Noninvasive Imaging of Conjunctival Goblet Cells as a Method for Diagnosing Dry Eye Disease in an Experimental Mouse Model.

Purpose: The purpose of this study was to evaluate a noninvasive conjunctival goblet cell (GC) imaging method for assessing dry eye disease (DED) in an experimental mouse model. Methods: Moxifloxacin-based fluorescence microscopy (MBFM) was used to examine GCs noninvasively in 56 mice. Forty-two (42) DED-induced mice were divided into 2 groups and treated topically for 14 days with cyclosporine (CsA) or normal saline (NS). In vivo MBFM imaging and clinical DED evaluations were performed and goblet cell density (GCD) and goblet cell area (GCA) were obtained and compared with histological GCD using periodic acid-Schiff (PAS) staining. Correlation and receiver operating characteristic (ROC) analyses showed MBFM's high diagnostic value. Results: The GCD and GCA of the DED mice obtained from in vivo MBFM imaging were highly correlated with clinical DED parameters and GCD obtained from PAS histology. The therapeutic effect of CsA, as observed by in vivo MBFM, was significant with respect to that of NS treatment. The ROC curves derived from in vivo MBFM showed high diagnostic value in assessing DED. Conclusions: The proposed noninvasive method has high diagnostic value in assessing the severity of DED and the effect of treatment for this disease. Translational Relevance: A noninvasive imaging method using moxifloxacin-based fluorescence microscopy was evaluated for assessing DED in an experimental mouse model. The method showed high diagnostic value in assessing the severity of DED and the effect of treatment, bridging the gap between basic research and clinical treatment. The study provides a promising tool for diagnosing and monitoring DED.

Deep learning framework for automated goblet cell density analysis in in-vivo rabbit conjunctiva.

Goblet cells (GCs) in the conjunctiva are specialized epithelial cells secreting mucins for the mucus layer of protective tear film and playing immune tolerance functions for ocular surface health. Because GC loss is observed in various ocular surface diseases, GC examination is important for precision diagnosis. Moxifloxacin-based fluorescence microscopy (MBFM) was recently developed for non-invasive high-contrast GC visualization. MBFM showed promise for GC examination by high-speed large-area imaging and a robust analysis method is needed to provide GC information. In this study, we developed a deep learning framework for GC image analysis, named dual-channel attention U-Net (DCAU-Net). Dual-channel convolution was used both to extract the overall image texture and to acquire the GC morphological characteristics. A global channel attention module was adopted by combining attention algorithms and channel-wise pooling. DCAU-Net showed 93.1% GC segmentation accuracy and 94.3% GC density estimation accuracy. Further application to both normal and ocular surface damage rabbit models revealed the spatial variations of both GC density and size in normal rabbits and the decreases of both GC density and size in damage rabbit models during recovery after acute damage. The GC analysis results were consistent with histology. Together with the non-invasive high-contrast imaging method, DCAU-Net would provide GC information for the diagnosis of ocular surface diseases.

Dual-energy computed tomography material decomposition improves prediction accuracy of hematoma expansion in traumatic intracranial hemorrhage.

OBJECTIVE: The angiographic spot sign (AS) on CT angiography (CTA) is known to be useful for predicting expansion in intracranial hemorrhage, but its use is limited due to its relatively low sensitivity. Recently, dual-energy computed tomography (DECT) has been shown to be superior in distinguishing between hemorrhage and iodine. This study aimed to evaluate the diagnostic performance of hematoma expansion (HE) using DECT AS in traumatic intracranial hemorrhage. METHODS: We recruited participants with intracranial hemorrhage confirmed via CTA for suspected traumatic cerebrovascular injuries. We evaluated AS on both conventional-like and fusion images of DECT. AS is grouped into three categories: intralesional enhancement without change, delayed enhancement (DE), and growing contrast leakage (GL). HE was evaluated by measuring hematoma size on DECT and follow-up CT. Logistic regression analysis was used to evaluate whether AS on fusion images was a significant risk factor for HE. Diagnostic accuracy was calculated, and the results from conventional-like and fusion images were compared. RESULTS: Thirty-nine hematomas in 24 patients were included in this study. Of these, 18 hematomas in 13 patients showed expansion on follow-up CT. Among the expanders, AS and GL on fusion images were noted in 13 and 5 hematomas, respectively. In non-expanders, 10 and 1 hematoma showed AS and GL, respectively. In the logistic regression model, GL on the fusion image was a significant independent risk factor for predicting HE. However, when AS was used on conventional-like images, no factors significantly predicted HE. In the receiver operating characteristic curve analysis, the area under the curve of AS on the fusion images was 0.71, with a sensitivity and specificity of 66.7% and 76.2%, respectively. CONCLUSIONS: GL on fusion images of DECT in traumatic intracranial hemorrhage is a significant independent radiologic risk factor for predicting HE. The AS of DECT fusion images has improved sensitivity compared to that of conventional-like images.

