Renal tubular function and morphology revealed in kidney without labeling using three-dimensional dynamic optical coherence tomography.

Renal tubule has distinct metabolic features and functional activity that may be altered during kidney disease. In this paper, we present label-free functional activity imaging of renal tubule in normal and obstructed mouse kidney models using three-dimensional (3D) dynamic optical coherence tomography (OCT) ex vivo. To create an obstructed kidney model, we ligated the ureter of the left kidney for either 7 or 14 days. Two different dynamic OCT (DOCT) methods were implemented to access the slow and fast activity of the renal tubules: a logarithmic intensity variance (LIV) method and a complex-correlation-based method. Three-dimensional DOCT data were acquired with a 1.3 [Formula: see text]m swept-source OCT system and repeating raster scan protocols. In the normal kidney, the renal tubule appeared as a convoluted pipe-like structure in the DOCT projection image. Such pipe-like structures were not observed in the kidneys subjected to obstruction of the ureter for several days. Instead of any anatomical structures, a superficial high dynamics appearance was observed in the perirenal cortex region of the obstructed kidneys. These findings suggest that volumetric LIV can be used as a tool to investigate kidney function during kidney diseases.

Effect of the COVID-19 pandemic on antibiotic consumption: A systematic review comparing 2019 and 2020 data.

Background: The coronavirus disease 2019 (COVID-19) pandemic has influenced antibiotic consumption over a long period, with variability in trends among studies. We conducted this systematic review to explore and compare the effect of the pandemic on overall and individual antibiotic consumption in 2020 with that in 2019. Methods: This systematic literature review was conducted using PubMed, EMBASE, and Web of Science databases. Data on antibiotic consumption in Japan was sourced from the Japan Surveillance of Antimicrobial Consumption. Results: A total of 1,442 articles and reports were screened, and 16 eligible articles were reviewed. The included studies were conducted in Jordan, Australia, Canada, UK, Japan, Brazil, India, China, and the EU. There was no study from African and Southeast Asian Countries. Overall, antibiotic consumption in the community consistently reduced in 2020. Studies from Australia, Canada, Portugal, Spain, the UK, Japan, and the European Union reported both decreases in overall and selected individual antibiotics consumption. In contrast, hospital-based studies reported both increases and decreases. Hospital-based studies in Lebanon, Spain, Italy, India, and the UK reported an increase in antibiotic consumption in 2020. Studies reporting an interruption of antibiotic stewardship programs during the pandemic also reported increases in antibiotic consumption for hospitalized patients in 2020 compared with that in 2019. Conclusion: Our results showed a different trend between communities and hospitals in antibiotic consumption during 2020 compared to 2019. The continuity of the antibiotic stewardship program might have influenced the antibiotic consumption trend variability among hospitals in 2020. Alongside this, the lack of information on antibiotic consumption from low-income countries and limited reports from middle-income countries revealed gaps that need to be urgently filled.

Label-free metabolic imaging of non-alcoholic-fatty-liver-disease (NAFLD) liver by volumetric dynamic optical coherence tomography.

Label-free metabolic imaging of non-alcoholic fatty liver disease (NAFLD) mouse liver is demonstrated ex vivo by dynamic optical coherence tomography (OCT). The NAFLD mouse is a methionine choline-deficient (MCD)-diet model, and two mice fed the MCD diet for 1 and 2 weeks are involved in addition to a normal-diet mouse. The dynamic OCT is based on repeating raster scan and logarithmic intensity variance (LIV) analysis that enables volumetric metabolic imaging with a standard-speed (50,000 A-lines/s) OCT system. Metabolic domains associated with lipid droplet accumulation and inflammation are clearly visualized three-dimensionally. Particularly, the normal-diet liver exhibits highly metabolic vessel-like structures of peri-vascular hepatic zones. The 1-week MCD-diet liver shows ring-shaped highly metabolic structures formed with lipid droplets. The 2-week MCD-diet liver exhibits fragmented vessel-like structures associated with inflammation. These results imply that volumetric LIV imaging is useful for visualizing and assessing NAFLD abnormalities.

Detection of Anterior Hyaloid Membrane Detachment Using Deep-Range Anterior Segment Optical Coherence Tomography.

