WAY-100635 Alleviates Corneal Lesions Through 5-HT1A Receptor-ROS-Autophagy Axis in Dry Eye

Purpose: To explore whether 5-HT1A receptors are involved in the dry eye disease (DED) mouse model and reveal its underlying mechanism.Methods: A C57BL/6J mouse DED model was established via the administration of 0.2% benzalkonium chloride twice a day for 14 days. Corneal fluorescein sodium staining score and Schirmer I test were checked before, and on days 7, 14, and 21 after treatment. The experiment was randomly divided into control, DED, 5-HT1A receptor agonist with or without N-acetylcysteine (NAC) and 5-HT1A receptor antagonist with or without NAC groups. The mRNA expression of inflammatory cytokines was measured by reverse transcription-quantitative polymerase chain reaction. Cellular reactive oxygen species (ROS) were detected by 2', 7'-dichlorodihydrofluorescein diacetate assays. Western blot analysis was used to measure the expression levels of autophagic proteins microtubule-associated protein 1 light chain 3 (LC3B-I/II) and autophagy-related gene 5 (ATG5).Results: 5-HT1A receptor agonist (8-OH-DPAT) increased corneal fluorescein sodium staining spots and 5-HT1A receptor antagonist (WAY-100635) decreased them. Treatment with 8-OH-DPAT was associated with the gene expression of more inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 10 (CXCL10) compared with treatment with WAY-100635. An increased expression of LC3B-I/II and ATG5 was observed in corneal epithelial cells in the mouse model of DED. 8-OH-DPAT significantly enhanced the expression of LC3B-I/II and ATG5 by disrupting ROS levels. WAY-100635 alleviates autophagy by inhibiting ROS production.Conclusion: Excessive ROS release through 8-OH-DPAT induction can lead to impaired autophagy and increased inflammatory response in DED. WAY-100635 reduces corneal epithelial defects and inflammation in DED, as well as alleviates autophagy by inhibiting ROS production. The activation of the 5-HT1A receptor-ROS-autophagy axis is critically involved in DED development.

FLGS-03. Fluorescein sodium-guided biopsy or resection in patients with contrast enhancing brainstem lesion in MRI

BACKGROUND It is challenging to resect or just biopsy the lesions in the brainstem, due to the essential function of the surfaces and limited space. Neuro-navigation is not always reliable and stereotatic biopsy is infrequently inconclusive due to small or inadequate samples. We want to share our experiences in the application of fluorescein sodium in surgery on patients with brainstem lesion which is contrast enhancing in MRI. METHODS Between January 2017 and June 2021, 5 patients with brainstem lesion underwent fluorescein sodium-guided surgery in neurosurgery department of Sun Yat-sen University Cancer Center. After injection of low dose of sodium fluorescein (3 mg/kg), the lesions with strong fluorescence staining were identified as the target area for biopsy or resection. RESULTS 5 consecutive patients (aged 6–47 years) with brainstem lesions prospectively underwent fluorescein sodium-guided surgery. The lesions were located in pontine in 3 patients and in the medulla in 2 patients. Gross total resection was achieved in 2 patients, and partial resection in the other 3 patients. In all patients, a pathological diagnosis was obtained (4 gliomas and 1 metastasis from non-small cell lung carcinoma) without severe complications, including mild facial or abduct nerve palsy in 3 patients. And all the specimens with strong fluorescence staining sent for pathology were proved to be tumorous. CONCLUSION Fluorescein sodium-guided technique was helpful to locate the lesion in brainstem which was contrast-enhanced in MRI. It was effective and safe to figure out an ideal trajectory to avoid damage of the crucial structures and improve the diagnostic rate.

Dual Fluorescence Emission by Irradiation of Single Optical Source for Fluorescein Sodium and 5-ALA Applications

Most brain surgeries aim to completely resection a tumor. However, the arrangement of blood vessels around brain tumors is often complex. Moreover, the tumors and blood vessels have similar colors, making it difficult to identify the boundaries between them with the naked eye. Fluorescent staining is a method used to distinguish the borders between brain tumors and blood vessels. The fluorescent contrast agents commonly used to observe tumors are 5-aminolevulinic acid (5-ALA) and fluorescein sodium (FS), which have different surgical sensitivities, depending on the type of tumor. In this article, a dual band band-pass filter (BPF) with dual-wavelength emission for 5-ALA and FS is designed, and the dual-band BPF capable of inducing simultaneous fluorescence emission of FS and 5-ALA was investigated experimentally to improve accuracy, speed, and energy efficiency in clinical settings. The possibility of dual fluorescence emission with a single irradiation is proposed. The proposed fluorescent dual-band filter has the advantage of saving energy, reducing auxiliary manpower and unit costs, and reducing operating room space requirements by producing two fluorescence diagnostic effects using a single equipment.

