Fluorescein Research Articles

Purification of Anti-OSH1 IgG and Immunohistochemical Staining of Rice Shoot Apical Meristem.

Immunohistochemical staining (IHC) enables the visualization of protein localization within tissues and organs. Here, we describe IHC to detect the ORYZA SATIVA HOMEOBOX1 (OSH1) protein in sections of rice shoot apical meristems (SAMs). First, anti-OSH1 IgG is purified from OSH1 polyclonal rabbit antisera. For subsequent IHC, rice shoot apices are fixed and embedded in paraffin and sections are prepared for IHC. In this IHC procedure, two different labeling methods, fluorescein isothiocyanate and alkaline phosphatase, are utilized for secondary antibody detection.

Utilizing the apical-out enteroids in vitro model to investigate intestinal glucose transport, barrier function, oxidative stress, and inflammatory responses in broiler chickens

IntroductionConventional 2D intestinal epithelial cell lines have been widely used in investigating intestinal functions, yet with limitations in recapitulating the in vivo gut physiology of chickens. A recently established chicken enteroid model with apical-out nature and the presence of leukocyte components represents intestinal mucosal functions. The objectives of this study were to 1) evaluate basic gut nutrient transport and barrier functions in this model and 2) identify the model’s effectiveness in studying inflammation and oxidative stress responses.MethodsEnteroids were generated from individual villus units isolated from the small intestine of Cobb500 broiler embryos. Enteroid viability, morphology, and epithelial cell markers were monitored; barrier function was evaluated based on the permeability to fluorescein isothiocyanate–dextran (FD4) with or without EDTA and lipopolysaccharide (LPS) challenges; nutrient transport was evaluated by fluorescence-labeled glucose (2NBD-G) with or without transporter blockade; the oxidative status was indicated by reactive oxygen species (ROS). Inflammatory and oxidative challenges were induced by LPS and menadione treatment, respectively. Selected marker gene expressions, including tight junction proteins (CLDN-1, CLDN-2, ZO-1, and OCCL), epithelial cell markers (Lgr-5, LYZ, and MUC-2), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, and INF-γ), and antioxidant enzymes (Nrf-2, catalase, and SOD), were determined by using RT-qPCR. Data were analyzed by one-way ANOVA among treatment groups.ResultsEnteroid cell activity was stable from day (d) 2 to d 6 and declined at d 7. Epithelial cell marker and cytokine expressions were stable from d 4 to d 6. FD4 permeability was increased after the EDTA treatment (P ≤ 0.05). Transporter-mediated 2NBD-G absorption was observed, which was reduced with glucose transporter blockade (P ≤ 0.05). Enteroids showed classic responses to LPS challenges, including upregulated gene expressions of IL-1β and IL-6, downregulated gene expressions of ZO-1 and OCCL, and increased FD4 permeability (P ≤ 0.05). Enteroids showed increased ROS generation (P ≤ 0.05) in response to oxidative stress.DiscussionIn conclusion, this apical-out enteroid model is a stable alternative in vitro model that exhibits intestinal barrier, nutrient transport, oxidation, and inflammation functions. With this enteroid model, we developed two challenge protocols for evaluating intestinal functions under oxidative stress and inflammation conditions.

Lethal versus surviving sepsis phenotypes displayed a partly differential regional expression of neurotransmitters and inflammation and did not modify the blood–brain barrier permeability in female CLP mice

