In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors.

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s-1 Analyzing swelling as osmotically driven water influx, we find the H2O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10-5 cm⋅s-1, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H2O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130.

BACKGROUND: Retinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice. METHODS: RD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor ß (gp-130) was injected intravitreally immediately after RD. RESULTS: Within one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade. CONCLUSIONS: Together, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.

Effect of scanning beam size on the lateral resolution of mouse retinal imaging with SLO.

Scanning laser ophthalmoscopy (SLO) employs the eye's optics as a microscope objective for retinal imaging in vivo. The mouse retina has become an increasingly important object for investigation of ocular disease and physiology with optogenetic probes. SLO imaging of the mouse eye, in principle, can achieve submicron lateral resolution thanks to a numerical aperture (NA) of ∼0.5, about 2.5 times larger than that of the human eye. In the absence of adaptive optics, however, natural ocular aberrations limit the available optical resolution. The use of a contact lens, in principle, can correct many aberrations, permitting the use of a wider scanning beam and, thus, achieving greater resolution then would otherwise be possible. In this Letter, using an SLO equipped with a rigid contact lens, we report the effect of scanning beam size on the lateral resolution of mouse retinal imaging. Theory predicts that the maximum beam size full width at half-maximum (FWHM) that can be used without any deteriorating effects of aberrations is ∼0.6 mm. However, increasing the beam size up to the diameter of the dilated pupil is predicted to improve lateral resolution, though not to the diffraction limit. To test these predictions, the dendrites of a retinal ganglion cell expressing YFP were imaged, and transverse scans were analyzed to quantify the SLO system resolution. The results confirmed that lateral resolution increases with the beam size as predicted. With a 1.3 mm scanning beam and no high-order aberration correction, the lateral resolution is ∼1.15 µm, superior to that achievable by most human AO-SLO systems. Advantages of this approach include stabilization of the mouse eye and simplified optical design.

Performance comparison of instrument automation pipelines using different programming languages.

The article presents a performance analysis of fully automated, in-house developed 2D ultrasound computerized tomography systems using different programming languages. The system is fully automated in four programming languages: LabVIEW, MATLAB, C and Python. It includes codes for sensors, instruments interfacing, real-time control, synchronized data acquisition, simultaneous raw data processing and analysis. Launch performance, eight performance indices and runtime performance are used for the analysis. It is found that C utilizes the least processing power and executes fewer I/O processes to perform the same task. In runtime analysis (data acquisition and real-time control), LabVIEW (365.69 s) performed best in comparison to MATLAB (623.83 s), Python (1505.54 s), and C (1252.03 s) to complete the experiment without data processing. However, in the experiment with data processing, MATLAB (640.33 s) performed best in comparison to LabVIEW (731.91 s), Python (1520.01 s) and C (1930.15 s). Python performed better in establishing faster interfacing and RAM usage. The study provides a methodology to select optimal programming languages for instrument automation-related aspects to optimize the available resources.

Gamma radiation detector selection for CT scanner.

Three types of gamma radiation detectors associated with distributed electronics namely, NaI (Tl), HPGe and LaBr3(Ce) are compared primarily focusing on electronic noise and scattering noise. Additionally, detectors of same make, material, size and electronics are also compared. A methodology is proposed to select the most suitable detector for computed tomography (CT) among the available options. Standard deviation parameter is employed to estimate electronic noise without performing CT experiment. Kanpur theorem-1(KT-1) is used to estimate the scattering noise quantitatively after verifying its sensitivity to scattering noise. The impact of scattering noise on CT profiles is evaluated using dice similarity dice coefficient. A good resemblance between KT-1 and dice coefficient is observed. A maximum difference of 56% in scattering noise is observed when five detectors used simultaneously instead of single detector whereas a discrepancy of 85% is observed between different types of radiation detectors. As far as ease of handling, operational and capital cost is concern one has to compromise minimum 12% of accuracy in CT reconstruction if NaI (Tl) detector is used with respect to best alternative available. The proposed methodology can be applied to measurement that require minimal scattering interference data other than CT experiments. The manufacturer can add noise level of detector as a characteristic parameter in the data sheet.

Design and Fabrication of a Dual Protein-Based Trilayered Nanofibrous Scaffold for Efficient Wound Healing.