Pituitary Apoplexy After Leuprolide Therapy in a Breast Cancer Patient: A Case Report.

Pituitary apoplexy (PA) is a clinical syndrome resulting from sudden hemorrhage and/or infarction of the pituitary gland. Recent reports documented the development of PA secondary to treatment with gonadotropin-releasing hormone (GnRH) agonists for prostate cancer. A 52-year-old woman visited our emergency room with a severe headache, occurred 1 day prior. She underwent breast-conserving surgery for breast cancer 1 month prior. She was currently undergoing radiation and hormone therapy, consisting of leuprorelin. Brain contrast-enhanced MRI revealed a pituitary adenoma with internal hemorrhage in the sellar and suprasellar areas. Pachymeningeal enhancement was observed along the retroclival and bilateral frontal areas. The patient was diagnosed with PA and aseptic meningitis. The patient underwent total excision via transsphenoidal surgery 8 days after admission. The patient was pathologically diagnosed with a pituitary adenoma with necrosis. On immunochemical staining, the tumor was positive for follicle-stimulating hormone. The follow-up MRI revealed no evidence of residual tumor or an improved pachymeningeal enhancement. She is currently undergoing follow-up at the neurosurgery and endocrinology outpatient departments with no noted complications. In breast cancer patients receiving GnRH agonist therapy, PA may be rare complication.

Feasibility of moxifloxacin and proflavine dual fluorescence imaging for detecting gastrointestinal neoplastic lesions: A prospective study.

OBJECTIVES: High-contrast and high-resolution imaging techniques would enable real-time sensitive detection of the gastrointestinal lesions. This study aimed to investigate the feasibility of novel dual fluorescence imaging using moxifloxacin and proflavine in the detection of neoplastic lesions of the human gastrointestinal tract. METHODS: Patients with the colonic and gastric neoplastic lesions were prospectively enrolled. The lesions were biopsied with forceps or endoscopically resected. Dual fluorescence imaging was performed by using custom axially swept wide-field fluorescence microscopy after topical moxifloxacin and proflavine instillation. Imaging results were compared with both confocal imaging with cell labeling and conventional histological examination. RESULTS: Ten colonic samples (one normal mucosa, nine adenomas) from eight patients and six gastric samples (one normal mucosa, five adenomas) from four patients were evaluated. Dual fluorescence imaging visualized detail cellular structures. Regular glandular structures with polarized cell arrangement were observed in normal mucosa. Goblet cells were preserved in normal colonic mucosa. Irregular glandular structures with scanty cytoplasm and dispersed elongated nuclei were observed in adenomas. Goblet cells were scarce or lost in the colonic lesions. Similarity analysis between moxifloxacin and proflavine imaging showed relatively high correlation values in adenoma compared with those in normal mucosa. Dual fluorescence imaging showed good detection accuracies of 82.3% and 86.0% in the colonic and the gastric lesions, respectively. CONCLUSIONS: High-contrast and high-resolution dual fluorescence imaging was feasible for obtaining detail histopathological information in the gastrointestinal neoplastic lesions. Further studies are needed to develop dual fluorescence imaging as an in vivo real-time visual diagnostic method.

Phosphatidylinositol 3-phosphate mediates Arc capsids secretion through the multivesicular body pathway.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene that plays a vital role in learning and memory. The recent discovery that Arc mediates the inter-neuronal RNA transfer implies its role in regulating neuronal functions across long distances. Arc protein has structural and functional properties similar to viral Group-specific antigen (Gag). By assembling into high-order, virus-like capsids, Arc mediates the intercellular RNA transfer. However, the exact secretion pathway through which Arc capsids maneuver cargos is unclear. Here, we identified that Arc capsids assemble and secrete through the endosomal-multivesicular body (MVB) pathway. Arc's endosomal entry is likely mediated by phosphatidylinositol-3-phosphate (PI3P). Indeed, reconstituted Arc protein preferably binds to PI3P. In mammalian cells, Arc forms puncta that colocalizes with FYVE, an endosomal PI3P marker, and competitive binding to PI3P via prolonged FYVE expression reduces the average number of Arc puncta per cell. Overexpression of MTMR1, a PI3P phosphatase, significantly reduces Arc capsid secretion. Arc capsids secrete through the endosomal-MVB axis as extracellular vesicles. Live-cell imaging shows that fluorescently labeled Arc primarily colocalizes Rab5 and CD63, early endosomal and MVB markers, respectively. Superresolution imaging resolves Arc accumulates within the intraluminal vesicles of MVB. CRISPR double knockout of RalA and RalB, crucial GTPases for MVB biogenesis and exocytosis, severely reduces Arc-mediated RNA transfer efficiency. These results suggest that, unlike the Human Immunodeficiency Virus Gag, which assembles on and bud off from the plasma membrane, Arc capsids assemble at the endocytic membranes of the endosomal-MVB pathway mediated by PI3P. Understanding Arc's secretion pathway helps gain insights into its role in intercellular cargo transfer and highlights the commonality and distinction of trafficking mechanisms between structurally resembled capsid proteins.

