Topical Solution for Retinal Delivery: Bevacizumab and Ranibizumab Eye Drops in Anti-Aggregation Formula (AAF) in Rabbits.

PURPOSE: Wet age-related macular degeneration (AMD) is a blinding retinal disease. Monthly intravitreal anti-VEGF antibody injections of bevacizumab (off-label) and ranibizumab (FDA approved) are the standard of care. Antibody aggregation may interfere with ocular absorption/distribution. This study assessed topical delivery of dilute antibodies to the posterior segment of rabbit eyes using a novel anti-aggregation formula (AAF). METHODS: Bevacizumab, or biosimilar ranibizumab was diluted to 5 mg/ml in AAF. All rabbits were dosed twice daily. Substudy 1 rabbits (bevacizumab, 100 µl eye drops): Group 1 (bevacizumab/AAF, n = 6); Group 2 (bevacizumab/PBS, n = 7) and Vehicle control (AAF, n = 1). Substudy 2 rabbits (ranibizumab biosimilar/AAF, 50 µl eye drops): (ranibizumab biosimilar/AAF, n = 8). At 14.5 days, serum was drawn from rabbits. Aqueous, vitreous and retina samples were recovered from eyes and placed into AAF aliquots. Tissue analyzed using AAF as diluent. RESULTS: Bevacizumab in AAF permeated/accumulated in rabbit aqueous, vitreous and retina 10 times more, than when diluted in PBS. AAF/0.1% hyaluronic acid eye drops, dosed twice daily, provided mean tissue concentrations (ng/g) in retina (29.50), aqueous (12.34), vitreous (3.46), and serum (0.28 ng/ml). Additionally, the highest concentration (ng/g) of ranibizumab biosimilar was present in the retina (18.0), followed by aqueous (7.82) and vitreous (1.47). Serum concentration was negligible (< 0.04 ng/ml). No irritation was observed throughout the studies. CONCLUSIONS: Bevacizumab and ranibizumab, in an AAF diluent eye drop, can be delivered to the retina, by the twice daily dosing of a low concentration mAb formulation. This may prove to be an adjunct to intravitreal injections.

Semi-automated micro-computed tomography lung segmentation and analysis in mouse models.

Computed Tomography (CT) is a standard clinical tool utilized to diagnose known lung pathologies based on established grading methods. However, for preclinical trials and toxicity investigations in animal models, more comprehensive datasets are typically needed to determine discriminative features between experimental treatments, which oftentimes require analysis of multiple images and their associated differential quantification using manual segmentation methods. Furthermore, for manual segmentation of image data, three or more readers is the gold standard of analysis, but this requirement can be time-consuming and inefficient, depending on variability due to reader bias. In previous papers, microCT image manual segmentation was a valuable tool for assessment of lung pathology in several animal models; however, the manual segmentation approach and the commercial software used was typically a major rate-limiting step. To improve the efficiency, the semi-manual segmentation method was streamlined, and a semi-automated segmentation process was developed to produce:•Quantifiable segmentations: using manual and semi-automated analysis methods for assessing experimental injury and toxicity models,•Deterministic results and efficiency through automation in an unbiased and parameter free process, thereby reducing reader variance, user time, and increases throughput in data analysis,•Cost-Effectiveness: portable with low computational resource demand, based on a cross-platform open-source ImageJ program.

CXCR3 deletion aggravates corneal neovascularization in a corneal alkali-burn model.

Corneal neovascularization can cause devastating consequences including vision impairment and even blindness. Corneal inflammation is a crucial factor for the induction of corneal neovascularization. Current anti-inflammatory approaches are of limited value with poor therapeutic effects. Therefore, there is an urgent need to develop new therapies that specifically modulate inflammatory pathways and inhibit neovascularization in the cornea. The interaction of chemokines and their receptors plays a key role in regulating leukocyte migration during inflammatory response. CXCR3 is essential for mediating the recruitment of activated T cells and microglia/macrophages, but the role of CXCR3 in the initiation and promotion of corneal neovascularization remains unclear. Here, we showed that the expression of CXCL10 and CXCR3 was significantly increased in the cornea after alkali burn. Compared with WT mice, CXCR3-/- mice exhibited significantly increased corneal hemangiogenesis and lymphangiogenesis after alkali burn. In addition, exaggerated leukocyte infiltration and leukostasis, and elevated expression of inflammatory cytokines and angiogenic factor were also found in the corneas of CXCR3-/- mice subjected to alkali burn. With bone marrow (BM) transplantation, we further demonstrated that the deletion of CXCR3 in BM-derived leukocytes plays a key role in the acceleration of alkali burn-induced corneal neovascularization. Taken together, our results suggest that upregulation of CXCR3 does not exhibit its conventional action as a proinflammatory cytokine but instead serves as a self-protective mechanism for the modulation of inflammation and maintenance of corneal avascularity after corneal alkali burn.

