Real-Time Video Microscopy of In Vitro Demodex Death by Intense Pulsed Light.

Objective: To directly observe the in vitro real-time effects of intense pulsed light (IPL) on a Demodex mite extracted from an eyelash of a patient with ocular rosacea. Background: Demodex is a risk factor in the pathogenesis of oculofacial rosacea, meibomian gland dysfunction (MGD), and dry eye disease (DED). Recent studies suggested IPL to control or eradicate Demodex organisms in the periocular area. Despite encouraging reports, the direct effect of IPL on Demodex is not well understood. Methods: An eyelash infested with Demodex was epilated from a 62-year-old female patient with oculofacial rosacea. Following isolation and adherence of a mite onto a microscope slide, real-time video microscopy was used to capture live images of the organism before, during, and after administration of IPL pulses. IPL pulses were delivered with the M22 IPL (Lumenis), with IPL settings used for treatment of DED due to MGD (the "Toyos protocol"). A noncontact digital laser infrared thermometer was used to measure the temperature of the slide. Results: Before the IPL pulses, legs of the Demodex mite spontaneously moved in a repetitive and semicircular motion. During administration of IPL, spontaneous movements of the legs continued. Immediately after administration of five IPL pulses, the temperature of the slide increased from room temperature to 49°C. Immediately afterward, the Demodex mite became completely immobilized. The legs appeared retracted, smoother, less corrugated, bulkier, and less well-defined. Movement of the Demodex mite was not observed at the hourly inspections for 5 h and after 24 h following the application of IPL pulses. Conclusions: Our video directly demonstrates the effect of IPL on a live Demodex mite extracted from a freshly epilated eyelash. The results suggest that IPL application with settings identical to those used for treatment of DED due to MGD causes a complete destruction of the organism.

Investigating the role of allergic contact dermatitis in residual ocular surface disease on dupilumab (ROSDD),.

BACKGROUND: The mechanisms underlying eye-related complications with dupilumab are poorly understood. OBJECTIVE: This study aimed to determine the incidence and characteristics of ocular complications with dupilumab and the prevalence of comorbid allergic contact dermatitis in the same subpopulation. METHODS: This is a retrospective chart review of 48 patients with atopic dermatitis who received dupilumab. For patients with eye involvement at first follow-up, we discuss the presence of eyelid dermatitis, blepharitis, or conjunctivitis and analyze available patch test findings in patients with ocular complications while treated with dupilumab. RESULTS: A total of 14 patients (29.2%) showed eye involvement while on dupilumab, all of whom experienced eye involvement prior to dupilumab. The results of the patch test were most commonly positive for emulsifier/surfactants (42.5%) and fragrances (30.4%). Nine patients experienced improvement with allergen avoidance subsequent to patch testing, and four of nine patients' conditions cleared almost entirely. This is a non-randomized study in a small cohort of patients. Only 18 patients had their disease confirmed by an ophthalmologist. CONCLUSION: All patients with eye involvement while on dupilumab had a history of eye involvement prior to dupilumab, suggest that dupilumab may encourage rather than cause ocular surface inflammation. Significant improvement after patch testing in nearly half of patients suggests that allergic contact dermatitis contributes to some cases of dupilumab-associated eye complications.

Carbon nanotube bucky paper as an artificial support membrane for retinal cell transplantation.

BACKGROUND AND OBJECTIVE: Transplantation of epithelial cells on a substrate to rescue diseased retinal cells is an experimental therapy for age-related macular degeneration. Carbon nanotube bucky paper was tested for cell transplantation into the retina. MATERIALS AND METHODS: Bucky paper was prepared and human RPE cells cultured on its surface demonstrating its utility as a cell transplantation substrate. Bucky paper was implanted underneath 9 rabbit retinas using a standard 3-port pars plana vitrectomy and subretinal bleb. A 1 mm retinotomy was created through which Bucky paper precut to fit was inserted with the subretinal forceps, into the subretinal bleb. The retina was reattached by airfluid exchange. RESULTS: By light microscopy, RPE cells demonstrated normal morphology and growth patterns on the bucky paper surface. Scanning electron microscopy confirmed a confluent monolayer of cells, and indicated the formation of microvilli on the apical surface. Bucky paper remained flat in the subretinal space after 2 weeks, the retina fully attached without edema or inflammation. CONCLUSION: Bucky paper possesses the necessary attributes for therapeutic cell transplantation in the eye.