BackgroundSeptic encephalopathy is frequent but its pathophysiology is enigmatic. We studied expression of neurotransmitters, inflammation and integrity of the blood–brain barrier (BBB) in several brain regions during abdominal sepsis. We compared mice with either lethal or surviving phenotype in the first 4 sepsis days. Mature CD-1 females underwent cecal ligation and puncture (CLP). Body temperature (BT) was measured daily and predicted-to-die (within 24 h) mice (for P-DIE; BT < 28 °C) were sacrificed together (1:1 ratio) with mice predicted-to-survive (P-SUR; BT > 35 °C), and healthy controls (CON). Brains were dissected into neocortex, cerebellum, midbrain, medulla, striatum, hypothalamus and hippocampus.ResultsCLP mice showed an up to threefold rise of serotonin in the hippocampus, 5-hydroxyindoleacetic and homovanillic acid (HVA) in nearly all regions vs. CON. Compared to P-SUR, P-DIE mice showed a 1.7 to twofold rise of HVA (386 ng/g of tissue), dopamine (265 ng/g) and 3,4-Dihydroxyphenylacetic acid (DOPAC; 140 ng/g) in the hippocampus, hypothalamus and medulla (174, 156, 82 ng/g of tissue, respectively). CLP increased expression of TNFα, IL-1β and IL-6 mRNA by several folds in the midbrain, cerebellum and hippocampus versus CON. The same cytokines were further elevated in P-DIE vs P-SUR in the midbrain and cerebellum. Activation of astrocytes and microglia was robust across regions but remained typically phenotype independent. There was a similar influx of sodium fluorescein across the BBB in both P-DIE and P-SUR mice.ConclusionsCompared to survivors, the lethal phenotype induced a stronger deregulation of amine metabolism and cytokine expression in selected brain regions, but the BBB permeability remained similar regardless of the predicted outcome.

Safety, efficacy, and side effects of sodium fluorescein-aided resection of glioblastoma: a quasi-experimental study

Background: The use of fluorescein sodium (FS) as a surgical adjunct in glioblastoma resection has shown promise in improving tumor visualization and resection outcomes. This study aimed to evaluate the safety, efficacy, and side effects of FS-aided resection in patients with glioblastoma. Methods: This is a prospective, single-center cohort study conducted at Ibrahim Cardiac Hospital and Research Institute from September 2021 to November 2023. Twelve patients with histologically confirmed glioblastoma underwent FS-guided resection. All participants received an intravenous dose of FS (5 mg/kg body weight) ~30 min before surgery. The study follows a quasi-experimental design, focusing on the outcomes of FS-aided surgery without a control group. Patients were selected based on specific inclusion and exclusion criteria, and all surgeries were performed by a single experienced neurosurgeon. The extent of tumor resection was classified as gross total resection (GTR), near-total resection (NTR), or partial resection (PR). Results: Gross total resection (GTR) was achieved in 66.6% of patients, near total resection (NTR) in 16.6%, and subtotal resection (STR) in 16.6%. No significant adverse effects were observed except for a single case of postoperative seizure, which was managed without long-term consequences. All patients showed normal liver and kidney function tests postoperatively. The low-dose FS protocol demonstrated both a high rate of GTR and a favorable safety profile, with only minor, transient side effects such as temporary yellow discoloration of the skin, sclera, and urine. No severe or long-term complications related to FS were observed during the follow-up period, which had a median duration of 13.4 months. Conclusion: FS appears to be a safe and effective aid in glioblastoma resection, achieving high rates of GTR with minimal side effects. The findings suggest that FS, particularly at a low dose, is a viable, cost-effective alternative to other fluorescent markers, especially in settings where resource constraints may limit the use of more expensive options like 5-ALA.

Phospholipid Nanoemulsion-Based Ocular Lubricant for the Treatment of Dry Eye Subtypes: A Multicenter and Prospective Study.

Dry eye disease (DED) is a multifactorial condition of the ocular surface (OS) characterized by loss of tear film homeostasis, ocular discomfort, and vision disturbances. Most available ocular lubricants target the aqueous deficiency of the tear, restoring only this layer, leaving the tear lipid stratum deficient, as occurs in most patients with evaporative DED. An innovative propylene glycol-hydroxypropyl guar enriched with a phospholipid nanoemulsion (PG-HPG-PH-N) is indicated to restore deficiencies in both the lipid and aqueous layers of the tear film, and its composition was designed to increase lubricant retention on the OS. The purpose of this study was to evaluate, through the Ocular Surface Disease Index (OSDI) and clinical assessment, the treatment of patients who had DED due to aqueous deficiency arising from mixed or evaporative DED subtypes with a PG-HPG-PH-N ocular lubricant at a reduced frequency of twice a day, in a prospective, multicenter, and single-arm study. Patients were screened from days - 7 to 0, and from day 1 (baseline and first day of treatment) to day 28 of treatment with this lubricant. After visit 1 (screening visit, days - 7 to 0), designed as pre-treatment OS assessment, patients returned to their research center on days 14 and 28 of treatment for a complete assessment, including anamnesis, the OSDI, corrected visual acuity, tear breakup time (TFBUT), OS staining evaluation, tolerability index, and environmental exposure questionnaire. Seventy patients were enrolled in this study (60 women, 10 men), with a mean age of 45 (range 27-64) years. TFBUT results showed an improvement in tear film stability as vital dyes sodium fluorescein and lysamine green showed a decrease in corneal staining after 14 and 28days of treatment. No significant adverse events were reported, demonstrating the good tolerability of the lubricant. The PG-HPG-PH-N nanoemulsion can be considered to be a safe and effective ocular lubricant for treating DED due to aqueous deficiency, both mixed and evaporative subtypes. Brazilian National Research Ethics Commission (ReBEC registration number 16055).

