Biochemistry and Molecular Biology Inverse Relationship Between the Intraretinal Concentration of Bioavailable Nitric Oxide and Blood Glucose in Early Experimental Diabetic Retinopathy

Citation: Guthrie MJ, Osswald CR, Kang-Mieler JJ. Inverse relationship between the intraretinal concentration of bioavailable nitric oxide and blood glucose in early experimental diabetic retinopathy. Invest Ophthalmol Vis Sci. 2015;56:37-44. DOI:10. 1167/iovs.14-15777 PURPOSE. To directly measure in vivo retinal nitric oxide (NO) concentration in experimental early diabetic retinopathy and correlate measurements with blood glucose to determine how intraretinal NO changes with severity of diabetes. METHODS. Long-Evans rats were made diabetic with streptozotocin (STZ). Three weeks post STZ injection, intraretinal NO concentration profiles were recorded using a dual NO/ electroretinogram microelectrode. Diabetic profiles were compared with profiles from healthy controls, healthy rats injected with the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME), and healthy rats that received acute glucose injections (''acute hyperglycemia''). The NO values at the retina/RPE boundary (100% retinal depth) and retinal surface (0% depth) were analyzed for correlation with blood glucose. RESULTS. The average NO concentrations in the outer retina, inner retina, and vitreous humor of mild diabetic rats (250-400 mg/dL) were significantly higher than controls by 73%, 47%, and 70%, respectively. The average NO concentrations in the outer retina, inner retina, and vitreous humor of severe diabetic rats (500-600 mg/dL) were lower than controls, with NO at 41%, 36%, and 36% of controls, respectively, similar to L-NAME-treated eyes (38%, 36%, 20% of control). The NO levels in moderate diabetic rats (400-500 mg/dL) and acute hyperglycemia rats were similar to controls. The NO was significantly and inversely correlated with blood glucose for diabetic rats at 100% depth (R ¼ À0.91) and 0% depth (R ¼ À0.79) but not for acute hyperglycemia rats. CONCLUSIONS. The higher-than-control level of NO in mild diabetic rats and lower-than-control level in severe diabetic rats show that severity of diabetes is an important factor when measuring the bioavailability of NO in diabetic retinopathy