Chronic spontaneous activity generated in the somata of primary nociceptors is associated with pain-related behavior after spinal cord injury.

Mechanisms underlying chronic pain that develops after spinal cord injury (SCI) are incompletely understood. Most research on SCI pain mechanisms has focused on neuronal alterations within pain pathways at spinal and supraspinal levels associated with inflammation and glial activation. These events might also impact central processes of primary sensory neurons, triggering in nociceptors a hyperexcitable state and spontaneous activity (SA) that drive behavioral hypersensitivity and pain. SCI can sensitize peripheral fibers of nociceptors and promote peripheral SA, but whether these effects are driven by extrinsic alterations in surrounding tissue or are intrinsic to the nociceptor, and whether similar SA occurs in nociceptors in vivo are unknown. We show that small DRG neurons from rats (Rattus norvegicus) receiving thoracic spinal injury 3 d to 8 months earlier and recorded 1 d after dissociation exhibit an elevated incidence of SA coupled with soma hyperexcitability compared with untreated and sham-treated groups. SA incidence was greatest in lumbar DRG neurons (57%) and least in cervical neurons (28%), and failed to decline over 8 months. Many sampled SA neurons were capsaicin sensitive and/or bound the nociceptive marker, isolectin B4. This intrinsic SA state was correlated with increased behavioral responsiveness to mechanical and thermal stimulation of sites below and above the injury level. Recordings from C- and Aδ-fibers revealed SCI-induced SA generated in or near the somata of the neurons in vivo. SCI promotes the entry of primary nociceptors into a chronic hyperexcitable-SA state that may provide a useful therapeutic target in some forms of persistent pain.

Long-lasting hyperexcitability induced by depolarization in the absence of detectable Ca2+ signals.

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.

A model neural interface based on functional chemical stimulation.

While functional electrical stimulation has been applied to treat a variety of neurological disorders, it cannot mimic function that is primarily achieved using neurochemical means. In this work, we present a neurotransmitter-based prosthetic interface in the form of a flexible microdevice that selectively releases chemical pulses through an aperture in a polymer membrane. The release profiles through the aperture are controlled by microfluidic switching in an underlying channel network. The profiles have been characterized using fluorescence microscopy as a function of pulse duration and frequency. Hippocampal neurons were cultured on the microdevices and cell stimulation via glutamate delivery was detected using calcium imaging. The release properties could be tuned to repeatedly elicit discrete action potentials in cells seeded proximate to the aperture, including single cell stimulation at 2 Hz. This model neural interface based on functional chemical stimulation may provide the biomimetic platform necessary to restore physiological pathways and function that electrical stimulation cannot fundamentally address.

Spatial cues for the enhancement of retinal pigment epithelial cell function in potential transplants.

Retinal pigment epithelial (RPE) cellular morphology and function are vital to the health of the retina. In age-related macular degeneration, RPE dysfunction and changes in Bruch's membrane occur. Thus, a potential cure is a dual-layer biomimetic transplant consisting of a layer of healthy RPE cells cultured on a support membrane. In this study, we investigated human anterior lens capsule as a replacement for Bruch's membrane and also explored different seeding methods as ways of inducing the desired cellular morphology and function. Using in vitro assays, we demonstrated that RPE cells cultured on lens capsule exhibited epithelial characteristics, such as the presence of actin belts and the formation of tight junctions in the monolayer. Bovine photoreceptor outer segments were also incubated with the RPE cells in order to quantify the binding and ingestion activity of the RPE cells. With these assays, we determined that cells seeded by centrifugation appeared to possess the most epithelial-like morphology, with the shortest overall length and the smallest elongation. They also exhibited enhanced metabolic activity, with a 1.5-fold increase over conventional gravity seeding. Thus, the spatial cues provided by centrifugation may assist cells in assuming native RPE function. Therefore, a dual-layer transplant, with RPE cells organized by centrifugation onto lens capsule, appears promising in achieving native retinal function.