Longitudinal characterization of retinal vasculature alterations with optical coherence tomography angiography in a mouse model of tauopathy.

Tauopathies are a family of neurodegenerative diseases which predominately afflict the rapidly growing aging population suffering from various brain disorders including Alzheimer's disease, frontotemporal dementia with parkinsonism-17 and Pick disease. As the only visually accessible region of the central nervous system, in recent years, the retina has attracted extensive attention for its potential as a target for visualizing and quantifying emerging biomarkers of neurodegenerative diseases. Our previous study has found that retinal vascular inflammation and leakage occur at the very early stage of tauopathic mouse model. Here, we aimed to non-invasively visualize age-dependent alterations of retinal vasculature assessing the potential for using changes in retinal vasculature as the biomarker for the early diagnosis of tauopathy. Optical coherence tomography angiography (OCTA), a non-invasive depth-resolved high-resolution imaging technique was used to visualize and quantify tauopathy-induced alterations of retinal vasculature in P301S transgenic mice overexpressing the P301S mutant form of human tau and age-matched wild type littermate mice at 3, 6 and 10 months of age. We observed significant alterations of vascular features in the intermediate capillary plexus (ICP) and deep capillary plexus (DCP) but not in the superficial vascular complex (SVC) of P301S mice at early stages of tauopathy. With aging, alterations of vascular features in P301S mice became more prominent in all three vascular plexuses. Staining of retinal vasculature in flatmounts and trypsin digests of P301S mice at 10 months of age revealed decreased vessel density and increased acellular capillary formation, indicating that vascular degeneration also occurs during tauopathy. Overall, our results demonstrate that the changes in retinal vascular features accelerate during the progression of tauopathy. Vessels in the ICP and DCP may be more susceptible to tauopathy than vessels in the SVC. Since changes in retinal vasculature often precede tau pathology in the brain, non-invasive identification of retinal vascular alterations with OCTA may be a useful biomarker for the early diagnosis of tauopathy and monitoring its progression.

Concentration-associated pathology of alkali burn in a mouse model using anterior segment optical coherence tomography with angiography.

Pathological features of alkali concentration-associated burn were studied using non-invasive anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA). Alkali burn was induced in C57BL/6J mice (n = 20) by placing filter paper soaked in 0.1, 0.25, 0.5, and 1 M NaOH for 30s on the right eye (left eye control). Longitudinal imaging was performed with AS-OCT/OCTA and fluorescein angiography over 14 days, after which eyes were enucleated at 7 and 14 days for histology and immunofluorescence. Concentration-associated corneal swelling was maximal at 0.5M, increasing linearly in a concentration-dependent fashion at 0.1, 0.25, and 0.5 M NaOH, to levels of 50%, 100%, and 175% of control, respectively. At 0.1M, corneal swelling and surface erosions were prominent, while at 0.25M, deep tissue damage, limbal neovascularization, and stromal haze were evident at 7 days. At 0.5M and 1M, severe exacerbation of the corneal swelling, angle closure, Descemet's membrane detachment, hyphema, and profuse central neovascularization were noted as early as day 3, which further progressed to inflammation, fibrosis, and opacity by day 7. We conclude that alkali concentration-dependent burn intensity biomarkers can be assessed by non-invasive AS-OCT/OCTA, distinguishing between mild, moderate, and severe ocular injury, with potential relevance toward clinical utilization in human eyes.

Spatial-Temporal Speckle Variance in the En-Face View as a Contrast for Optical Coherence Tomography Angiography (OCTA).