Characterizing the photoluminescence of fluorescein-labeled cellulose in aqueous and alcohol solutions: influence of the cellulose backbone

Although many dyes have been introduced into cellulose, whether bound to its backbone or within a cellulose matrix, few studies have determined whether the backbone statically or dynamically quenches the photoluminescence of the dye. To advance cellulosic fluorescent films, the influence of the cellulose backbone on photoluminescence must be understood. We determined the fluorescence properties of fluorescein isothiocyanate (FITC) and fluorescein-labeled cellulose (FLC) in water and alcohol, including their quantum yields \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\boldsymbol{\\phi}_{\ extit{\ extbf{PL}}}$$\\end{document}, lifetimes \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\\boldsymbol{\ au}}$$\\end{document}, and rates of radiative \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extit{\ extbf{k}}}_{\ extit{\ extbf{r}}}$$\\end{document} and nonradiative \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extit{\ extbf{k}}}_{\ extit{\ extbf{nr}}}$$\\end{document} decay. Dissolved FLC had a ~ 30× lower \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\boldsymbol{\\phi}_{\ extit{\ extbf{PL}}}$$\\end{document} than FITC, suggesting that incorporating FITC into the cellulose backbone remarkably reduces the fluorescence efficiency. The FLC solutions had a six-fold lower \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extit{\ extbf{k}}}_{\ extit{\ extbf{r}}}$$\\end{document} than their FITC counterparts but a 10–20 times higher \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extit{\ extbf{k}}}_{\ extit{\ extbf{nr}}}$$\\end{document}. Presumably, this was because the cellulose backbone interacted weakly with the fluorescein moieties, suggesting a quenching mechanism that can be termed quasi-static, corresponding to static quenching between the fluorescein moieties and cellulose backbone, in addition to the fluorescence quenching caused by the intramolecular nonradiative processes of fluorescein, as observed in conventional molecules. Using the Strickler‒Berg formula, we deduced the analytical radiative decay rate constants \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extit{\ extbf{k}}}_{\ extit{\ extbf{r}}}^{\ extit{\ extbf{S.B.}}}$$\\end{document} and eventually estimated the number of very short-lived fluorescein moieties per single fluorescent fluorescein moiety, corresponding well with static quenching.

Changes in the Tissue Barrier after Exposure to Lipopolysaccharide on the Apical Side of Enterocytes and the Follicle-Associated Epithelium in Peyer's Patches of the Rat Intestine.

To study the para- and transcellular permeability of columnar epithelium and follicle-associated epithelium of Peyer's patches in the rat intestine, LPS was applied from the mucosal side to simulate the action of endotoxins from gram-negative bacteria of gut microbiota. LPS did not affect transepithelial resistance or sodium fluorescein permeability, but increased the levels of claudin-3 and claudin-4 in enterocytes, suggesting strengthening of the paracellular intestinal barrier. Transcellular permeability was evaluated by electron microscopy based on the number of vesicular structures in the cytoplasm of different cell types. LPS increased the number of small vesicles in follicle-associated epithelium of Peyers' patches. In columnar epithelial cells, LPS reduced the number of smaller vesicles and increased the number of larger ones. LPS did not damage the tissue barrier, but enhanced transcytosis, which could potentiate the effects of endotoxin on its receptors in the intestinal mucosa.