Thin collagen film scaffolds for retinal epithelial cell culture.

Collagen films have been used in biological implantation and surgical grafts. The development of thin collagen films on the order of 10 microm thick that ensure a planar distribution of implanted cells is a necessary step towards surgical grafts for treatment of age-related macular degeneration (AMD). Here, collagen films were manufactured on a Teflon support to a thickness of 2.4+/-0.2 microm, comparable to that of native Bruch's membrane. Because one important function of Bruch's membrane is allowing the flow of nutrients and waste to and from the retinal pigment epithelium the diffusion properties of the collagen films were studied using blind-well chambers. The diffusion coefficient of the collagen film was determined to be 4.1 x 10(-10)cm(2)/s for 71,200 Da dextran molecules. Viability studies utilizing the blind-well chambers also confirmed that nutrient transport through the films was sufficient to sustain retinal pigment epithelial (RPE) cells. The films were bioassayed in a RPE cell culture model to confirm cell attachment and viability. RPE cells were shown to form an epithelial phenotype and were able to phagocytize photoreceptor outer segments.

Neural stimulation with a carbon nanotube microelectrode array.

We present a novel prototype neural interface using vertically aligned multiwalled carbon nanotube (CNT) pillars as microelectrodes. Functionalized hydrophilic CNT microelectrodes offer a high charge injection limit (1-1.6 mC/cm2) without faradic reactions. The first repeated in vitro stimulation of hippocampal neurons with CNT electrodes is demonstrated. These results suggest that CNTs are capable of providing far safer and more efficacious solutions for neural prostheses than previous metal electrode approaches.

A model retinal interface based on directed neuronal growth for single cell stimulation.

In this work, we use cell micropatterning technologies to direct neuronal growth to individual electrodes, and demonstrate that such an approach can achieve selective stimulation and lower stimulation thresholds than current field-effect based retinal prostheses. Rat retinal ganglion cells (RGCs) were purified through immunopanning techniques, and microcontact printing (microCP) was applied to align and pattern laminin on a microelectrode array, on which the RGCs were seeded and extended neurites along the pattern to individual electrodes. The stimulation threshold currents of RGCs micropatterned to electrodes were found to be significantly less than those of non-patterned RGCs over a wide range of electrode-soma distances, as determined with calcium imaging techniques. Moreover, the stimulation threshold for micropatterned cells was found to be independent of electrode-soma distance, and there was no significant effect of microCP on cell excitability. The effects of additional stimulation parameters, such as electrode size and pulse duration, on threshold currents were determined. The stimulation results quantitatively demonstrate the potential benefits of a retinal prosthetic interface based on directed neuronal growth.

Determination of human lens capsule permeability and its feasibility as a replacement for Bruch's membrane.

We have investigated human anterior lens capsule as a potential replacement for Bruch's membrane as a treatment for age-related macular degeneration. Any substrate to replace Bruch's membrane should possess certain characteristics to maintain proper function of the overlying retina. One of the important properties of Bruch's membrane is allowing the flow of nutrients and waste between the retinal pigment epithelium and the choriocapillaris. Here, we measured the permeability of the lens capsule by studying the diffusion of various molecular weight FITC-dextran molecules. Expressions for extraction of diffusion coefficients from concentration vs. time data from a blind-well chamber apparatus were derived for both a single and double membrane experiments. The diffusion coefficients in the lens capsule were found to be in the range of 10(-6) to 10(-10)cm2/s. We demonstrated a power law relationship, with the diffusion coefficient possessing a -0.6 order dependence on molecular weight. The molecular weight exclusion limit was determined to be 150+/-40 kDa. We have compared this value with reported values of Bruch's membrane molecular weight exclusion limit and find that the lens capsule has the potential to act as a substitute Bruch's membrane.

Critical interval of somal calcium transient after neurite transection determines B 104 cell survival.