MICROELECTRODE ARRAY FOR CAPACITIVE TRANSDUCTION OF RETINAL RESPONSES

Neural degenerative diseases and traumatic injuries to the eye affect millions of people worldwide, motivating the development of neural prosthetic interfaces to restore sensory or motor function in affected individuals. Advances in neural sensing and stimulation interface technology will allow a more comprehensive understanding of neural function while leading to the development of hybrid biological-electronic sensor devices for robust, functioning neural prosthetic systems. Current techniques of neural activity sensing employ multi-electrode arrays (MEAs) that typically incorporate metal electrodes and measure currents via an electrochemical junction, leading to corrosion and charge transfer across the electrode-tissue interface. High-density neural interface technology will require active circuitry within the implant; the device must withstand corrosion and induce minimal damage at the electrode/tissue interface. The work shown here demonstrates a prototype neural interface device based on capacitive coupling through hafnium oxide encapsulation of a novel 3D device architecture, advancing neural sensing technology toward long-term implantable neural interfaces. The functionalization of biosensors interfaced with neural tissue is important to ensure that the active components of the sensor are fully protected from the surrounding biological environment. Self-assembled monolayers (SAMs) have been extensively studied as coatings for implantable devices due to their ability to tailor surface properties and relative ease of film formation. We report a series of studies aimed at investigating the stability of phosphonate self-assembled monolayers, octdecylphosphonic acid (ODPA) or perfluorophosphonic acid (PFPA) on various oxide surfaces (SiO2, TiO2, Al2O3 and HfO2) to serve as the biotic-abiotic interface of the prototype neural device developed here. The monolayers were deposited by a series of techniques including self-assembly from solution, tethering by aggregation and growth and Langmuir-Blodgett (LB). SAMs prepared by LB were primarily used in our stability investigations because they were found to be the most uniform and reproducible. All films deposited on oxide-coated substrates were characterized by means of water contact angle measurements, spectroscopic ellipsometry, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). XPS data conclusively showed covalent phosphonate formation on all substrates except SiO2, which had background spectra that interfered with the data analysis. AFM images of SAMs formed on SiO2 and TiO2 showed significant surface reorganization upon exposure to water within 30 minutes. SAMs formed on Al2O3 and HfO2 were much more stable upon exposure to water. PFPA SAMs on HfO2 were found to be the most stable SAMs studied here in either water or phosphate buffer at room temperature. This is the first report of a SAM-oxide system showing stability for an extended period of time, greater than 20 days. These data suggest that phosphonate SAMs should be considered for implantable neural devices that require longer-term stability under aqueous conditions. To examine the encoding and processing of information by networks of neurons, microelectrode arrays (MEAs) have been developed and applied, but evolving scientific questions and biomedical applications require higher density sampling and wider spatial coverage. The integration of 3D electrodes can provide closer contact with neurons to facilitate detection and resolution of single cell action potentials. The fabrication methods implemented here allows reliable fabrication of a novel MEA consisting of probes with dimensions of a few microns, unlike most other approaches to 3D electrode arrays, which produce structures on the scale of tens of microns or more. The device incorporates over 3,800 micro pillar electrodes, grouped into 60 independent sensors for compatibility with existing electronics, spread over an area of 750 μm2; each sensor site consists of an 8x8 array of micropillars, interconnected by a lead to an output pad of the device. Individual 3D pillars are 3 μm in diameter with a height of 8 μm. Our experience has suggested that such microstructured probes can achieve more intimate contact with the surface of neural tissue, and enhance the quality of neuronal recordings. Electrochemical impedance spectroscopy (EIS) at 1 kHz measured average magnitude and phase shift of 710 W and 17°, respectively, for a single sensor site. These values confirm the robustness of our fabrication process for developing highly conductive 3D microelectrodes. The results shown here demonstrate high-density, three-dimensional microfabrication technology that was applied to the development of an advanced capacitive sensor array for neural tissue. Applications in sensing technology now require electro-neural interface devices to withstand corrosion and induce minimal damage at the electrode/tissue interface. We have developed a platform suitable for hermetic sealing and have shown encapsulation through atomic layer deposition of hafnium oxide over the active components of the device to overcome the direct current limitations of existing MEA technology. EIS was used to study the oxide deposition on the 3D micro pillar sensor array to ensure a pinhole-free dielectric coating. The characteristic impedance magnitudes increase up to 3 orders of magnitude upon oxide deposition and the phase indicates fully capacitive sensor sites. The fabrication process and electrochemical impedance study shown here, demonstrates the usefulness of such techniques for building high-density 3D arrays that can be fully encapsulated with a protective dielectric coating. This work advances the technology towards capacitive sensing of retinal neurons with a robust, non-invasive sensing device. Sensing retinal neurons with the 3D micropillar array developed here was performed for direct current and capacitive configurations of the device. Electroretinograms (ERGs) were recorded and the overall performance of the device was analyzed. The devices showed good consistency across all 60 Pt electrode clusters during characterization and when interfaced with retinal tissue. ERGs were recorded by more than 80% of the direct current electrode sites and the performance was evenly distributed around the mean response. This performance surpasses previous reports of 3D electrode arrays interfaced with retinal tissue, where typically 1-6 electrode signals are recorded successfully. Encapsulation of the device platform was achieved and successful recordings of ERG signals were shown. This work is the first report of sensing the overall electrical behavior of retinal tissue with a coupled capacitive MEA

Glaucoma

This book addresses the basic and clinical science of glaucomas, a group of diseases that affect the optic nerve and visual fields and is usually accompanied by increased intraocular pressure. The book incorporates the latest development as well as future perspectives in glaucoma, since it has expedited publication. It is aimed for specialists in glaucoma, researchers, general ophthalmologists and trainees to increase knowledge and encourage further progress in understanding and managing these complicated diseases

Electrophysiology

The outstanding evolution of recording techniques paved the way for better understanding of electrophysiological phenomena within the human organs, including the cardiovascular, ophthalmologic and neural systems. In the field of cardiac electrophysiology, the development of more and more sophisticated recording and mapping techniques made it possible to elucidate the mechanism of various cardiac arrhythmias. This has even led to the evolution of techniques to ablate and cure most complex cardiac arrhythmias. Nevertheless, there is still a long way ahead and this book can be considered a valuable addition to the current knowledge in subjects related to bioelectricity from plants to the human heart

A study of the retinal vascular pathology in the Royal College of Surgeons rat: A model of human retinal degeneration.