Cathepsin B- and L-like Protease Activities Are Induced During Developmental Barley Leaf Senescence

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC50 values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC50 in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves.

Brain-to-blood transport of fluorescein in vitro

Investigating blood-brain barrier (BBB) dysfunction has become a pre-clinical and clinical research focus as it accompanies many neurological disorders. Nevertheless, knowledge of how diagnostic BBB tracers cross the endothelium from blood-to-brain or vice versa often remains incomplete. In particular, brain-to-blood transport (efflux) may reduce tracer extravasation of intravascularly (i.v.) applied tracers. Conversely, impaired efflux could mimic phenotypic extravasation. Both processes would affect conclusions on BBB properties primarily attributed to blood-to-brain leakage. Here, we specifically investigated efflux of fluorescent BBB tracers, focusing on the most common non-toxic marker, sodium fluorescein, which is applicable in patients. We used acute neocortical slices from mice and applied fluorescein, sulforhodamine-B, rhodamine-123, FITC dextran to the artificial cerebrospinal fluid. Anionic low molecular weight (MW) fluorescein and sulforhodamine-B, but not ~ 10-fold larger FITC-dextran and cationic low MW rhodamine-123, showed efflux into the lumen of blood vessels. Our data suggest that fluorescein efflux depends on organic anion transporter polypeptides (Oatp) rather than P-glycoprotein. Furthermore, sodium-potassium ATPase inhibition and incomplete oxygen-glucose deprivation (OGD, 20% O2) reduced fluorescein efflux, while complete OGD (0% O2) abolished efflux. We provide evidence for active efflux of fluorescein in vitro. Impaired efflux of fluorescein could thus contribute to the frequently observed BBB dysfunction in neuropathologies in addition to blood-to-brain leakage.

Moxifloxacin HCl -loaded Cellulose Acetate Butylate In Situ Forming Gel for Periodontitis Treatment.

Periodontitis presents significant treatment challenges due to its complexity and potential complications. In response, an in situ forming gel (ISG) loaded with moxifloxacin HCl (Mx) and cellulose acetate butyrate (CAB) was developed for targeted periodontitis therapy. Mx-loaded 10-45% CAB-based ISGs were developed, and their physicochemical properties such as rheology, viscosity, contact angle, gel morphology and gel formation, interface interaction were investigated. Moreover, the formulation performance studies including drug release and kinetics, in vitro degradation, and antimicrobial activities were also evaluated. The Mx-loaded ISGs containing 25-45% CAB demonstrated rapid matrix formation in both macroscopic and microscopic examinations and presented plastic deformation matrix. Tracking with sodium fluorescein and Nile red fluorescence probes indicated delayed solvent movement owing to CAB matrix formation. Adequate CAB content sustained Mx release for one week, following Peppas-Sahlin model and indicating a predominantly Fickian diffusion mechanism. Higher CAB content likely contributed to a denser matrix structure, leading to a slower in vitro degradation rate. Synchrotron radiation X-ray tomographic and SEM imaging provided insights into the CAB matrix structure and porous network formation. These ISG formulations effectively inhibited Staphylococcus aureus, Escherichia coli, Candida albicans, and Porphyromonas gingivalis. The Mx-loaded 40% CAB-based ISG shows promise as a dosage form for treating periodontitis. Further clinical trials are necessary to ensure the safety of this new ISG formulation, despite existing safety data for other medicinal uses of CAB. HIGHLIGHTS: Moxifloxacin HCl-loaded 10-45% cellulose acetate butyrate (CAB)-based in situ forming gels (ISG) were developed. They were evaluated for physicochemical properties, drug release, in vitro degradation, and antimicrobial activities. ISGs with 25-45% CAB showed swift matrix formation and plastic deformation Adequate CAB content sustained Mx release with Fickian diffusion mechanism They promise for periodontitis treatment because of effective inhibition of related pathogens.