Nerve cells may survive or die after axonal or dendritic transection. After neurite transection near (<50 mum) the cell body of Fura-2-loaded B 104 neuroblastoma (rat brain-derived) cells, the somal calcium concentration (SCC) undergoes a three-phase transient change: a rapid (0-0.15-min post-transection [PT]) rise phase, followed by an early (0.15--1.5-min PT) rapid decay phase, and succeeded by a late (1.5-60-min PT) slower decay phase that restores SCC to preinjury levels. The SCC in a critical interval (1.5-12.5 min PT) of the third transient phase correlates with cell fate, i.e., most transected cells that exclude dye (restore a barrier) and die have a significantly higher (P<0.005) SCC in this critical interval than do transected cells that exclude dye and survive at 24-hr PT. Loading BAPTA (chelation of somal Ca(2+)) before, but not after, the critical interval increases the percentage of cells that survive compared to that of cells transected without BAPTA loading. Furthermore, most transected cells that die despite successful barrier restoration exhibit characteristics consistent with apoptosis initiated during the critical interval of the SCC, including caspase activation and plasmalemmal phosphatidylserine translocation. These data suggest that decreased cell survival for injuries near the soma is due to Ca(2+)-initiated apoptosis during the critical interval of the third phase of the SCC transient. (c) 2005 Wiley-Liss, Inc.

Directed retinal nerve cell growth for use in a retinal prosthesis interface.

PURPOSE: Retinal prosthetic devices that use microelectrode arrays to stimulate retinal nerve cells may provide a viable treatment for degenerative retinal diseases. Current devices are based on electrical field-effect stimulation of remaining functional neural elements. However, the distance between target neurons and electrodes limits the potential density of electrodes and the ability to stimulate specific types of retinal neurons that contribute to visual perceptions. This study was conducted to investigate the use of microcontact printing (muCP) to direct cultured or explant retinal ganglion cell (RGC) neurites to precise and close stimulation positions and to evaluate the cell types that grow from a retinal explant. METHODS: RGCs and whole retinal explants were isolated from postnatal day-7 Sprague-Dawley rats using immunopanning purification and microdissection, respectively. Aligned muCP was used to direct the growth of RGC neurites from pure cultures (n=105) and retinal explants (n=64) along laminin patterns and to individual microelectrodes. Immunofluorescence stains (n=39) were used to determine the cell types that grew out from the retinal explants. RESULTS: RGC neurite growth was directed reproducibly along aligned laminin micropatterns to individual microelectrodes in pure RGC cultures and from full-thickness explanted rat retinas in 92% of experiments, neurites from pure RGC cultures extended along the laminin lines with an average length of 263 +/- 118 microm (SD; n=27) after 24 hours. Neurites from retinal explants extended in more than 80% of experiments and were observed to grow to an average length of 279 +/- 78 microm (n=64) after 2 days in culture. These neurites grew up to 3 mm after 1 month of culture on the laminin micropatterns. Immunohistochemical stains demonstrated that extended processes from both RGCs and glial cells grew out of retinal explants onto stamped laminin lines. CONCLUSIONS: Using muCP to pattern surfaces with growth factors, individual neuronal processes from pure RGC culture and whole retinal explants can be directed to discrete sites on a microelectronic chip surface. By directing RGC neurite processes to specific sites, single cell stimulation becomes possible. This may allow discrete populations of retinal neurons to be addressed so that physiologic retinal processing of visual information can be achieved.

Migration of retinal cells through a perforated membrane: implications for a high-resolution prosthesis.

PURPOSE: One of the critical difficulties in design of a high-resolution retinal implant is the proximity of stimulating electrodes to the target cells. This is a report of a phenomenon of retinal cellular migration into a perforated membrane that may help to address this problem. METHODS: Mylar membranes with an array of perforations (3-40 microm in diameter) were used as a substrate for in vitro retinal culture (chicken, rats) and were also transplanted into the subretinal space of adult RCS rats. A membrane was also constructed with a seal on one side to restrict the migration. RESULTS: Retinal tissue in vitro grew within 3 days through perforations of greater than 5 microm in diameter when the membranes were positioned on the photoreceptor side, but no migration occurred if the implant was placed on the inner retinal surface. Histology with light microscopy and transmission electron microscopy (TEM) demonstrated that migrating cells retain neuronal structures for signal transduction. Similar growth of RCS rat retinal cells occurred in vivo within 5 days of implantation. A basal seal kept the migrating tissue within a small membrane compartment. CONCLUSIONS: Retinal neurons migrate within a few days into perforations (> 5 microm in diameter) of a membrane placed into the subretinal space. This may provide a means of gaining close proximity between electrodes in a retinal prosthetic chip and target cells, and thus allow a greater density of stimulating elements to subserve higher resolution. Further studies are needed to explore the long-term stability of the retinal migration.