PerfuPul—A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia

Complex in vitro models, especially those based on human cells and tissues, may successfully reduce or even replace animal models within pre-clinical development of orally inhaled drug products. Microfluidic lung-on-chips are regarded as especially promising models since they allow the culture of lung specific cell types under physiological stimuli including perfusion and air-liquid interface (ALI) conditions within a precisely controlled in vitro environment. Currently, though, such models are not available to a broad user community given their need for sophisticated microfabrication techniques. They further require systematic comparison to well-based filter supports, in analogy to traditional Transwells®. We here present a versatile perfusable platform that combines the advantages of well-based filter supports with the benefits of perfusion, to assess barrier permeability of and aerosol deposition on ALI cultured pulmonary epithelial cells. The platform as well as the required technical accessories can be reproduced via a detailed step-by-step protocol and implemented in typical bio-/pharmaceutical laboratories without specific expertise in microfabrication methods nor the need to buy costly specialized equipment. Calu-3 cells cultured under liquid covered conditions (LCC) inside the platform showed similar development of transepithelial electrical resistance (TEER) over a period of 14 days as cells cultured on a traditional Transwell®. By using a customized deposition chamber, fluorescein sodium was nebulized via a clinically relevant Aerogen® Solo nebulizer onto Calu-3 cells cultured under ALI conditions within the platform. This not only allowed to analyze the transport of fluorescein sodium after ALI deposition under perfusion, but also to compare it to transport under traditional static conditions.

Redosing of Fluorescein Sodium Improves Image Interpretation During Intraoperative Ex Vivo Confocal Laser Endomicroscopy of Brain Tumors

BackgroundFluorescein sodium (FNa) is a fluorescence agent used with a wide-field operating microscope for intraoperative guidance and with confocal laser endomicroscopy (CLE) to evaluate brain tissue. Susceptibility of FNa to degradation over time may affect CLE image quality during prolonged surgeries. This study describes improved characteristics of CLE images after intraoperative redosing with FNa.MethodsA retrospective analysis was performed using CLE images obtained ex vivo from samples obtained during tumor resections with FNa-based fluorescence guidance with a wide-field operating microscope. The comparison groups included CLE images acquired after FNa redosing (redose imaging group), images from the same patients acquired after the initial FNa dose (initial-dose imaging group), and images from patients in whom redosing was not used (single-dose imaging group). A detailed assessment of image quality and interpretation regarding different FNa dosage and timing of imaging after FNa administration was conducted for all comparison groups.ResultsThe brightest and most contrasting images were observed in the redose group compared to the initial-dose and single-dose groups (P<0.001). The decay of FNa signal negatively correlated with brightness (rho = -0.52, P<0.001) and contrast (rho = -0.57, P<0.001). Different doses of FNa did not significantly affect the brightness (P=0.15) or contrast (P=0.09) in CLE images. As the mean timing of imaging increased, the percentage of accurately diagnosed images decreased (P=0.03).ConclusionsThe decay of the FNa signal is directly associated with image brightness and contrast. The qualitative interpretation scores of images were highest for the FNa redose imaging group. Redosing with FNa to improve the utility of CLE imaging should be considered a safe and beneficial strategy during prolonged surgeries.

The EyeFlowCell: Development of a 3D-Printed Dissolution Test Setup for Intravitreal Dosage Forms

An in vitro dissolution model, the so-called EyeFlowCell (EFC), was developed to test intravitreal dosage forms, simulating parameters such as the gel-like consistency of the vitreous body. The developed model consists of a stereolithography 3D-printed flow-through cell with a polyacrylamide (PAA) gel as its core. This gel needed to be coated with an agarose sheath because of its low viscosity. Drug release from hydroxypropyl methylcellulose-based implants containing either triamcinolone acetonide or fluorescein sodium was studied in the EFC using a schematic eye movement by the EyeMovementSystem (EyeMoS). For comparison, studies were performed in USP apparatus 4 and USP apparatus 7. Significantly slower drug release was observed in the PAA gel for both model drugs compared with the compendial methods. Drug release from fluorescein sodium-containing model implants was completed after 40 min in USP apparatus 4, whereas drug release in the gel-based EFC lasted 72 h. Drug release from triamcinolone acetonide-containing model implants was completed after 35 min in USP apparatus 4 and after 150 min in USP apparatus 7, whereas this was delayed until 96 h in the EFC. These results suggest that compendial release methods may overestimate the drug release rate in the human vitreous body. Using a gel-based in vitro release system such as the EFC may better predict drug release.