The visibility of anterior hyaloid membrane (AHM) and Berger's space in phakic eyes was investigated. In 624 eyes of 624 patients, the retrolental space was scanned with the deep-range anterior segment optical coherence tomography (AS-OCT, CASIA2, Tomey). Subgroup analysis was conducted in 223 eyes undergoing cataract surgery. The logistic regression analysis using the backward-elimination method was performed to evaluate the influence of various factors on the visibility of AHM (dependent variable). Explanatory variables included age, gender, axial length, corneal power, corneal cylinder, and the Co III gradings. Intrarater repeatability for AHM visibility was excellent with the prevalence-adjusted bias-adjusted kappa (κ coefficient) of 0.90. AHM was observed in 43 eyes (6.9%). The patients with visible AHM (68.1 ± 8.8 years) were significantly older than those without visible AHM (52.6 ± 25.6 years) (p < 0.001). The logistic regression analysis in the cataract surgery subgroup revealed that axial length (p = 0.030) and corneal power (p = 0.043) were significantly associated with AHM visibility. The mean absolute refractive error from target was significantly larger in eyes with visible AHM (0.670 ± 0.384 D) than with invisible AHM (0.494 ± 0.412 D) (p = 0.037). The postoperative refractive prediction was less accurate in eyes with visible AHM, but no significant tendency existed in terms of myopic or hyperopic shifts.

Subretinal injection in mice to study retinal physiology and disease.

Subretinal injection (SRI) is a widely used technique in retinal research and can be used to deliver nucleic acids, small molecules, macromolecules, viruses, cells or biomaterials such as nanobeads. Here we describe how to undertake SRI of mice. This protocol was adapted from a technique initially described for larger animals. Although SRI is a common procedure in eye research laboratories, there is no published guidance on the best practices for determining what constitutes a 'successful' SRI. Optimal injections are required for reproducibility of the procedure and, when carried out suboptimally, can lead to erroneous conclusions. To address this issue, we propose a standardized protocol for SRI with 'procedure success' defined by follow-up examination of the retina and the retinal pigmented epithelium rather than solely via intraoperative endpoints. This protocol takes 7-14 d to complete, depending on the reagent delivered. We have found, by instituting a standardized training program, that trained ophthalmologists achieve reliable proficiency in this technique after ~350 practice injections. This technique can be used to gain insights into retinal physiology and disease pathogenesis and to test the efficacy of experimental compounds in the retina or retinal pigmented epithelium.

The Learning Curve of Murine Subretinal Injection Among Clinically Trained Ophthalmic Surgeons.

Purpose: Subretinal injection (SRI) in mice is widely used in retinal research, yet the learning curve (LC) of this surgically challenging technique is unknown. Methods: To evaluate the LC for SRI in a murine model, we analyzed training data from three clinically trained ophthalmic surgeons from 2018 to 2020. Successful SRI was defined as either the absence of retinal pigment epithelium (RPE) degeneration after phosphate buffered saline injection or the presence of RPE degeneration after Alu RNA injection. Multivariable survival-time regression models were used to evaluate the association between surgeon experience and success rate, with adjustment for injection agents, and to calculate an approximate case number to achieve a 95% success rate. Cumulative sum (CUSUM) analyses were performed and plotted individually to monitor each surgeon's simultaneous performance. Results: Despite prior microsurgery experience, the combined average success rate of the first 50 cases in mice was only 27%. The predicted SRI success rate did not reach a plateau above 95% until approximately 364 prior cases. Using the 364 training cases as a cutoff point, the predicted probability of success for cases 1 to 364 was 65.38%, and for cases 365 to 455 it was 99.32% (P < 0.0001). CUSUM analysis showed an initial upward slope and then remained within the decision intervals with an acceptable success rate set at 95% in the late stage. Conclusions: This study demonstrates the complexity and substantial LC for successful SRI in mice with high confidence. A systematic training system could improve the reliability and reproducibility of SRI-related experiments and improve the interpretation of experimental results using this technique. Translational Relevance: Our prediction model and monitor system allow objective quantification of technical proficiency in the field of subretinal drug delivery and gene therapy for the first time, to the best of our knowledge.

Label-free functional and structural imaging of liver microvascular complex in mice by Jones matrix optical coherence tomography.

We demonstrate label-free imaging of the functional and structural properties of microvascular complex in mice liver. The imaging was performed by a custom-built Jones-matrix based polarization sensitive optical coherence tomography (JM-OCT), which is capable of measuring tissue's attenuation coefficient, birefringence, and tiny tissue dynamics. Two longitudinal studies comprising a healthy liver and an early fibrotic liver model were performed. In the healthy liver, we observed distinctive high dynamics beneath the vessel at the initial time point (0 h) and reappearance of high dynamics at 32-h time point. In the early fibrotic liver model, we observed high dynamics signal that reveals a clear network vascular structure by volume rendering. Longitudinal time-course imaging showed that these high dynamics signals faded and decreased over time.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Nucleoside reverse transcriptase inhibitors and Kamuvudines inhibit amyloid-ß induced retinal pigmented epithelium degeneration.

