Hepatocyte-specific METTL3 ablation by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and postnatal lethality.

AIM: In 2019, to examine the functions of METTL3 in liver and underlying mechanisms, we generated mice with hepatocyte-specific METTL3 homozygous knockout (METTL3Δhep) by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT) or Alb-Cre mice (JAX), respectively. In this study, we explored the potential reasons why hepatocyte-specific METTL3 homozygous disruption by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), resulted in acute liver failure (ALF) and then postnatal lethality. MAIN METHODS: Mice with hepatocyte-specific METTL3 knockout were generated by simultaneously crossing METTL3fl/fl mice with Alb-iCre mice (GPT; Strain No. T003814) purchased from the GemPharmatech Co., Ltd., (Nanjing, China) or with Alb-Cre mice (JAX; Strain No. 003574) obtained from The Jackson Laboratory, followed by combined-phenotype analysis. The publicly available RNA-sequencing data deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession No.: GSE198512 (postnatal lethality), GSE197800 (postnatal survival) and GSE176113 (postnatal survival) were mined to explore the potential reasons why hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), leads to ALF and then postnatal lethality. KEY FINDINGS: Firstly, we observed that hepatocyte-specific METTL3 homozygous deficiency by Alb-iCre mice (GPT) or by Alb-Cre mice (JAX) caused liver injury, abnormal lipid accumulation and apoptosis. Secondly, we are surprised to find that hepatocyte-specific METTL3 homozygous deletion by Alb-iCre mice (GPT), but not by Alb-Cre mice (JAX), led to ALF and then postnatal lethality. Our findings clearly demonstrated that METTL3Δhep mice (GPT), which are about to die, exhibited the severe destruction of liver histological structure, suggesting that METTL3Δhep mice (GPT) nearly lose normal liver function, which subsequently contributes to ALF, followed by postnatal lethality. Finally, we unexpectedly found that as the compensatory growth responses of hepatocytes to liver injury induced by METTL3Δhep (GPT), the proliferation of METTL3Δhep hepatocytes (GPT), unlike METTL3Δhep hepatocytes (JAX), was not evidenced by the significant increase of Ki67-positive hepatocytes, not accompanied by upregulation of cell-cycle-related genes. Moreover, GO analysis revealed that upregulated genes in METTL3Δhep livers (GPT), unlike METTL3Δhep livers (JAX), are not functionally enriched in terms associated with cell cycle, cell division, mitosis, microtubule cytoskeleton organization, spindle organization, chromatin segregation and organization, and nuclear division, consistent with the loss of compensatory proliferation of METTL3Δhep hepatocytes (GPT) observed in vivo. Thus, obviously, the loss of the compensatory growth capacity of METTL3Δhep hepatocytes (GPT) in response to liver injury might contribute to, at least partially, ALF and subsequently postnatal lethality of METTL3Δhep mice (GPT). SIGNIFICANCE: These findings from this study and other labs provide strong evidence that these phenotypes (i.e., ALF and postnatal lethality) of METTL3Δhep mice (GPT) might be not the real functions of METTL3, and closely related with Alb-iCre mice (GPT), suggesting that we should remind researchers to use Alb-iCre mice (GPT) with caution to knockout gene in hepatocytes in vivo.

Perivenular Capillary Rarefaction in Diabetic Retinopathy: Interdevice Characterization and Association to Clinical Staging.

Purpose: Geometric perfusion deficit (GPD) is a newly described OCT angiography (OCTA) parameter identifying the total area of presumed retinal ischemia. The aim of our study is to characterize differences in GPD and other common quantitative OCTA parameters between macular full field, perivenular zones, and periarteriolar zones for each clinical stage of nonproliferative diabetic retinopathy (DR) and to assess the influence of ultrahigh-speed acquisition and averaging on the described differences. Design: Prospective observational study. Participants: Forty-nine patients, including 11 (22.4%) with no sign of DR, 12 (24.5%) with mild DR, 13 (26.5%) with moderate DR, and 13 (26.5%) with severe DR. Patients with diabetic macular edema, proliferative DR, media opacity, head tremor, and overlapping retinal diseases or systemic diseases influencing OCTA were excluded. Methods: OCT angiography was performed 3 times for each patient: 1 using Solix Fullrange single volume (V1) mode, 1 using Solix Fullrange 4 volumes mode with automatically averaged scan (V4), and 1 using AngioVue. Main Outcome Measures: Full macular, periarteriolar, and perivenular perfusion density (PD), vessel length density (VLD), vessel density index, and GPD for both the superficial capillary plexus (SCP) and deep capillary plexus (DCP). Results: In patients showing no sign of DR, PD and VLD were significantly lower in the perivenular area in both the DCP and SCP using V1 and V4, whereas GPD was significantly higher in the perivenular zone in the DCP and SCP with all 3 devices. In patients with mild DR, all 3 measurements (PD, VLD, and GPD) were significantly different in the perivenular zone with all 3 devices. In patients with moderate DR, PD and VLD were lower in the DCP and SCP when measured with V1 and V4. Moreover, GPD was higher in the perivenular zone in the DCP with all 3 devices, whereas only V4 detected a difference in the SCP. In severe DR, only V4 detected a lower PD and VLD and a higher GPD in the DCP of the perivenular zone. V4 also detected a higher GPD in the SCP. Conclusions: Geometric perfusion deficit highlights prevalent perivenular location of macular capillary ischemia in all stages of DR. In severe DR patients, only averaging technology allows detection of the same finding. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Long-term follow-up of PRPH2-associated retinal dystrophy.