Chronic wound healing is a major threat all over the world. There are currently a plethora of biomaterials-based wound dressings available for wound healing applications. In this study, a dual protein-based (silk fibroin and sericin) nanofibrous scaffold from a natural source (B.mori silkworm cocoons) with antibacterial and antioxidative properties for wound healing was investigated. An electrospun layer-by-layer silk protein-based nanofibrous scaffold was fabricated with a top layer of hydrophobic silk fibroin protein blended with polyvinyl alcohol (PVA), a middle layer of waste protein silk sericin loaded with silver(I) sulfadiazine as an antibacterial agent, and a bottom layer using silk fibroin blended with polycaprolactone (PCL). The trilayered nanofibrous scaffold with a smooth and bead-free morphology demonstrated excellent wettability, slow in vitro degradation, controlled drug release, and potent antibacterial and antioxidant properties. In vitro, the scaffold also demonstrated excellent hemocompatibility and biocompatibility. Furthermore, in vivo wound contraction, histological, and micro-CT investigations show complete wound healing and the formation of new skin tissue in a male Balb/c mouse model treated with the scaffold. The antioxidant properties of the sericin protein and SSD-based triple-layered nanofibrous scaffold protect the wound from bacterial infection and improve wound healing in a mouse model. The current study develops a dual protein-based nanofibrous scaffold with antibacterial and antioxidant properties as a promising wound dressing material.

Deep learning models for benign and malign ocular tumor growth estimation.

Relatively abundant availability of medical imaging data has provided significant support in the development and testing of Neural Network based image processing methods. Clinicians often face issues in selecting suitable image processing algorithm for medical imaging data. A strategy for the selection of a proper model is presented here. The training data set comprises optical coherence tomography (OCT) and angiography (OCT-A) images of 50 mice eyes with more than 100 days follow-up. The data contains images from treated and untreated mouse eyes. Four deep learning variants are tested for automatic (a) differentiation of tumor region with healthy retinal layer and (b) segmentation of 3D ocular tumor volumes. Exhaustive sensitivity analysis of deep learning models is performed with respect to the number of training and testing images using eight performance indices to study accuracy, reliability/reproducibility, and speed. U-net with UVgg16 is best for malign tumor data set with treatment (having considerable variation) and U-net with Inception backbone for benign tumor data (with minor variation). Loss value and root mean square error (R.M.S.E.) are found most and least sensitive performance indices, respectively. The performance (via indices) is found to be exponentially improving regarding a number of training images. The segmented OCT-Angiography data shows that neovascularization drives the tumor volume. Image analysis shows that photodynamic imaging-assisted tumor treatment protocol is transforming an aggressively growing tumor into a cyst. An empirical expression is obtained to help medical professionals choose a particular model given the number of images and types of characteristics. We recommend that the presented exercise should be taken as standard practice before employing a particular deep learning model for biomedical image analysis.

Non-invasive multimodal imaging of Diabetic Retinopathy: A survey on treatment methods and Nanotheranostics.

Diabetes Retinopathy (DR) is one of the most prominent microvascular complications of diabetes. It is one of the pre-eminent causes for vision impairment followed by blindness among the working-age population worldwide. The de facto cause for DR remains challenging, despite several efforts made to unveil the mechanism underlying the pathology of DR. There is quite less availability of the low cost pre-emptive theranostic imaging tools in terms of in-depth resolution, due to the multiple factors involved in the etiology of DR. This review work comprehensively explores the various reports and research works on all perspectives of diabetic retinopathy (DR), and its mechanism. It also discusses various advanced non-destructive imaging modalities, current, and future treatment approaches. Further, the application of various nanoparticle-based drug delivery strategies used for the treatment of DR are also discussed. In a nutshell, the present review work bolsters the pursuit of the development of an advanced non-invasive optical imaging modal with a nano-theranostic approach for the future diagnosis and treatment of DR and its associated ocular complications.

Novel window for cancer nanotheranostics: non-invasive ocular assessments of tumor growth and nanotherapeutic treatment efficacy in vivo.

In cancer research there is a fundamental need for animal models that allow the in vivo longitudinal visualization and quantification of tumor development, nanotherapeutic delivery, the tumor microenvironment including blood vessels, macrophages, fibroblasts, immune cells, and extracellular matrix, and the tissue response to treatment. To address this need, we developed a novel mouse ocular xenograft model. Green fluorescent protein (GFP) expressing human glioblastoma cells (between 500 and 10,000) were implanted into the subretinal space of immunodeficient mice (56 eyes). The resultant xenografts were imaged in vivo non-invasively with combined fluorescence scanning laser ophthalmoscopy (SLO) and volumetric optical coherence tomography (OCT) for a period up to several months. Most xenografts exhibited a latent phase followed by a stable or rapidly increasing volume, but about 1/3 underwent spontaneous remission. After prescribed growth, a population of tumors was treated with intravenously delivered doxorubicin-containing porphyrin and cholic acid-based nanoparticles ("nanodox"). Fluorescence resonance energy transfer (FRET) emission (doxorubicin → porphyrin) was used to localize nanodox in the xenografts, and 690 nm light exposure to activate it. Such photo-nanotherapy was highly effective in reducing tumor volume. Histopathology and flow cytometry revealed CD4 + and CD8 + immune cell infiltration of xenografts. Overall, the ocular model shows potential for examining the relationships between neoplastic growth, neovascularization and other features of the immune microenvironment, and for evaluating treatment response longitudinally in vivo.