Creation of 3D chitin/chitosan composite scaffold from naturally pre-structured verongiid sponge skeleton

This study represents the first creation and characterization of a 3D chitin/chitosan composite scaffold derived from the naturally pre-structured skeleton of the cultivated marine demosponge Aplysina aerophoba, aiming to preserve the intricate architecture of the unique tube-like chitin while incorporating chitosan layers. Advanced staining methods, including the use of iodine and Cibacron Brilliant Red (CBR), were employed to distinguish these polysaccharides. ATR-FTIR spectroscopy confirmed the system's structural integrity and identified the optimal chitin/chitosan balance, achieved after 60-minute treatment in 38 % NaOH at 95 °C. Fluorescent microscopy using fluorescein isothiocyanate (FITC) effectively confirmed the presence of chitosan layers in the created chitin/chitosan scaffolds. Scanning electron microscopy analysis further elucidated significant morphological distinctions, where chitin fibers displayed a smooth, uniform surface, contrasting with the ragged and irregular texture of chitosan-containing fibers, indicating significant surface modifications. Zeta potential measurements confirmed the partial transformation of chitin into chitosan. The dual-layer configuration, consisting of a resilient chitin core and a versatile chitosan exterior, not only provides structural support, but also enhances the scaffold's functionality for potential technological and biomedical applications. The preferential metallization of the chitosan phase by copper nanoparticles in the created 3D chitin/chitosan composite opens the way to the potential use of such scaffolds in catalysis.

A pH-responsive fluorescent film with the smartphone-assistance for real-time and visual detection of food freshness

Monitoring food freshness is considerably important for food safety. In this study, a smart pH-responsive fluorescence hydroxypropyl methyl cellulose-κ-carrageenan-fluorescein isothiocyanate-NH2-CaAl2O4 (H-K-F-N) film was prepared. Taking synergetic advantage of the pH-dependent behavior of fluorescein isothiocyanate dye and the luminescence characteristics of calcium aluminate phosphor, the film exhibited a unique strong pH-responsive fluorescence with an exceptional linear relationship (correlation coefficient, R2 = 0.9993) across a wide pH rang of 2.0–12.0. Moreover, the H-K-F-N film, as a smart sensor, could be used to estimate the total volatile basic nitrogen and total viable count through fluorescence intensity based on the partial least squares regression model and support vector machine regression, respectively. Leveraging the relationship between the fluorescent image's digital signals and food freshness indicators, a smartphone-assisted system was developed. These results demonstrated that H-K-F-N film is promising for applications in intelligent food packaging and food safety monitoring.

Efficacy of Administration Routes in Crayfish: Comparative Analysis of Intracoelomic and Intrapericardial Techniques Using Fluorescein Dye.

Crayfish are emerging as model organisms for various disciplines. Moreover, decapod crustaceans also exhibit pain-like reactions and heightened anxiety when exposed to harmful stimuli, leading to short-term or persistent behavioral shifts. Awareness of decapod crustacean sentience and thus, suffering calls for refinement of current laboratory protocols. This study aims to enhance the standard methodology for injecting substances into crayfish by minimizing stress-inducing manipulation. We examined the impacts of various administration routes on the persistence of injected chemicals in marbled crayfish, its excretion, and animal survival. Fluorescein dye was used as a visual marker. It was administered via three alternative injection routes-intracoelomic (IC), intrapericardial administration through areola (IP-A), and intrapericardial administration through arthrodial membrane (IP-AM). Continuous video observations were made for a 4-h period under UV light, followed by intermittent observations at 12-h intervals over 48 h. The highest mortality (20%) was observed in IP-A administration. The IP-A method also provided the fastest systemic distribution of the dye in the body. Results indicated visibly higher urination frequency in IP-AM compared to IP-A. IC mirrored IP-AM outcomes without any observed mortality. We conclude that IC administration proved superior to intrapericardial methods, offering the least harmful but effective approach for crayfish injections.

Microvascular Regeneration and Perfusion of Partially Decellularized Tracheal Grafts.