Association of cerebral white matter hyperintensities with coronary artery calcium in a healthy population: a cross-sectional study.

In brain magnetic resonance imaging (MRI), white matter hyperintensity (WMH) is a commonly encountered finding and is known to reflect cerebral small vessel disease. The aim of our study was to investigate the association of coronary artery calcium (CAC) with WMH and elucidate the relationship between WMH and atherosclerotic risk factors in a large-scale healthy population. This retrospective study included 1337 individuals who underwent brain MRI and CAC scoring computed tomography at healthcare centers affiliated with a tertiary hospital. Cerebral WMH was defined as Fazekas score greater than 2 on brain MRI. Intracranial artery stenosis (ICAS) was also assessed and determined to be present when stenosis was more than 50% on angiography. The associations of risk factors, CAC score, and ICAS with cerebral WMH were assessed by multivariable regression analysis. In multivariable analysis, categories of higher CAC scores showed increased associations with both periventricular and deep WMHs in a dose-dependent relationship. The presence of ICAS was also significantly related to cerebral WMH, and among the clinical variables, age and hypertension were independent risk factors. In conclusion, CAC showed a significant association with cerebral WMH in a healthy population, which might provide evidence for referring to the CAC score to identify individuals with risk of cerebral WMH.

Severe headache in a middle-aged woman after device closure of a ventricular septal defect.

Reversible cerebral vasoconstriction syndrome presents with thunderclap headache and represents a group of conditions that show reversible multifocal narrowing of cerebral arteries. Some patients who undergo device closure of an atrial septal defect complain of headache, which are posited as a migraine. Here we report a case of severe headache due not to migraine but reversible cerebral vasoconstriction syndrome after device closure of a ventricular septal defect.

Multiple De Novo Cerebral Arteriovenous Malformations in a Patient with Alcoholic Liver Cirrhosis.

Cerebral arteriovenous malformations (cAVMs) are traditionally considered congenital anomalies. The literature includes only two reported cases of de novo solitary cAVMs associated with liver cirrhosis (LC). Here, we report a unique case of multiple de novo cAVMs in a patient with alcoholic LC. Teaching point: In patients with potential risks for de novo cAVM, including liver cirrhosis, the possibility of multiple de novo cAVMs should be considered.

Moxifloxacin-Based Extended Depth-of-Field Fluorescence Microscopy for Real-Time Conjunctival Goblet Cell Examination.

Conjunctival goblet cells (CGCs) are mucin-secreting cells in the eye and play essential roles for ocular surface homeostasis. Since various ocular surface pathologies are related to CGC dysfunction, CGC examination is important for the evaluation of ocular surface conditions. Recently we introduced moxifloxacin-based fluorescence microscopy (MBFM) for non-invasive CGC imaging. However, the imaging speed was up to 1 frame per second (fps) and needed to be improved for clinical applications. In this study, we developed a high-speed moxifloxacin-based, extended depth-of-field (EDOF) microscopy system that operates at a maximum imaging speed of 15 fps. The system used a deformable mirror for the high-speed axial sweeping of focal plane during single-frame acquisitions. The acquired images contained both in-focus and out-of-focus information, and deconvolution was used to filter the in-focus information. The system had a DOF of 800 [Formula: see text], field-of-view of 1.2 mm ×1.2 mm, and resolution of [Formula: see text]. Its performance was demonstrated by real-time, breathing-motion-insensitive CGC imaging of mouse and rabbit models, in vivo. High-speed EDOF microscopy has potentials for non-invasive, real-time CGC examinations of human subjects.

Multi-Focus Image Fusion Using Focal Area Extraction in a Large Quantity of Microscopic Images.