PURPOSE: To report a thirteen-year follow-up of PRPH2-associated retinal dystrophy. METHODS: A 54-year-old female patient presented with decreased vision and mild metamorphopsia in both eyes since the age of 40. A complete evaluation was performed using multimodal imaging techniques. RESULTS: At presentation, fundus examination revealed multiple irregular pisciform flecks in the posterior pole sparing the peripapillary area in both eyes, as well as some mildly atrophic zones in the perifoveal area. The mildly atrophic areas evolved and merged into a central atrophic zone in the following ten years, leading to a decreased vision of less than 20/400 in both eyes. The genetic molecular diagnosis revealed a mutation in PRPH2/RDS gene (NM_000322.4:c.421T>C (p.Tyr141His)). Based on genetics, imaging, and clinical findings, a diagnosis of multifocal pattern dystrophy simulating STGD1/fundus flavimaculatus (MPDSFF) was evoked. Her mother was found to have the same gene mutation, with multiple irregular pisciform flecks in the posterior pole associated with central areolar choroidal dystrophy. CONCLUSIONS: This report demonstrated the thirteen-year progression of MPDSFF in a patient with a pathogenic variant of the PRPH2/RDS gene (NM_000322.4:c.421T>C (p.Tyr141His)).

Optical coherence tomography angiography for quantitative microvascular assessment in diabetic retinopathy: inter-device and intra-device agreement and correlation with clinical staging.

AIM: To compare acquisitions from single-volume Solix protocol (V1), multi-volume averaged Solix protocol (V4), and AngioVue in patients with diabetic retinopathy (DR) to assess differences in quantitative parameters introduced by high-speed scanning and averaging. METHODS: Thirty-eight diabetic patients were divided into 4 groups showing either no sign, mild, moderate, or severe DR at fundus examination. For optical coherence tomography angiography (OCTA) acquisitions both AngioVue, Solix V1, and V4 were used on each patient. Outcome measures were macular perfusion density (PD), vessel length density (VLD), and vessel density index (VDI) for both superficial capillary plexus (SCP) and deep capillary plexus (DCP) and flow deficits (FD) in the choriocapillaris (CC). RESULTS: Our study revealed a good agreement in SCP parameters measured with all 3 devices. DCP measurements with Solix V1 showed moderate agreement with V4 and poor agreement with AngioVue measurements. Inter-device agreement in CC-FD assessment was relatively poor considering all 3 devices. The averaging process led to an underestimation of DCP and SCP parameters in all stages of DR (more evident in mild DR). AngioVue measurements compared to Solix V1 led to overestimation of SCP-PD (more pronounced in severe DR patients) and of DCP parameters (more evidently in moderate DR). The regression model derived from Solix V1 parameters was the best for categorization into the different stages. CONCLUSIONS: Averaging and high-speed scanning introduce changes in OCTA quantitative parameters in DR. Solix V1 is the most suitable for early diagnosis of DR, while averaged protocol could be the preferred choice in advanced stages of the disease.

Deep learning to distinguish Best vitelliform macular dystrophy (BVMD) from adult-onset vitelliform macular degeneration (AVMD).