Nonfibrillar amyloid-ß oligomers (AßOs) are a major component of drusen, the sub-retinal pigmented epithelium (RPE) extracellular deposits characteristic of age-related macular degeneration (AMD), a common cause of global blindness. We report that AßOs induce RPE degeneration, a clinical hallmark of geographic atrophy (GA), a vision-threatening late stage of AMD that is currently untreatable. We demonstrate that AßOs induce activation of the NLRP3 inflammasome in the mouse RPE in vivo and that RPE expression of the purinergic ATP receptor P2RX7, an upstream mediator of NLRP3 inflammasome activation, is required for AßO-induced RPE degeneration. Two classes of small molecule inflammasome inhibitors-nucleoside reverse transcriptase inhibitors (NRTIs) and their antiretrovirally inert modified analog Kamuvudines-both inhibit AßOs-induced RPE degeneration. These findings crystallize the importance of P2RX7 and NLRP3 in a disease-relevant model of AMD and identify inflammasome inhibitors as potential treatments for GA.

Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

Optical coherence tomography-based tissue dynamics imaging for longitudinal and drug response evaluation of tumor spheroids.

We present optical coherence tomography (OCT)-based tissue dynamics imaging method to visualize and quantify tissue dynamics such as subcellular motion based on statistical analysis of rapid-time-sequence OCT signals at the same location. The analyses include logarithmic intensity variance (LIV) method and two types of OCT correlation decay speed analysis (OCDS). LIV is sensitive to the magnitude of the signal fluctuations, while OCDSs including early- and late-OCDS (OCDS e and OCDS l , respectively) are sensitive to the fast and slow tissue dynamics, respectively. These methods were able to visualize and quantify the longitudinal necrotic process of a human breast adenocarcinoma spheroid and its anti-cancer drug response. Additionally, the effects of the number of OCT signals and the total acquisition time on dynamics imaging are examined. Small number of OCT signals, e.g., five or nine suffice for dynamics imaging when the total acquisition time is suitably long.

Repurposing anti-inflammasome NRTIs for improving insulin sensitivity and reducing type 2 diabetes development.

Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P < 0.0001; 95% prediction interval, 0.618 to 0.734). Meanwhile, an NRTI, lamivudine, improves insulin sensitivity and reduces inflammasome activation in diabetic and insulin resistance-induced human cells, as well as in mice fed with high-fat chow; mechanistically, inflammasome-activating short interspersed nuclear element (SINE) transcripts are elevated, whereas SINE-catabolizing DICER1 is reduced, in diabetic cells and mice. These data suggest the possibility of repurposing an approved class of drugs for prevention of diabetes.

Chronic Dicer1 deficiency promotes atrophic and neovascular outer retinal pathologies in mice.

Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

Comparison of anterior segment and lens biometric measurements in patients with cataract.

PURPOSE: This study was performed to compare anterior ocular biometric measurements of deep-range swept-source anterior segment optical coherence tomography (AS-OCT) (CASIA2) versus short-range swept-source AS-OCT (CASIA SS-1000), ultrasonography (AL-4000), and Scheimpflug camera analysis (Pentacam and EAS-1000) in patients with cataract. METHODS: One hundred eighty-five eyes of 128 participants with mild refractive error or cataract were examined. The central corneal thickness (CCT), aqueous depth (AQD), and lens thickness were obtained. The repeatability of CASIA2 measurements was assessed. RESULTS: In patients with cataract, the CCT, AQD, lens thickness, and lens anterior curvature by CASIA2 showed high intraclass correlation coefficients (ICCs) of > 0.99. Conversely, measurements of the posterior part of the lens such as lens posterior curvature showed lower ICCs. The ICCs were higher in healthy young participants than in patients with cataract. The ICCs tended to be lower in patients with mild than dense cataract. There was no statistically significant difference in the CCT and AQD between the CASIA2 and CASIA SS-1000 or in the lens thickness measurements between the CASIA2 and AL-4000 and between the CASIA2 and EAS-1000. There was a significant linear correlation in the biometric measurements between the CASIA2 and the other instruments. CONCLUSION: We evaluated the biometric measurements of the anterior eye segment by the CASIA2. The CASIA2 yielded results comparable with those of the CASIA SS-1000, ultrasonography, and Scheimpflug camera. However, mild cataract decreased the repeatability of measurements of the posterior part of the lens.

Comparison of intensity, phase retardation, and local birefringence images for filtering blebs using polarization-sensitive optical coherence tomography.

