Surgical procedure of intratympanic injection and inner ear pharmacokinetics simulation in domestic pigs

Frontiers in Pharmacology, Journal Year: 2024, Volume and Issue: 15

Published: Jan. 26, 2024

Introduction: One major obstacle in validating drugs for the treatment or prevention of hearing loss is limited data available on distribution and concentration human inner ear. Although small animal models offer some insights into ear pharmacokinetics, their smaller organ size different barrier (round window membrane) permeabilities compared to humans can complicate study interpretation. Therefore, developing a reliable large model drug delivery crucial. The middle anatomy domestic pigs closely resembles that humans, making them promising candidates studying pharmacokinetics. However, unlike anatomical orientation tortuosity porcine external canal frustrates local Methods: In this study, we developed surgical technique access tympanic membrane pigs. To assess pre- post-surgery, auditory brainstem responses click pure tones were measured. Additionally, performed 3D segmentation images used simulate diffusion dexamethasone within through fluid simulation software (FluidSim). Results: We have successfully delivered sodium phosphate via intratympanic injection. recorded measurements revealed no adverse effects thresholds attributable surgery. also simulated rates confirmed accuracy simulations using in-vivo data. Discussion: characterized method conducting pharmacokinetic studies This mirrors cochlea thickness its barriers. time concentrations reported align with from studies. demonstrated potential as

Language: Английский

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Intelligent Beam Optimization for Light-Sheet Fluorescence Microscopy through Deep Learning

Intelligent Computing, Journal Year: 2024, Volume and Issue: 3

Published: Jan. 1, 2024

Light-sheet fluorescence microscopy (LSFM) provides the benefit of optical sectioning coupled with rapid acquisition times, enabling high-resolution 3-dimensional imaging large tissue-cleared samples. Inherent to LSFM, quality heavily relies on characteristics illumination beam, which only illuminates a thin section sample. Therefore, substantial efforts are dedicated identifying slender, nondiffracting beam profiles that yield uniform and high-contrast images. An ongoing debate concerns identification optimal beams for different samples: Gaussian, Bessel, Airy patterns, and/or others. However, comparisons among challenging as their optimization objectives often different. Given our datasets (approximately 0.5 TB images per sample) already analyzed using deep learning models, we envisioned approach problem by designing an tailored boost performance model. We hypothesized integrating physical LSFM model (after passing it through variable phase mask) into training cell detection network would achieve this goal. Here, report joint continuously updates mask results in improved image better detection. The efficacy method is demonstrated both simulations experiments reveal enhancements compared traditional Gaussian light sheet. discuss how systems computational novel insights advancing design models analysis datasets.

Language: Английский

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COMBINe enables automated detection and classification of neurons and astrocytes in tissue-cleared mouse brains

Cell Reports Methods, Journal Year: 2023, Volume and Issue: 3(4), P. 100454 - 100454

Published: April 1, 2023

Tissue clearing renders entire organs transparent to accelerate whole-tissue imaging; for example, with light-sheet fluorescence microscopy. Yet, challenges remain in analyzing the large resulting 3D datasets that consist of terabytes images and information on millions labeled cells. Previous work has established pipelines automated analysis tissue-cleared mouse brains, but focus there was single-color channels and/or detection nuclear localized signals relatively low-resolution images. Here, we present an workflow (COMBINe, Cell detectiOn Mouse BraIN) map sparsely neurons astrocytes genetically distinct forebrains using mosaic double markers (MADM). COMBINe blends modules from multiple RetinaNet at its core. We quantitatively analyzed regional subregional effects MADM-based deletion epidermal growth factor receptor (EGFR) neuronal astrocyte populations forebrain.

Language: Английский

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Three-dimensional mouse cochlea imaging based on the modified Sca/eS using confocal microscopy

Anatomical Science International, Journal Year: 2023, Volume and Issue: 98(3), P. 309 - 317

Published: Feb. 11, 2023

The three-dimensional stria vascularis (SV) and cochlear blood vessel structure is essential for inner ear function. Here, modified Sca/eS, a sorbitol-based optical-clearing method, was reported to visualize SV vascular in the intact mouse cochlea. Cochlear macrophages as well perivascular-resident macrophage-like melanocytes were detected GFP-positive cells of CX3CR1+/GFP mice. This study's method effective elucidating function under both physiological pathological conditions.

Language: Английский

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Tissue clearing and three-dimensional imaging of the whole cochlea and vestibular system from multiple large-animal models

STAR Protocols, Journal Year: 2023, Volume and Issue: 4(2), P. 102220 - 102220

Published: April 14, 2023

The inner ear of humans and large animals is embedded in a thick dense bone that makes dissection challenging. Here, we present protocol enables three-dimensional (3D) characterization intact ears from large-animal models. We describe steps for decalcifying bone, using solvents to remove color lipids, imaging tissues 3D confocal light sheet microscopy. then detail pipeline count hair cells antibody-stained imaged cochleae open-source software. For complete details on the use execution this protocol, please refer (Moatti et al., 2022).1

Language: Английский

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A gene edited pig model for studying LGR5+ stem cells: implications for future applications in tissue regeneration and biomedical research