Optical Coherence Tomography (OCT) is an adaptable depth-resolved imaging modality capable of creating a non-invasive 'digital biopsy' of the eye. One of the latest advances in OCT is optical coherence tomography angiography (OCTA), which uses the speckle variance or phase change in the signal to differentiate static tissue from blood flow. Unlike fluorescein angiography (FA), OCTA is contrast free and depth resolved. By combining high-density scan patterns and image processing algorithms, both morphometric and functional data can be extracted into a depth-resolved vascular map of the retina. The algorithm that we explored takes advantage of the temporal-spatial relationship of the speckle variance to improve the contrast of the vessels in the en-face OCT with a single frame. It also does not require the computationally inefficient decorrelation of multiple A-scans to detect vasculature, as used in conventional OCTA analysis. Furthermore, the spatial temporal OCTA (ST-OCTA) methodology tested offers the potential for post hoc analysis to improve the depth-resolved contrast of specific ocular structures, such as blood vessels, with the capability of using only a single frame for efficient screening of large sample volumes, and additional enhancement by processing with choice of frame averaging methods. Applications of this method in pre-clinical studies suggest that the OCTA algorithm and spatial temporal methodology reported here can be employed to investigate microvascularization and blood flow in the retina, and possibly other compartments of the eye.

Longitudinal Assessment of Alkali Injury on Mouse Cornea Using Anterior Segment Optical Coherence Tomography.

Purpose: Chemical burns due to alkalis cause extensive damage to the ocular surface leading to blindness. Assessment of ocular burn could be challenging due to severe opacity, inflammation, and angiogenesis. Anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA) may provide fast, non-invasive deep tissue visualization of pathology with high sensitivity in conjunction with slit-lamp analysis. Methods: C57-BL/6J mice were anesthetized with ketamine/dexmedetomidine, and corneal alkali burn was induced (n = 6) by placing filter paper soaked in 1-M sodium hydroxide for 30 seconds on the right eye while the left eye was kept as control. Longitudinal imaging was done with AS-OCT/OCTA and fluorescein angiography at various time intervals for 14 days. Results: AS-OCT showed characteristic pathological changes in alkali-burned eyes with high sensitivity. Although OCT/OCTA showed three-dimensional and cross-sectional views of the anterior chamber and angiogenesis, fluorescein angiography showed nascent vessels with active leakage. Corneal swelling progressively increased by 125.26% on day 12 with a high prevalence of epithelial bullae, stromal cysts, stromal splitting, and Descemet's membrane detachment. Neovascularization was noted as early as day 4 in the burned eyes by both methods. Severe corneal opacity and anterior chamber inflammation were also detected by AS-OCT/OCTA. Conclusions: AS-OCT/OCTA is a promising, noninvasive, high-resolution imaging modality that can provide both qualitative and quantitative information regarding deep tissue pathology at a structural level. Translational Relevance: Noninvasive AS-OCT/OCTA and fluorescein methods show promise in clinical pathology evaluation for ocular injury management and prognostic indications, as the early presence of Descemet's membrane detachment and corneal swelling appears to be correlated with the severity and localization of corneal neovascularization.

Early alterations of neurovascular unit in the retina in mouse models of tauopathy.

The retina, as the only visually accessible tissue in the central nervous system, has attracted significant attention for evaluating it as a biomarker for neurodegenerative diseases. Yet, most of studies focus on characterizing the loss of retinal ganglion cells (RGCs) and degeneration of their axons. There is no integrated analysis addressing temporal alterations of different retinal cells in the neurovascular unit (NVU) in particular retinal vessels. Here we assessed NVU changes in two mouse models of tauopathy, P301S and P301L transgenic mice overexpressing the human tau mutated gene, and evaluated the therapeutic effects of a tau oligomer monoclonal antibody (TOMA). We found that retinal edema and breakdown of blood-retina barrier were observed at the very early stage of tauopathy. Leukocyte adhesion/infiltration, and microglial recruitment/activation were constantly increased in the retinal ganglion cell layer of tau transgenic mice at different ages, while Müller cell gliosis was only detected in relatively older tau mice. Concomitantly, the number and function of RGCs progressively decreased during aging although they were not considerably altered in the very early stage of tauopathy. Moreover, intrinsically photosensitive RGCs appeared more sensitive to tauopathy. Remarkably, TOMA treatment in young tau transgenic mice significantly attenuated vascular leakage, inflammation and RGC loss. Our data provide compelling evidence that abnormal tau accumulation can lead to pathology in the retinal NVU, and vascular alterations occur more manifest and earlier than neurodegeneration in the retina. Oligomeric tau-targeted immunotherapy has the potential to treat tau-induced retinopathies. These data suggest that retinal NVU may serve as a potential biomarker for diagnosis and staging of tauopathy as well as a platform to study the molecular mechanisms of neurodegeneration.