Initial stages of Best vitelliform macular dystrophy (BVMD) and adult vitelliform macular dystrophy (AVMD) harbor similar blue autofluorescence (BAF) and optical coherence tomography (OCT) features. Nevertheless, BVMD is characterized by a worse final stage visual acuity (VA) and an earlier onset of critical VA loss. Currently, differential diagnosis requires an invasive and time-consuming process including genetic testing, electrooculography (EOG), full field electroretinogram (ERG), and visual field testing. The aim of our study was to automatically classify OCT and BAF images from stage II BVMD and AVMD eyes using a deep learning algorithm and to identify an image processing method to facilitate human-based clinical diagnosis based on non-invasive tests like BAF and OCT without the use of machine-learning technology. After the application of a customized image processing method, OCT images were characterized by a dark appearance of the vitelliform deposit in the case of BVMD and a lighter inhomogeneous appearance in the case of AVMD. By contrast, a customized method for processing of BAF images revealed that BVMD and AVMD were characterized respectively by the presence or absence of a hypo-autofluorescent region of retina encircling the central hyperautofluorescent foveal lesion. The human-based evaluation of both BAF and OCT images showed significantly higher correspondence to ground truth reference when performed on processed images. The deep learning classifiers based on BAF and OCT images showed around 90% accuracy of classification with both processed and unprocessed images, which was significantly higher than human performance on both processed and unprocessed images. The ability to differentiate between the two entities without recurring to invasive and expensive tests may offer a valuable clinical tool in the management of the two diseases.

Correlations Between Subjective Evaluation of Quality of Life, Visual Field Loss, and Performance in Simulated Activities of Daily Living in Glaucoma Patients.

PRECIS: Glaucoma patients displayed alterations in their quality of life (QoL) and their ability to perform activities of daily living. The visual field (VF) of the worse eye might serve as a good marker for QoL evaluation. PURPOSE: The purpose of this study was to explore the correlations between VF defects, performance in simulated activities of daily living, and subjective evaluation of QoL in glaucoma patients. METHODS: Thirty-two patients with glaucoma and 10 age-matched control subjects were included. All participants answered a QoL questionnaire and underwent an assessment of visual function including monocular and binocular best-corrected visual acuity, binocular contrast sensitivity test (LogCS), and monocular and binocular VF. All subjects also carried out a series of simulated activities of daily living in a controlled environment. RESULTS: Glaucoma patients had lower QoL scores compared with controls for the composite score, near and distance activities, social functioning, mental health, role difficulties, dependency, and color vision. With regard to performance in the simulated mobility task, the number of mobility incidents was higher for glaucoma patients than for control subjects. For the reaching and grasping tasks, the overall movement duration for small objects was significantly longer in glaucoma patients compared with controls. The VF mean deviation of the worse eye was correlated with most of the QoL subscores. Mobility incidents as well as the reaching and grasping task parameters were not significantly correlated with QoL scores. CONCLUSIONS: Glaucoma patients showed an alteration of performance in simulated daily living activities, associated with a decreased QoL. There was no clear correlation between alterations in QoL and ability to perform activities of daily living. The QoL related to vision was mostly correlated to the visual function of the worse eye.

Optical Coherence Tomography Angiography of Perilimbal Vasculature in Port-Wine Stain and Sturge-Weber Syndrome Patients.

Purpose: To evaluate episcleral vasculature in corneal limbus with optical coherence tomography angiography (OCTA) in normal controls, port-wine stain (PWS) patients, and Sturge-Weber syndrome (SWS) patients. Methods: Unilateral eyes from 18 normal controls (25.41 ± 4.00 years), 16 PWS patients (21.35 ± 11.05 years), and 8 SWS patients with ipsilateral late-onset glaucoma (22.13 ± 7.82 years). Each subject underwent slit-lamp examination, applanation tonometry, and OCTA. All OCTA scans were performed using an OCTA system operating at a wavelength of 1050-nm in four quadrants (superior, inferior, nasal, and temporal). The scans were delineated into conjunctival and episcleral layers using IMAGEnet6 for analysis. Results: Slit-lamp and OCTA images demonstrated dense dilated episcleral vessels in PWS and SWS patients, particularly in the SWS group. The mean limbal involvements of episcleral vascular anomalies under slit lamp were respectively 0.00 ± 0.00, 5.44 ± 2.92, and 8.88 ± 2.70 clock hours in the control, PWS, and SWS groups (F = 58.46, P < 0.01). Quantitative analysis of OCTA scans showed that the episcleral vessel density in controls, PWS, and SWS groups were 25.03% ± 1.47%, 28.28% ± 1.96%, and 33.59% ± 3.00%, respectively (F = 18.17, P < 0.01). We also observed higher episcleral vessel diameter index in the SWS and PWS groups in comparison with the controls, particularly in the SWS group (P < 0.01). The vessel measurements, including density and diameter, were significantly correlated with the increased IOP and cup-to-disc (C/D) in SWS patients (P < 0.01). Conclusions: To our knowledge, this is the first demonstration of OCTA in PWS and SWS patients and represents direct pathoanatomic evidence for episcleral alterations in SWS patients. The episcleral vessel measurements correlated with the increased IOP and C/D in SWS patients, indicating the episcleral vascular hypertrophy may be a risk factor for glaucoma in adult SWS patients.