The non-invasive examination of conjunctival goblet cells using a microscope is a novel procedure for the diagnosis of ocular surface diseases. However, it is difficult to generate an all-in-focus image due to the curvature of the eyes and the limited focal depth of the microscope. The microscope acquires multiple images with the axial translation of focus, and the image stack must be processed. Thus, we propose a multi-focus image fusion method to generate an all-in-focus image from multiple microscopic images. First, a bandpass filter is applied to the source images and the focus areas are extracted using Laplacian transformation and thresholding with a morphological operation. Next, a self-adjusting guided filter is applied for the natural connections between local focus images. A window-size-updating method is adopted in the guided filter to reduce the number of parameters. This paper presents a novel algorithm that can operate for a large quantity of images (10 or more) and obtain an all-in-focus image. To quantitatively evaluate the proposed method, two different types of evaluation metrics are used: "full-reference" and "no-reference". The experimental results demonstrate that this algorithm is robust to noise and capable of preserving local focus information through focal area extraction. Additionally, the proposed method outperforms state-of-the-art approaches in terms of both visual effects and image quality assessments.

Neural stem cells derived from human midbrain organoids as a stable source for treating Parkinson's disease: Midbrain organoid-NSCs (Og-NSC) as a stable source for PD treatment.

Successful clinical translation of stem cell-based therapy largely relies on the scalable and reproducible preparation of donor cells with potent therapeutic capacities. In this study, midbrain organoids were yielded from human pluripotent stem cells (hPSCs) to prepare cells for Parkinson's disease (PD) therapy. Neural stem/precursor cells (NSCs) isolated from midbrain organoids (Og-NSCs) expanded stably and differentiated into midbrain-type dopamine(mDA) neurons, and an unprecedentedly high proportion expressed midbrain-specific factors, with relatively low cell line and batch-to-batch variations. Single cell transcriptome analysis followed by in vitro assays indicated that the majority of cells in the Og-NSC cultures are ventral midbrain (VM)-patterned with low levels of cellular senescence/aging and mitochondrial stress, compared to those derived from 2D-culture environments. Notably, in contrast to current methods yielding mDA neurons without astrocyte differentiation, mDA neurons that differentiated from Og-NSCs were interspersed with astrocytes as in the physiologic brain environment. Thus, the Og-NSC-derived mDA neurons exhibited improved synaptic maturity, functionality, resistance to toxic insults, and faithful expressions of the midbrain-specific factors, in vitro and in vivo long after transplantation. Consequently, Og-NSC transplantation yielded potent therapeutic outcomes that are reproducible in PD model animals. Collectively, our observations demonstrate that the organoid-based method may satisfy the demands needed in the clinical setting of PD cell therapy.

Brainstem Venous Infarction from Thrombosis on the Jugular Bulb as a Serious Complication of Blunt Head Trauma Injury: A Case Report.

The incidence of dural venous sinus thrombosis (DVST) and the cerebral venous infarct have not exactly known, but DVST is closely related to the skull fracture around the venous sinus. A 56-year-old man experienced massive watery discharge after hitting on his face by a falling machine. He was alert and no cerebrospinal fluid discharge on admission. Air-density was shown on the jugular fossa in the brain computed tomography. On the 3th day of trauma, he suddenly had dyspnea and loss of consciousness and became comatose. Acute edema on medulla, pons and right cerebellar hemisphere and focal infarct on right medulla were visualized on the brain magnetic resonance imaging. And the sigmoid sinus and the jugular vein were occluded and venous circulation on the right posterior fossa was diminished on the cerebral angiography. Air-density on the sinus may be an indicator into developing venous thrombosis and brainstem venous infarct could be followed by the DVST round the jugular bulb.

Evaluation of Reproducibility of Brain Volumetry between Commercial Software, Inbrain and Established Research Purpose Method, FreeSurfer.

BACKGROUND AND PURPOSE: We aimed to determine the intermethod reproducibility between the commercial software Inbrain (MIDAS IT) and the established research-purpose method FreeSurfer, as well as the effect of MRI resolution and the pathological condition of subjects on their intermethod reproducibility. METHODS: This study included 45 healthy volunteers and 85 patients with mild cognitive impairment (MCI). In 43 of the 85 patients with MCI, three-dimensional, T1-weighted MRI data were obtained at an in-plane resolution of 1.2 mm. The data of the remaining 42 patients with MCI and the healthy volunteers were obtained at an in-plane resolution of 1.0 mm. The within-subject coefficient of variation (CoV), intraclass correlation coefficient (ICC), and effect size were calculated, and means were compared using paired t-tests. The parameters obtained at 1.0-mm and 1.2-mm resolutions in patients with MCI were compared to evaluate the effect of the in-plane resolution on the intermethod reproducibility. The parameters obtained at a 1.0-mm in-plane resolution in patients with MCI and healthy volunteers were used to analyze the effect of subject condition on intermethod reproducibility. RESULTS: Overall the two methods showed excellent reproducibility across all regions of the brain (CoV=0.5-3.9, ICC=0.93 to >0.99). In the subgroup of healthy volunteers, the intermethod reliability was only good in some regions (frontal, temporal, cingulate, and insular). The intermethod reproducibility was better in the 1.0-mm group than the 1.2-mm group in all regions other than the nucleus accumbens. CONCLUSIONS: Inbrain and FreeSurfer showed good-to-excellent intermethod reproducibility for volumetric measurements. Nevertheless, some noticeable differences were found based on subject condition, image resolution, and brain region.