En-Face Optical Coherence Tomography Angiography for Longitudinal Monitoring of Retinal Injury.

A customized Optical Coherence Tomography Angiography (OCTA) algorithm and Orthogonal OCT (en-face and B-Scans) were used for longitudinal assessment of retina murine vascular and tissue remodeling comparing photoreceptor ablation and laser-induced Choroidal Neovascularization (CNV). In the mouse model, we utilized a combined OCTA/OCT technique to image and quantify morphological and vascular features of laser lesions over time. This approach enabled us to monitor and correlate the dynamics of retina vascular and tissue remodeling as evidenced by swelling, edema, and scarring. From the OCT B-Scans, three stages of inflammatory progression were identified: the early response occurring within hours to day 3, the transition phase from 3-7 days, and the late stage of 7-21 days entering either the resolving phase or chronic phase, respectively. For the case of CNV, en-face OCTA revealed a transient non-perfusion of inner retina capillaries, specifically Deep Vascular Plexus (DVP), which corresponded to growth in lesions of a height of 200 µm or greater. Non-perfusion first occurred at 24 hours, persisted during edema and CNV formation days 7-14. In contrast, the acute inflammation induced photoreceptor damage, but no detectable alterations to the microvasculature were observed. We demonstrated that the en-face OCTA system is capable of visualizing capillary networks (~5 µm) and the corresponding tissue remodeling and growth dynamics allowing for separating acute injury from CNV. For the first time, by using OCTA we observed the presence of the 5-10 µm capillary non-perfusion present in DVP as part of CNV formation and the associated wound healing in the retina.

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses.

Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

OCT-Angiography for Non-Invasive Monitoring of Neuronal and Vascular Structure in Mouse Retina: Implication for Characterization of Retinal Neurovascular Coupling.

PURPOSE: Optical coherence tomography angiography (OCT-A) is a newly developed technique to visualize retinal vasculature non-invasively based on interferometry. Although OCT-A has been used clinically, its applications in small animal studies have been limited. This study is designed to develop and demonstrate the feasibility of a protocol for the use of an en-face OCT-based method to visualize and quantify retinal microvasculature in mice that can be used for in vivo assessment of retina ischemia. METHODS: A customized algorithm was developed to extract angiographic profiles of the mouse retina from en-face OCT using an unmodified Bioptigen Envisu R-Class OCT imaging system. En-face OCT images were collected in living animals and then compared to images acquired following termination of blood flow to the retina. The images were processed with ImageJ using the raw file importer. The vessel enhancement algorithm was developed based on a combination of local contrast enhancement, Laplacian of Gaussian peak detection and background subtraction methods. For comparison, fluorescein angiography (FA) was performed using Heidelberg Spectralis® HRA+OCT imaging system. RESULTS: By vessel enhancement algorithm, we successfully extracted retinal vasculature and quantified retinal vessel branch points, vascular area and vessel lengths with AngioTool. While the retinal neuronal structure could be simultaneously identified and quantified using B-scan and volumetric OCT run in the annular scanning model, the retinal vasculature in OCT-A was dramatically diminished after the animals were sacrificed, indicating en-face OCT-A signal is a measure of the blood flow. CONCLUSIONS: These studies indicate that a novel approach to extract angiographs from en-face OCT images by utilizing local structure enhancement can be used to provide depth-resolved retinal vasculature distributions. Simultaneous non-invasive analysis of retinal vessels and neurons by OCT-A and OCT may provide a novel approach to characterize retinal ischemia accompanied by neurovascular coupling.

The Nav1.2 channel is regulated by GSK3.

BACKGROUND: Phosphorylation plays an essential role in regulating voltage-gated sodium (Na(v)) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Na(v) channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Na(v) channels. METHODS: We used patch-clamp electrophysiology to record sodium currents from Na(v)1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. RESULTS: We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Na(v)1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3ß suppressed Na(v)1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Na(v)1.2 channel indicates that cell surface expression of CD4-Na(v)1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3ß phosphorylates T(1966) at the C-terminal tail of Na(v)1.2. CONCLUSION: These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail. GENERAL SIGNIFICANCE: These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.