Frontiers in Genome Editing, Journal Year: 2024, Volume and Issue: 6

Published: June 6, 2024

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era biomedical research, significantly enhancing our understanding of disease mechanisms helping develop novel therapies. These technologies been instrumental creating precise animal models for use stem cell research regenerative medicine. For instance, we developed transgenic pig model to enable the investigation LGR5-expressing cells. The was designed induce expression H2B-GFP under regulatory control LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, identified distinct subpopulations cells within adult human, mouse, tissues. LGR5, leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling these + demonstrate varied roles anatomical distributions, underscoring necessity suitable translational models. This facilitates tracking has provided valuable insights into across different tissues species. pulmonary tissue, Lgr5 mice are predominantly located alveolar compartments, driving differentiation epithelial progenitors Wnt pathway activation. In contrast, pigs humans, situated unique sub-basal position adjacent airway epithelium. fetal stages pattern during lung bud tip formation is evident humans but lacking mice. Species differences with respect also observed skin, intestines, cochlea further reinforcing need careful selection appropriate paper discusses potential utility exploring role tissue development regeneration goal translating findings human clinical applications.

Language: Английский

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Enhancing Light-Sheet Fluorescence Microscopy Illumination Beams through Deep Design Optimization

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Dec. 1, 2023

Light sheet fluorescence microscopy (LSFM) provides the benefit of optical sectioning coupled with rapid acquisition times for imaging tissue-cleared specimen. This allows high-resolution 3D large tissue volumes. Inherently to LSFM, quality heavily relies on characteristics illumination beam, notion that beam only illuminates a thin section is being imaged. Therefore, substantial efforts are dedicated identifying slender, non-diffracting profiles can yield uniform and high-contrast images. An ongoing debate concerns employment most optimal beam; Gaussian, Bessel, Airy patterns and/or others. Comparisons among different challenging as their optimization objective often different. Given our datasets (~0.5TB images per sample) already analyzed using deep learning models, we envisioned approach this problem by hypothesizing tailor boost models performance. We achieve integrating physical LSFM model after passing through variable phase mask into training cell detection network. Here report joint continuously updates mask, improving image better detection. Our method's efficacy demonstrated both simulations experiments, revealing enhancements in compared traditional Gaussian light sheet. offer valuable insights designing systems computational exhibits significant potential advancing optics design analysis datasets.

Language: Английский

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T-CLEARE: a pilot community-driven tissue clearing protocol repository

Frontiers in Bioengineering and Biotechnology, Journal Year: 2024, Volume and Issue: 12

Published: Sept. 16, 2024

Selecting and implementing a tissue clearing protocol is challenging. Established more than 100 years ago, still rapidly evolving field of research. There are currently many published protocols to choose from, each performs better or worse across range key evaluation factors (e.g., speed, cost, stability, fluorescence quenching). Additionally, often optimized for specific experimental contexts, applying an existing new problem can require lengthy period adaptation by trial error. Although the primary literature review articles provide useful starting point optimization, there growing recognition that results vary dramatically with changes type antibody used. To help address this issue, we have developed novel, freely available repository named T-CLEARE (Tissue CLEAring REpository; https://doryworkspace.org/doryviz ). incorporates community responses open survey designed capture details not commonly found in scientific literature, including modifications required use cases instances when did perform well (negative results). The goal share evaluations various types potentially identify best-in-class methods given application.

Language: Английский

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COMBINe: Automated Detection and Classification of Neurons and Astrocytes in Tissue Cleared Mouse Brains

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: Dec. 6, 2022

Summary Tissue clearing renders entire organs transparent to enable combination with light sheet fluorescence microscopy and accelerate whole tissue imaging. Yet, challenges remain in analyzing the large resulting 3D datasets that consist of terabytes images information on millions labeled cells. Previous work has established pipelines for automated analysis cleared mouse brains. However, they have focused single color channels and/or detection nuclear localized signals, relatively low-resolution images. To address this gap, we present an workflow map neurons astrocytes genetically distinct Mosaic Analysis Double Markers (MADM) forebrains. We named COMBINe (Cell detectiOn Mouse BraIN) as it combines modules from multiple pipelines. With RetinaNet its core, quantitatively analyzed regional subregional effects MADM-based deletion Epidermal growth factor receptor neuronal astrocyte populations forebrain.

Language: Английский

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T-CLEARE: A Pilot Community-Driven Tissue-Clearing Protocol Repository

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: March 12, 2023

ABSTRACT Selecting and implementing a tissue-clearing protocol is challenging. Established more than 100 years ago, tissue clearing still rapidly evolving field of research. There are currently many published protocols to choose from, each performs better or worse across range key evaluation factors (e.g., speed, cost, stability, fluorescence quenching). Additionally, often optimized for specific experimental contexts, applying an existing new problem can require lengthy period adaptation by trial error. Although the primary literature review articles provide useful starting point optimization, there growing recognition that do not sufficient detail replicate reproduce results. To help address this issue, we have developed novel, freely available repository named T-CLEARE (Tissue CLEAring REpository; https://doryworkspace.org/doryviz ). incorporates community responses open survey designed capture details commonly found in scientific literature, including modifications required use cases instances when did perform well (negative results). The goal forum share evaluations various types potentially identify best-in-class methods given application.

Language: Английский

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