Corneal Reinnervation and Sensitivity Recovery after Pterygium Excision.

PURPOSE: To evaluate changes in corneal sensitivity and subbasal nerve density after pterygium excision. METHODS: This prospective trial included 22 eyes with nasal primary pterygium and 18 controls. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer in the nasal, superior, temporal, inferior, and center quadrants of the cornea before surgery and 10 days, 1 month, and 3months after surgery. The central cornea was analyzed using in vivo confocal microscopy (IVCM) before surgery and 1 and 3 months after surgery. Subbasal nerve density and other nerve parameters were analyzed using NeuronJ. Nerve tortuosity was evaluated and graded in individual IVCM scans. The tear film break-up time (TBUT) test and Schirmer's test were performed before surgery, as well as 1 and 3 months after surgery. All the same tests were performed in the controls. RESULTS: All affected eyes showed a significant increase in corneal sensitivity in the nasal corneal quadrant after surgery when compared with preoperative data (F = 37.3; P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (F = 37.3; P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (F = 37.3; P < 0.01). Compared with controls, pterygium patients demonstrated decreased corneal subbasal nerve density (. CONCLUSION: Pterygium patients demonstrated deteriorated corneal subbasal nerve fibers when compared with healthy controls in terms of nerve length, nerve trunks, and nerve branches. Therefore, pterygium excision improves corneal sensitivity and increases corneal subbasal nerve density.

Optical coherence tomography angiography for marginal corneal vascular remodelling after pterygium surgery with limbal-conjunctival autograft.

PURPOSE: To demonstrate the marginal corneal vascular remodelling using optical coherence tomography angiography (OCTA) after pterygium surgery. METHODS: Twenty-two eyes of 19 patients (8 males, 11 females; age, 58.68 ± 0.34 years) with primary grade-T3 nasal pterygium were enroled in this study. The eyes underwent excision of the pterygium followed by a free limbal-conjunctival autograft. OCTA was performed in the nasal limbal area before surgery and at 10 days, 1 month, and 3 months after surgery. The scans were analyzed in terms of postoperative vascular remodelling of the autograft and marginal corneal vascular arcades (MCAs). RESULTS: Preoperatively, the pterygium presented as abnormal centripetal vascular growth in OCTA scans. The conjunctival vessel density in the nasal quadrant was 29.26% ± 1.00%, 15.80% ± 0.83%, 19.80% ± 0.88%, and 20.26% ± 0.89% before and 10 days, 1 month, and 3 months, respectively, after surgery (F = 1.55, P < 0.01). The vessel density of MCAs was 28.33% ± 0.88%, 42.09% ± 0.41%, and 42.46% ± 0.31% 10 days, 1 month, and 3 months, respectively, after surgery (F = 188.2, P < 0.01). CONCLUSIONS: We describe a new application of OCTA for MCA vasculature imaging. Vascular remodelling of the graft and MCAs appeared at 1 month and continued for 3 months after surgery.

Chronic dry eye induced corneal hypersensitivity, neuroinflammatory responses, and synaptic plasticity in the mouse trigeminal brainstem.