A critical barrier to successful tracheal transplantation is poor vascularization. Despite its importance, little is known about microvascular regeneration in tissue-engineered grafts. We have demonstrated that partially decellularized tracheal grafts (PDTG) support neotissue formation including new submucosal microvasculature (CD31+). However, the perfusion of this neovasculature is unknown. In this study, we used a mouse model of tracheal replacement to measure the microvascular regeneration and perfusion of PDTG. PDTG and syngeneic tracheal grafts (STG, surgical control) (n = 5 for each group) were orthotopically transplanted into C5BL/6 J mice. We quantified vascularity of STG and PDTG samples at 1 and 3 months with conventional histology (N = 3 ~ 10/group). At 1, 3, and 6 months, animals were injected with fluorescein isothiocyanate (FITC) tomato lectin into the left ventricle. After perfusion, tracheas were fixed, harvested, mounted, stained for CD31 expression, and imaged with resonant scanning confocal microscopy. Percent CD31+, FITC area was compared between groups and endpoints compared with native trachea. Microvascular intersections were quantified using Sholl analysis. Functional microvasculature was seen in both groups. Although percent vascularization (CD31) in PDTG was restored by 3 months, microvascular pattern in PDTG displayed a unique morphology compared with control. Surgery alone appeared to globally change microvascular pattern and perfusion. PDTG demonstrated equivalent perfusion to surgical control by 6 months. Sholl analysis revealed a reduction of microvessel intersectionality that persisted in PDTG and was not seen in surgical or native controls. PDTG exhibited microvascular regeneration. Perfusion was present in PDTG, improved, and persisted over long-term time points. NA Laryngoscope, 2024.

Novel ophthalmic hyaluronic acid-hydrogel with curcumin nanoparticles for enhanced healing of ulcerative keratitis in rabbit model

Corneal ulcers, whether melting or indolent, are common in humans and companion animals. Treatment involves local administration of antibiotic eye drops and corneal healing drugs. Compared to traditional treatments for ulcerative keratitis, herbal medicines offer unique advantages, such as potent anti-inflammatory effects and inhibition of proinflammatory cytokines. Curcumin, extracted from the Curcuma Longa plant, possesses extensive pharmacological properties, such as anti-inflammatory, anti-cancer, and antioxidant properties, and is used in various medicines. In this study, we developed a novel ophthalmic drop hydrogel using a formulation of Curcumin NPs encapsulated with β-cyclodextrin and hyaluronic acid, to accelerate corneal healing and improve the quality of healed structures. The formation of Curcumin NPs into Hyaluronic acid-based hydrogels was characterized by zeta, FTIR, and scanning electron microscope (SEM) analyses. A total of 25 healthy male New Zealand Albino rabbits were experimentally induced with ulcerative keratitis and treated individually with topical medication. Rabbits were divided into five groups. Fluorescein dye staining, corneal clarity score, Schirmer tear test, proinflammatory cytokine measurement, and pathologic factors assessments were used to evaluate the optimised Curcumin NPs with β-cyclodextrin in Hyaluronic acid hydrogel. Our results demonstrated that the optimized Curcumin NPs with β-cyclodextrin in hyaluronic acid hydrogel significantly reduced the frequency of medication administration compared to conventional therapies, enhancing the quality of healed structures and effectively treating ulcerative keratitis. All findings in this study provide new insight into designing and fabricating novel ophthalmic medicine for ulcerative keratitis for topical usage.

The penetration efficiency of a dissolved model drug into hair follicles depends on the concentration of added nanoparticles.

Hair follicles have recently emerged as promising drug delivery targets and gates for skin penetration. The so-called ratchet effect, which is based on an interaction between the hair shaft surface, the intrafollicular stratum corneum and nanoparticles, has proven to be very effective for the transport of active ingredients. Especially the nanoparticle-assisted decolonization of hair follicles constitutes an interesting new area of application. In a recently published work it was shown that small molecules as well as macromolecules solved in an outer phase of a formulation can be transported into the deeper parts of the hair follicles by adding nanoparticles to the formulation. In this case the nanoparticles constitute an entity independent of the drug and the transport is hypothesized to be based on an adhesion effect. In the present work, we focused on the impact of the particle concentration in the formulation on the transport efficiency of the model drug fluorescein sodium into hair follicles utilizing an ex vivo porcine skin model. It was observed that a particle concentration of 4% significantly enhances the transport efficiency of fluorescein as compared to 2% particle concentration. Doubling the concentration to 8% did not significantly increase the penetration depth. The effect evolved more efficiently when using 4Hz circular motion massage as compared to 100Hz oscillating massage. These results deliver interesting information on the optimal formulation as well as application parameters for a future application in clinical studies for e.g. skin antisepsis purposes.