Survival of mammalian B104 cells following neurite transection at different locations depends on somal Ca2+ concentration.

We report that cell survival after neurite transection in a mammalian neuronal model (cultured B104 cells) critically depends on somal [Ca2+]i, a novel result that reconciles separate long-standing observations that somal survival decreases with more-proximal axonal transections and that increased somal Ca2+ is cytotoxic. Using fluorescence microscopy, we demonstrate that extracellular Ca2+ at the site of plasmalemmal transection is necessary to form a plasmalemmal barrier, and that other divalent ions (Ba2+, Mg2+) do not play a major role. We also show that extracellular Ca2+, rather than injury per se, initiates the formation of a plasmalemmal barrier and that a transient increase in somal [Ca2+]i significantly decreases the percentage of cells that survive neurite transection. Furthermore, we show that the increased somal [Ca2+]i and decreased cell survival following proximal transections are not due to less frequent or slower plasmalemmal sealing or Ca2+ entry through plasmalemmal Na+ and Ca2+ channels. Rather, the increased somal [Ca2+]i and lethality of proximal neurite injuries may be due to the decreased path length/increased diameter for Ca2+ entering the transection site to reach the soma. A ryanodine block of Ca2+ release from internal stores before transection has no effect on cell survival; however, a ryanodine- or thapsigargin-induced buildup of somal [Ca2+]i before transection markedly reduces cell survival, suggesting a minor involvement of Ca2+-induced release from internal stores. Finally, we show that cell survival following proximal injuries can be enhanced by increasing intracellular Ca2+ buffering capacity with BAPTA to prevent the increase in somal [Ca2+]i.

Localized chemical release from an artificial synapse chip.

A device that releases chemical compounds in small volumes and at multiple, well defined locations would be a powerful tool for clinical therapeutics and biological research. Many biomedical devices such as neurotransmitter-based prostheses or drug delivery devices require precise release of chemical compounds. Additionally, the ability to control chemical gradients will have applications in basic research such as studies of cell microenvironments, stem cell niches, metaplasia, or chemotaxis. We present such a device with repeatable delivery of chemical compounds at multiple locations on a chip surface. Using electroosmosis to drive flow through microfluidic channels, we pulse minute quantities of a bradykinin solution through four 5-microm apertures onto PC12 cells and show stimulation of individual cells using a Ca(2+)-sensitive fluorescent dye. We also present basic computational results with experimental verification of both fluid ejection and fluid withdrawal by imaging pH changes by using a fluorescent dye. This "artificial synapse chip" is a prototype neural interface that introduces a new paradigm for neural stimulation, with eventual application in treating macular degeneration and other neurological disorders.

Fluid flow past an aperture in a microfluidic channel.

Electroosmotically driven flow in neurotransmitter-based retinal prostheses offers a novel approach to interfacing the nervous system. Here, we show that electroosmotically driven flow in a microfluidic channel can be used either to eject or to withdraw fluid through a small aperture in the channel wall. We study this fluid movement numerically using a finite-element method and experimentally using microfabricated channels and apertures. Two devices are used to test the concept of fluid ejection and withdrawal: (1) a single, large channel with four apertures and (2) a prototype neural interface with four individually addressable apertures. We compared experimental and numerical results in microchannels using the observed pH dependence of the fluorescent dye fluorescein, finding good agreement between the results. Because of the simplicity and rapid response of electroosmotic flow, this technique may be useful for neurotransmitter-based neural interfaces.