BACKGROUND: Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches. METHODS: A mouse model of DED was obtained by unilateral excision of the extraorbital lachrymal gland (ELG) and Harderian gland (HG) of adult female C57BL/6 mice. In vivo tests were conducted at 7, 14, and 21 days (d) after surgery. Tear production was measured by a phenol red test and corneal alterations and inflammation were assessed by fluorescein staining and in vivo confocal microscopy. Corneal nerve morphology was evaluated by nerve staining. Mechanical corneal sensitivity was monitored using von Frey filaments. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous corneal nerve activity. RT-qPCR and immunostaining were used to determine RNA and protein levels at d21. RESULTS: We observed a marked reduction of tear production and the development of corneal inflammation at d7, d14, and d21 post-surgery in DED animals. Chronic DE induced a reduction of intraepithelial corneal nerve terminals. Behavioral and electrophysiological studies showed that the DED animals developed time-dependent mechanical corneal hypersensitivity accompanied by increased spontaneous ciliary nerve fiber electrical activity. Consistent with these findings, DED mice exhibited central presynaptic plasticity, demonstrated by a higher Piccolo immunoreactivity in the ipsilateral trigeminal brainstem sensory complex (TBSC). At d21 post-surgery, mRNA levels of pro-inflammatory (IL-6 and IL-1ß), astrocyte (GFAP), and oxidative (iNOS2 and NOX4) markers increased significantly in the ipsilateral trigeminal ganglion (TG). This correlated with an increase in Iba1, GFAP, and ATF3 immunostaining in the ipsilateral TG of DED animals. Furthermore, pro-inflammatory cytokines (IL-6, TNFα, IL-1ß, and CCL2), iNOS2, neuronal (ATF3 and FOS), and microglial (CD68 and Itgam) markers were also upregulated in the TBSC of DED animals at d21, along with increased immunoreactivity against GFAP and Iba1. CONCLUSIONS: Overall, these data highlight peripheral sensitization and neuroinflammatory responses that participate in the development and maintenance of dry eye-related pain. This model may be useful to identify new analgesic molecules to alleviate ocular pain.

Degeneration of Corneal Sensation and Innervation in Patients with Facial Paralysis: A Cross-Sectional Study Using in Vivo Confocal Microscopy.

Purpose: Facial paralysis (FP) leads to diverse periocular complications which threats visual acuity and affects corneal nerve functionally and morphologically. This study aims to summarize the clinical ophthalmic outcomes, corneal sensation, and morphological alterations of subbasal nerve and dendritic cells (DCs) in patients with facial paralysis.Methods: We performed a cross-sectional study of 48 consecutive patients with facial paralysis at one tertiary hospital. Forty-eight healthy participants were enrolled as controls. The images of corneal nerves and epithelial DCs were detected by in vivo confocal microscopy (IVCM). Each patient received thorough ophthalmic examination, tear film function tests, corneal fluorescence staining and Cochet-Bonnet esthesiometry test. Clinical and morphologic data were compared with controls.Results: Forty patients (83.3%) showed corneal injuries from punctate epithelial defects to corneal ulcers and scars. Visual impairment and eyelid malposition were observed. Corneal sensitivity remarkably decreased (25.1 ± 23.8 mm) in the affected eyes and was correlated to diminished subbasal nerve density (P = .019, r = 0.387). Numbers of corneal main nerve trunks and branches were significantly reduced (P < .0001) while DCs were increased (P < .0001) in patients with FP when compared with controls. Nerve fiber density showed inverse association with DC density (P = .019, r = -0.389).Conclusions: Ocular complications including corneal erosions, loss of corneal sensation, visual impairment and eyelid malposition have largely affected patients with facial paralysis. Morphological changes of diminished corneal subbasal nerve and increased DCs were detected by IVCM. Corneal epithelial defect, corneal opacity, corneal sensation, dendritic cell density are factors associated with corneal subbasal nerve density. Patients with FP are suggested to have complete ophthalmic evaluation and early instruction on ocular prevention.

Assessment of Ocular Surface Damage during the Course of Type 2 Diabetes Mellitus.

PURPOSE: To investigate the impact of disease duration on the ocular surface during the course of type 2 diabetes mellitus compared with nondiabetic controls. METHODS: One hundred twenty diabetic patients were divided into three groups according to disease duration: less than 5 years, 5-10 years, and over 10 years. All eyes were imaged using a corneal topographer (Oculus Keratograph 5M). Tear film measurements and meibography were also recorded. Meibomian gland changes were scored from 0 to 6 (meiboscore). RESULTS: The noninvasive breakup time first (NIKBUT-1st) and noninvasive breakup time average (NIKBUT-avg) were significantly shorter in the over 10 years diabetic group compared with the control group (P=0.0056 and P=0.010, resp.). Tear meniscus height (TMH) was significantly lower in the over 10 years diabetic group compared with the control group (P=0.0016) and the 5 years group (P=0.0061). We also found that more patients in the over 10 years diabetic group showed bulbar and limbal hyperemia compared with the control group (bulbar hyperemia: P=0.049; limbal hyperemia: P=0.026). The meiboscore in the over 10 years diabetic group was significantly higher compared with the other three groups (P < 0.05). Bulbar hyperemia showed a significant negative correlation with NIKBUT-1st in the over 10 years diabetic group (r=-0.35 and P < 0.05). CONCLUSION: Ocular surface damage in long-term type 2 diabetes is more severe than that in patients with shorter disease duration.