Effect of epiretinal electrical stimulation on the glial cells in a rabbit retinal eyecup model.

Introduction: We examined how pulse train electrical stimulation of the inner surface of the rabbit retina effected the resident glial cells. We used a rabbit retinal eyecup preparation model, transparent stimulus electrodes, and optical coherence tomography (OCT). The endfeet of Müller glia processes line the inner limiting membrane (ILM). Methods: To examine how epiretinal electrode stimulation affected the Müller glia, we labeled them post stimulation using antibodies against soluble glutamine synthetase (GS). After 5 min 50 Hz pulse train stimulation 30 µm from the surface, the retina was fixed, immunostained for Müller glia, and examined using confocal microscopic reconstruction. Stimulus pulse charge densities between 133-749 µC/cm2/ph were examined. Results: High charge density stimulation (442-749 µC/cm2/ph) caused significant losses in the GS immunofluorescence of the Müller glia endfeet under the electrode. This loss of immunofluorescence was correlated with stimuli causing ILM detachment when measured using OCT. Müller cells show potassium conductances at rest that are blocked by barium ions. Using 30 msec 20 µA stimulus current pulses across the eyecup, the change in transretinal resistance was examined by adding barium to the Ringer. Barium caused little change in the transretinal resistance, suggesting under low charge density stimulus pulse conditions, the Müller cell radial conductance pathway for these stimulus currents was small. To examine how epiretinal electrode stimulation affected the microglia, we used lectin staining 0-4 h post stimulation. After stimulation at high charge densities 749 µC/cm2/ph, the microglia under the electrode appeared rounded, while the local microglia outside the electrode responded to the stimulated retina by process orientation inwards in a ring by 30 min post stimulation. Discussion: Our study of glial cells in a rabbit eyecup model using transparent electrode imaging suggests that epiretinal electrical stimulation at high pulse charge densities, can injure the Müller and microglia cells lining the inner retinal surface in addition to ganglion cells.

Toward a wearable monitor of local muscle fatigue during electrical muscle stimulation using tissue Doppler imaging.

Electrical muscle stimulation (EMS) is widely used in rehabilitation and athletic training to generate involuntary muscle contractions. However, EMS leads to rapid muscle fatigue, limiting the force a muscle can produce during prolonged use. Currently available methods to monitor localized muscle fatigue and recovery are generally not compatible with EMS. The purpose of this study was to examine whether Doppler ultrasound imaging can assess changes in stimulated muscle twitches that are related to muscle fatigue from electrical stimulation. We stimulated five isometric muscle twitches in the medial and lateral gastrocnemius of 13 healthy subjects before and after a fatiguing EMS protocol. Tissue Doppler imaging of the medial gastrocnemius recorded muscle tissue velocities during each twitch. Features of the average muscle tissue velocity waveforms changed immediately after the fatiguing stimulation protocol (peak velocity: -38%, p = .022; time-to-zero velocity: +8%, p = .050). As the fatigued muscle recovered, the features of the average tissue velocity waveforms showed a return towards their baseline values similar to that of the normalized ankle torque. We also found that features of the average tissue velocity waveform could significantly predict the ankle twitch torque for each participant (R2 = 0.255-0.849, p < .001). Our results provide evidence that Doppler ultrasound imaging can detect changes in muscle tissue during isometric muscle twitch that are related to muscle fatigue, fatigue recovery, and the generated joint torque. Tissue Doppler imaging may be a feasible method to monitor localized muscle fatigue during EMS in a wearable device.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses.

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Access resistance of stimulation electrodes as a function of electrode proximity to the retina.

OBJECTIVE: Epiretinal prostheses seek to effectively stimulate the retina by positioning electrode arrays close to its surface so current pulses generate narrow retinal electric fields. Our objective was to evaluate the use of the electrical impedance of insulated platinum electrodes as a measure of the proximity of insulated platinum electrodes to the inner surface of the retina. APPROACH: We examined the impedance of platinum disk electrodes, 0.25 mm in diameter, insulated with two widths (0.8 and 1.6 mm outer diameter) of transparent fluoropolymer in a rabbit retinal eyecup preparation. Optical coherence tomography measured the electrode's proximity to the retinal surface which was correlated with changes in the voltage waveform at the electrode. Electrode impedance changes during retinal deformation were also studied. MAIN RESULTS: When the 1.6 mm diameter insulated electrodes advanced towards the retinal surface from 1000 µm, their voltage step at current pulse onset increased, reflecting an access resistance increase of 3880 ± 630 Ω, with the 50% midpoint averaging 30 µm, while thin 0.8 mm insulated electrode advancement showed an access resistance increase 50% midpoint averaging 16 µm. Using impedance spectroscopy, electrode-retina proximity differences were seen in the 1.6 mm insulated electrode impedance modulus between 1 and 100 kHz and the waveform phase angle at 0.3-10 kHz, while thin 0.8 mm insulated electrode advancement produced smaller impedance modulus changes with retinal proximity between 3 and 100 kHz. These impedance changes with retinal proximity may reflect different sized zones of eye wall being coupled in series with the insulated platinum electrode. SIGNIFICANCE: The proximity of stimulus electrodes to neural tissue in fluid-filled spaces can be estimated from access resistance changes in the stimulus pulse waveform. Because many prosthetic devices allow back telemetry communication of the stimulus electrode waveform, it is possible these series resistance increases observed with retinal proximity could be used as a metric of stimulus electrode placement.

The use of time-lapse optical coherence tomography to image the effects of microapplied toxins on the retina.

PURPOSE: We developed a novel technique for accelerated drug screening and retinotoxin characterization using time-lapse optical coherence tomography (OCT) and a drug microapplication device. METHODS: Using an ex vivo rabbit eyecup preparation, we studied retinotoxin effects in real-time by microperfusing small retinal areas under a transparent fluoropolymer tube. Known retinotoxic agents were applied to the retina for 5-minute periods, while changes in retinal structure, thickness, and reflectance were monitored with OCT. The OCT images of two agents with dissimilar mechanisms, cyanide and kainic acid, were compared to their structural changes seen histologically. RESULTS: We found the actions of retinotoxic agents tested could be classified broadly into two distinct types: (1) agents that induce neuronal depolarization, such as kainic acid, causing increases in OCT reflectivity or thickness of the inner plexiform and nuclear layers, and decreased reflectivity of the outer retina; and (2) agents that disrupt mitochondrial function, such as cyanide, causing outer retinal structural changes as evidenced by a reduction in the OCT reflectivity of the photoreceptor outer segment and pigment epithelium layers. CONCLUSIONS: Retinotoxin-induced changes in retinal layer reflectivity and thickness under the microperfusion tube in OCT images closely matched the histological evidence of retinal injury. Time-lapse OCT imaging of the microperfused local retina has the potential to accelerate drug retinotoxicological screening and expand the use of OCT as an evaluation tool for preclinical animal testing.