Polarization-sensitive optical coherence tomography (PS-OCT) allows the recording of depth-resolved polarimetric measurements. It has been reported that phase retardation and local birefringence images can noninvasively detect fibrotic area in blebs after glaucoma surgery. Evaluation of scar fibrosis in blebs is important not only for predicting bleb function, but also for planning revision trabeculectomy. Herein, we characterize the intensity, phase retardation, and local birefringence images of blebs using PS-OCT. A total of 85 blebs from 85 patients who had undergone trabeculectomy were examined. Both phase retardation and local birefringence images detected fibrotic changes in blebs after glaucoma surgery. Phase retardation images detected slight fibrotic change during the early stage after surgery, whereas local birefringence images showed localized fibrotic tissue. There are two main patterns of local birefringence image changes in blebs: plate-like birefringence changes and diffuse changes. The area of plate-like birefringence change was significantly larger in poorly functioning blebs and is thus correlated with bleb function. These data suggest that the plate-like fibrotic change evaluation by PS-OCT may be useful not only for noninvasive evaluation of fibrotic scar tissue in blebs, but also for developing strategies for revision trabeculectomy.

cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits DICER1 deficiency, resultant accumulation of endogenous Alu-retroelement RNA, and NLRP3-inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human-cell-culture and mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-ß production and gasdermin D-dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-ß, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of cGAS in responding to mobile-element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial-damage-induced inflammasome activation, expand the immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness.

Two-color pump-probe interferometry of ultra-fast light-matter interaction.

Two-color side-view probing of light-matter interaction from minute focal volume of a tightly focused fs-laser pump pulse reveals charge dynamics with high 0.9 µm optical resolution and approximately ~45fs temporal resolution defined by pulse duration. Use of two colors is advantageous for probing optically excited plasma regions with different density. Holographical digital focusing and spatial filtering were implemented to obtain the same resolution images for subsequent Fourier analysis. Fast plasma density decay with time constant ~150 fs was resolved and is consistent with self-trapping. Potential applications of an optical control over light-induced defects with deep-sub-wavelength resolution is discussed.

Functional visual acuity in patients with successfully treated amblyopia: a pilot study.

PURPOSE: The aim of this study was to use conventional visual acuity measurements to quantify the functional visual acuity (FVA) in eyes with successfully treated amblyopia, and to compare the findings with those for contralateral normal eyes. METHODS: Nineteen patients (7 boys, 12 girls; age 7.5 ± 2.2 years) with successfully treated unilateral amblyopia and the same conventional decimal visual acuity in both eyes (better than 1.0) were enrolled. FVA, the visual maintenance ratio (VMR), maximum and minimum visual acuity, and the average response time were recorded for both eyes of all patients using an FVA measurement system. The differences in FVA values between eyes were analyzed. RESULTS: The mean LogMAR FVA scores, VMR (p < 0.001 for both), and the LogMAR maximum (p < 0.005) and minimum visual acuity (p < 0.001) were significantly poorer for the eyes with treated amblyopia than for the contralateral normal eyes. There was no significant difference in the average response time. CONCLUSIONS: Our results indicate that FVA and VMR were poorer for eyes with treated amblyopia than for normal eyes, even though the treatment for amblyopia was considered successful on the basis of conventional visual acuity measurements. These results suggest that visual function is impaired in eyes with amblyopia, regardless of treatment success, and that FVA measurements can provide highly valuable diagnosis and treatment information that is not readily provided by conventional visual acuity measurements.

Quantitative Evaluation of Phase Retardation in Filtering Blebs Using Polarization-Sensitive Optical Coherence Tomography.

PURPOSE: Polarization-sensitive optical coherence tomography (PS-OCT) can detect and evaluate scar fibrosis of the filtering blebs after glaucoma surgery. Although the change in phase retardation reportedly reflects bleb function, quantitative assessment of phase retardation in ocular tissues has not been conducted. We aimed to establish quantitative methods to investigate changes in phase retardation in the blebs after surgery using PS-OCT. METHODS: Twenty-two blebs of 22 patients who had undergone glaucoma filtration surgery were consecutively examined for 4 months. Phase retardation was measured by PS-OCT and quantitatively analyzed to evaluate its relationship with bleb function based on intraocular pressure and medication use. Cross-sectional re-evaluation was also performed for a previous data set of 153 blebs of 122 patients. RESULTS: In consecutive measurements, all blebs showed a low phase retardation value and good bleb function until 2 weeks. One month postoperatively, the phase retardation value was significantly increased, whereas bleb function was still good. The phase retardation value at 1 month postoperatively was significantly correlated with bleb function at 4 months postoperatively. While 55.6% of blebs with a high phase retardation value at 1 month subsequently lost function, only 7.7% with a low phase retardation value had bleb failure. In the cross-sectional re-evaluation, the quantitatively evaluated phase retardation value was highly correlated with bleb function (ß = 0.770, P < 0.001). CONCLUSIONS: An increase in phase retardation preceded deterioration of bleb function. The change in phase retardation may provide a prognostic metric for bleb function in the early stage after surgery.