Moxifloxacin based axially swept wide-field fluorescence microscopy for high-speed imaging of conjunctival goblet cells.

Goblet cells (GCs) in the conjunctiva are specialized epithelial cells producing mucins on the ocular surface. GCs play important roles in maintaining homeostasis of the ocular surface, and GC dysfunction is associated with various complications including dry eye diseases. Current GC examination methods, which are conjunctival impression cytology and confocal reflection microscopy, have limitations for routine examination. Fluorescence microscopy using moxifloxacin was recently introduced as a non-invasive and high-contrast imaging method, but further development is needed to be used for GC examination. Here we developed a non-invasive high-speed high-contrast GC imaging method, called moxifloxacin based axially swept wide-field fluorescence microscopy (MBAS-WFFM). This method acquired multiple fluorescence images with the axial sweeping of the focal plane to capture moxifloxacin labeled GCs on the tilted conjunctival surface in focus and generated all-in-focus images by combining the acquired images. The imaging field of view and imaging speed were increased to 1.6 mm × 1.6 mm and 30 fps. An image processing method was developed for the analysis of GC density. MBAS-WFFM was applied to alkali burn mouse models and detected GC damage and recovery via longitudinal imaging. MBAS-WFFM could assess the status of GCs rapidly and non-invasively. We anticipate MBAS-WFFM to be a starting point for non-invasive GC examination and the diagnosis of GC associated diseases. For example, MBAS-WFFM could be used to classify dry eye diseases into detail categories for effective treatment.

Cerebral White Matter Lesions on Diffusion-Weighted Images and Delayed Neurological Sequelae after Carbon Monoxide Poisoning: A Prospective Observational Study.

INTRODUCTION: Carbon monoxide (CO) poisoning can result in delayed neurological sequelae (DNS). Factors predicting DNS are still controversial. This study aims to determine whether acute brain lesions observed using diffusion-weighted magnetic resonance imaging (MRI) following acute CO poisoning are related to the subsequent development of DNS. METHODS: This prospective study was conducted on patients with CO poisoning treated at a university hospital in Bucheon, Korea. From August 2016 to July 2019, a total of 283 patients visited the hospital because of CO poisoning. Exclusion criteria included age under 18 years, refusing hyperbaric oxygen therapy, refusing MRI, being discharged against medical advice, being lost to follow-up, having persistent neurological symptoms at discharge, and being transferred from another hospital 24 h after exposure. RESULTS: Of the 154 patients included in the final study, acute brain lesions on MRI (ABLM) were observed in 49 patients (31.8%) and DNS occurred in 30 patients (19.5%). In a logistic regression analysis, lower Glasgow coma scale score and higher exposure time were associated with DNS, and the presence of ABLM in white matter was significantly associated with DNS (OR 6.741; 95% CI, 1.843-24.660; p = 0.004). CONCLUSION: The presence of ABLM in white matter was significantly related to the occurrence of DNS. Early prediction of the risk of developing DNS through MRI may be helpful in treating patients with CO poisoning.

Characterization of early-stage cutaneous radiation injury by using optical coherence tomography angiography.

Cutaneous radiation injury (CRI) is a skin injury caused by exposure to high dose ionizing radiation (IR). Diagnosis and treatment of CRI is difficult due to its initial clinically latent period and the following inflammatory bursts. Early detection of CRI before clinical symptoms will be helpful for effective treatment, and various optical methods have been applied with limitations. Here we show that optical coherence tomography angiography (OCTA) could detect changes in the skin during the latent period in CRI mouse models non-invasively. CRI was induced on the mouse hindlimb with exposure to various IR doses and the injured skin regions were imaged longitudinally by OCTA until the onset of clinical symptoms. OCTA detected several changes in the skin including the skin thickening, the dilation of large blood vessels, and the irregularity in vessel boundaries. Some of OCTA findings were confirmed by histology. The study results showed that OCTA could be used for early CRI detection.