Protective Effect of Intravitreal Administration of Exosomes Derived from Mesenchymal Stem Cells on Retinal Ischemia.

PURPOSE: Exosomes derived from human mesenchymal stem cells (hMSCs) cultured under hypoxic conditions contain proteins and growth factors that promote angiogenesis. This study investigated the effect of intravitreal administration of these exosomes on retinal ischemia using a murine model. METHODS: Oxygen-induced retinopathy (OIR) was induced by exposing one-week-old male C57BL/6J mice to 5 days of 75% hyperoxic conditioning, and returning to room air. After hyperoxic conditioning, the right eye of each mouse was injected intravitreally with 1 µl saline or exosomes derived from hMSCs and compared to control mice of the same age raised in room air without OIR injected intravitreally with saline. Two weeks post-injection, fluorescein angiography (FA) and phase-variance optical coherence tomography angiography (pvOCTA) were used to assess retinal perfusion. Retinal thickness was determined by OCT. The extent of retinal neovascularization was quantitated histologically by counting vascular nuclei on the retinal surface. RESULTS: Among eyes with OIR, intravitreal exosome treatment partially preserved retinal vascular flow in vivo and reduced associated retinal thinning; retinal thickness on OCT was 111.1 ± 7.4µm with saline versus 132.1 ± 11.6µm with exosome, p < 0.001. Retinal neovascularization among OIR eyes was reduced with exosome treatment when compared to saline-treated eyes (7.75 ± 3.68 versus 2.68 ± 1.35 neovascular nuclei per section, p < 0.0001). No immunogenicity or ocular/systemic adverse effect was associated with intravitreal exosome treatment. CONCLUSIONS: Intravitreal administration of exosomes derived from hMSCs was well tolerated without immunosuppression and decreased the severity of retinal ischemia in this murine model. This appealing novel non-cellular therapeutic approach warrants further exploration.

The Photosensitivity of Rhodopsin Bleaching and Light-Induced Increases of Fundus Reflectance in Mice Measured In Vivo With Scanning Laser Ophthalmoscopy.

PURPOSE: To quantify bleaching-induced changes in fundus reflectance in the mouse retina. METHODS: Light reflected from the fundus of albino (Balb/c) and pigmented (C57Bl/6J) mice was measured with a multichannel scanning laser ophthalmoscopy optical coherence tomography (SLO-OCT) optical system. Serial scanning of small retinal regions was used for bleaching rhodopsin and measuring reflectance changes. RESULTS: Serial scanning generated a saturating reflectance increase centered at 501 nm with a photosensitivity of 1.4 × 10-8 per molecule µm2 in both strains, 2-fold higher than expected were irradiance at the rod outer segment base equal to that at the retinal surface. The action spectrum of the reflectance increase corresponds to the absorption spectrum of mouse rhodopsin in situ. Spectra obtained before and after bleaching were fitted with a model of fundus reflectance, quantifying contributions from loss of rhodopsin absorption with bleaching, absorption by oxygenated hemoglobin (HbO2) in the choroid (Balb/c), and absorption by melanin (C57Bl/6J). Both mouse strains exhibited light-induced broadband reflectance changes explained as bleaching-induced reflectivity increases at photoreceptor inner segment/outer segment (IS/OS) junctions and OS tips. CONCLUSIONS: The elevated photosensitivity of rhodopsin bleaching in vivo is explained by waveguide condensing of light in propagation from rod inner segment (RIS) to rod outer segment (ROS). The similar photosensitivity of rhodopsin in the two strains reveals that little light backscattered from the sclera can enter the ROS. The bleaching-induced increases in reflectance at the IS/OS junctions and OS tips resemble results previously reported in human cones, but are ascribed to rods due to their 30/1 predominance over cones in mice and to the relatively minor amount of cone M-opsin in the regions scanned.

Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.