The leading causes of loss of vision in the developed world are the degenerative diseases of photoreceptors in particular, age-related macular degeneration (AMD) and retinitis pigmentosa (RP). A common characteristic of these diseases is secondary damage affecting the vascular network, which is apparently initiated by photoreceptor loss. One problem with investigating the vascular consequences of these diseases has been the lack of a suitable animal model that can be used to investigate various potential treatments. This study has developed methods of quantifying retinal vascular damage in the pigmented Royal College of Surgeons (RCS) rat, which is characterised by the formation of vascular complexes and these methods have been used to explore strategies to retard or reverse this damage. This was done with the view to improving the retinal environment, thereby assisting other therapeutic strategies that target the primary defect causing the loss of photoreceptors. The work was divided into three areas: 1) investigation of the vascular effects of progressive photoreceptor loss and development of computerised image analysis to quantify changes, 2) pharmaceutical intervention to modify the normal sequence of events, 3) examination of the effects of RPE sub-retinal transplantation on the vascular network to determine how the retinal vasculature would react to the presence of transplanted human RPE cells at different time-points. These three areas of study validate the use of naturally occurring events in the RCS rat to provide a model of vascular pathology in human retinal degenerative diseases. This contrasts with previous models, which have relied on creating wounds to simulate conditions that occurring in the diseased human retina

The Drosophila Homolog of the Intellectual Disability Gene ACSL4 Acts in Glia to Regulate Morphology and Neuronal Activity: A Dissertation

Recent developments in neurobiology make it clear that glia play fundamental and active roles, in the adult and in development. Many hereditary cognitive disorders have been linked to developmental defects, and in at least two cases, Rett Syndrome and Fragile X Mental Retardation, glia are important in pathogenesis. However, most studies of developmental disorders, in particular intellectual disability, focus on neuronal defects. An example is intellectual disability caused by mutations in ACSL4, a metabolic enzyme that conjugates long-chain fatty acids to Coenzyme A (CoA). Depleting ACSL4 in neurons is associated with defects in dendritic spines, a finding replicated in patient tissue, but the etiology of this disorder remains unclear. In a genetic screen to discover genes necessary for visual function, I identified the Drosophila homolog of ACSL4, Acsl, as a gene important for the magnitude of neuronal transmission, and found that it is required in glia. I determined that Acsl is required in a specific subtype of glia in the Drosophila optic lobe, and that depletion of Acsl from this population causes morphological defects. I demonstrated that Acsl is required in development, and that the phenotype can be rescued by human ACSL4. Finally, I discovered that ACSL4 is expressed in astrocytes in the mouse hippocampus. This study is highly significant for understanding glial biology and neurodevelopment. It provides information on the role of glia in development, substantiates a novel role for Acsl in glia, and advances our understanding of the potential role that glia play in the pathogenesis of intellectual disability

Aerospace Medicine and Biology - A continuing bibliography with indexes

Annotated bibliography and indexes on Aerospace Medicine and Biology - Dec. 196

Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds

A Flight Sensory-Motor to Olfactory Histamine Circuit Mediates Olfactory Processing of Ecologically and Behaviorally Natural Stimuli

Environmental pressures have conferred species specific behavioral and morphological traits to optimize reproductive success. To optimally interact with their environment, nervous systems have evolved motor-to-sensory circuits that mediate the processing of its own reafference. Moth flight behavioral patterns to odor sources are stereotyped, presumably to optimize the likelihood of interacting with the odor source. In the moth Manduca sexta wing beating causes oscillatory flow of air over the antenna; because of this, odorant-antennal interactions are oscillatory in nature. Electroantennogram recordings on antennae show that the biophysical properties of their spiking activity can effectively track odors presented at the wing beat frequency. Psychophysical experiments using Manduca show that when odors are pulsed, as opposed to presented as a continuous stream, detection and discrimination thresholds are lowered. In this study, we characterized histamine immunoreactivity in the thoracic ganglia and brain of Manduca. We generated antibodies for and characterized the distribution of the histamine B receptor, the first known antibody for this receptor protein. Our results show an elaborate pair of neurons projecting from the mesothoracic ganglion to the brain, including axon innervation of the antennal lobe and antennal mechanosensory and motor centers. Additionally, histamine B receptor labeling overlapped with a subset of GABAergic and peptidergic local interneurons. Next, we characterized the response properties of these cells within the context of fictive flight behavior and found a tonic increase in activity. Furthermore, disrupting this circuit, with surgical ablation and pharmacology, disrupts antennal lobe projection neurons from entraining to odors presented at a natural 20 Hz frequency, as well as behavioral measures of detection and discrimination thresholds. Finally, we characterized the relationship between motor patterns/behaviors, and circuit structure of this pair of histamine immunoreactive neurons. Specifically, presence of MDHn axon collaterals entering the antennal lobe is correlated with olfactory-guided target approach behaviors in crepuscular and nocturnal moths who require stereotyped zigzagging and wing beating behaviors for locating an olfactory target have axonal ramifications in the antennal lobe. This study is the first characterization of a motor to olfactory corollary discharge circuit in invertebrates and may represent the first characterization of a higher order corollary discharge circuit in an invertebrate model