival, respectively, at 2 weeks, and 13%, 54%, and 81% survival, respectively, at 4 weeks. Removing the pumps at 2 weeks resulted in ganglion cell survival levels of 76% and 74% in the AC at 4 and 6 weeks postcrush, respectively. The PERGs from 2-week treated, but 4- and 6-week survival animals were comparable to those of the 2-week animals. CONCLUSIONS: Treating the entire central visual pathway is important following optic nerve trauma. Long-term preservation of central vision may be achieved with as little as 2 weeks of treatment using this approach.

Autocrine and paracrine interactions and neuroprotection in glaucoma.

Retinal ganglion cells represent the output neurons of the retina. They are responsible for integrating electrical signals that originate with the photoreceptors and, via their axons that comprise the optic nerve, transmit that information to higher visual centers of the brain. The retinal ganglion cells reside on the inner surface of the retina and their axons course across the inner surface to exit at the back of the eye through a region known as the optic nerve head. Within this region, initiation of the degenerative processes associated with glaucoma are thought to occur, leading to degeneration of not only the optic nerve but also the retinal ganglion cells themselves. Studies aimed at understanding the mechanisms behind glaucoma have identified diverse cellular components and molecular events that occur in response to nerve injury. The challenge to date has been to identify and promote pro-survival events while suppressing those that support further degradation and loss of vision. Complicating this process is the fact that the cells and molecules involved can play multiple roles. An understanding of the players and their complex relationships is central to the development of a successful treatment strategy.

Combined application of BDNF to the eye and brain enhances ganglion cell survival and function in the cat after optic nerve injury.

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.

BDNF preserves the dendritic morphology of alpha and beta ganglion cells in the cat retina after optic nerve injury.

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.

Structure-function relations of parasol cells in the normal and glaucomatous primate retina.

PURPOSE: The purpose of this study was to examine the effect that chronic elevation of intraocular pressure has on the intrinsic and visual response properties of parasol cells in the primate retina. METHODS: A primate model of experimental glaucoma was combined with intracellular recording and staining techniques using an isolated retina preparation. Intrinsic electrical properties were examined by injection of depolarizing and hyperpolarizing currents. Visual responses were studied using drifting and counterphased gratings. Morphologic comparisons were made by injecting recorded cells with Neurobiotin and analyzing them quantitatively with a computer-based neuron reconstruction system. RESULTS: Structurally, parasol cells from glaucomatous eyes had smaller somata and smaller, less complex dendritic arbors, resulting in a significant reduction in total dendrite length and surface area. Functionally, these neurons did not differ from normal in their mean resting membrane potentials, input resistances, or thresholds to electrical activation, but did differ in membrane time constants and spike duration. Parasol cells from both normal and glaucomatous eyes preferred low-spatial-frequency stimuli, but significantly fewer glaucoma-related cells were driven visually-in particular, by patterned stimuli. Glaucomatous cells also did not respond as well to visual stimuli presented at increased temporal frequencies. CONCLUSIONS: Ganglion cells in the glaucomatous eye retain most of their normal intrinsic electrical properties, but are less responsive, both spatially and temporally, to visual stimuli. The reduction in visual responsiveness most likely results from significant changes in dendritic architecture, which affects their level of innervation by more distal retinal neurons.

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.

A quantitative assessment of TPP-induced delayed neuropathy in the retina and lateral geniculate nucleus of the European ferret (Mustela putorius furo).

Triphenyl phosphite (TPP) has been examined extensively in our lab to assess its degenerative effects on the visual pathway of the European ferret. Tanaka et al. [Fundam. Appl. ToxicoL 22 (1994) 577; J. Toxicol. Environ. Health 58 (1999) 215] reported an age-related pattern of fiber and cell body degeneration progressing from retinal axons and lateral geniculate nucleus (LGN) neurons to the visual cortex. These studies, however, did not address whether TPP exposure results in retinal ganglion cell (RGC) degeneration, nor did they quantify the degenerative effects in the LGN. The purpose of this study was to quantify the effects of TPP on RGCs and LGN neurons. We administered single subcutaneous injections of TPP (1184 mg/kg) to 13 ferrets for histological analysis. The retinae were examined as whole-mounts and the brains sectioned parasagittally (50 microm). RGC countsfrom matched areas of nasal retina showed significantly fewer (21%) neurons in the TPP-treated ferrets (Sd = 282 +/- 52S.D.; 7d = 284 +/- 12S.D.) compared with control (359 +/- 42S.D.). No significant difference in cell number was found in temporal retina, even though this region contained, on average, 13% fewer ganglion cells in TPP-treated ferrets (Sd = 3344 +/- 44S.D.; 7d = 357 +/- 39S.D. versus control = 394 +/- 72S.D.). The mean soma sizes and RGC cell size distributions for nasal and temporal retinae were not significantly different for any group. LGN neurons were significantly smaller (28%) than control in the TPP-treated ferrets (Sd = 155 microm2 +/- 23S.D.; 7d = 152 microm2 +/- 28S.D. versus control = 214 microm2 +/- 9S.D.). Cell size distributions for LGN neurons were shifted toward smaller cell sizes in both TPP-treated groups compared to control.

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 microg). GFAP protein levels were normal at 1 day post-crush, but increased approximately 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.