Tyramine-assisted fluorescent labeling of polysaccharides: Characterization, and interfacial properties

Fluorescence labeling has been widely used in various fields, including fluorescent sensors, biochemistry, medical and chemical research. This study proposed an efficient strategy for the detection and microanalysis of polysaccharides. Soy hull polysaccharide (SHP) was successfully labeled with fluorescein isothiocyanate (FITC) via an amination reaction using tyramine as a linker. The labeled polysaccharide (FTSHP) was characterized by fluorescence, UV–visible, flourier transform infrared (FT-IR), 1H NMR, differential scanning calorimetry (DSC) and interfacial tension. The results indicated that the labeling efficiency of FTSHP with different concentration of FITC (0.20–0.35 wt%) was 1.51 %, 1.58 %, 1.63 %, and 1.67 %, respectively, with fluorescence intensity increasing as FITC concentration increased. Moreover, the interfacial adsorption capacity and thermal stability of FTSHP were significantly improved compared to SHP. This labeling method offers a promising approach for detection and analysis of polysaccharides, providing new insights into the study of other natural polysaccharides.

A confocal laser scanning microscopic analysis of efficacy of different activated irrigants in the removal of calcium hydroxide medicament and subsequent penetrability of Bio-C sealer – An in vitro study

Abstract Aim: The aim of the study was to investigate the efficacy of different irrigants activated by pro-agitator tip system (PATS) Vario on the removal of calcium hydroxide medicament and subsequent penetration depth of Bio-C sealer. Materials and Methods: Fifty single-rooted mandibular premolars were selected. Access cavities were prepared; biomechanical preparation was done. Metapex that had been combined with rhodamine B dye was used to fill each sample. All the samples were divided into five groups (n = 10) – Group I: chitosan–citrate, Group II: intracanal heated sodium hypochlorite (NaOCl), Group III: phytic acid, Group IV: SmearClear, and Group V: saline. All samples were obturated using gutta-percha and Bio-C sealer (combined with fluorescein dye). Later, all samples were sectioned at 3, 6, and 9 mm from the apex and observed under a confocal microscope for residual Ca(OH)2 and sealer penetration into dentinal tubules. Results: The saline group exhibited the least amount of sealer penetration and high residual Ca(OH)2, both of which were statistically significant (P ≤ 0.05). Conclusion: The use of PATS Vario for irrigant activation enhanced the calcium hydroxide removal efficacy and penetration of Bio-C sealer into dentinal tubules. The elimination of calcium hydroxide and sealer penetration, from the apical region of the tooth, can be accelerated by intracanal heating of NaOCl.

Boron Nitride‐Supported Fluorescein Isothiocyanate for Improving Optical Performance and Stability

AbstractOrganic luminogens play an indispensable role in optoelectronic devices, yet there is a pressing need to address their aggregation‐caused quenching (ACQ) and stability. Here we designed and prepared multilayered boron nitride nanosheets/fluorescein isothiocyanate (MBNNSs/FITC) composite phosphors with tunable photoluminescence by controlling the FITC content and annealing temperature. The research results indicate that MBNNSs/FITC with low or high loading of FITC is not conducive to enhancing the emission intensity of FITC, which can be attributed to the fact that low loading leads to a decrease in the content of fluorescent groups, while high loading promotes intermolecular aggregation of FITC dyes. Interestingly, the emission intensity of FITC in MBNNSs/FITC‐6 composite phosphors can be further enhanced by annealing treatment (200 °C), which can be ascribed to the suppression of the ACQ and effective energy transfer from the MBNNSs to the FITC. Moreover, the resultant composite phosphor displays outstanding thermal stability, remarkable photo stability and exceptional water‐resistance. This work provides a new design space for the development of non‐rare‐earth fluorescent materials using organic fluorescent dyes.

Evaluation of the safety and efficacy of skin penetration enhancer Putocrin®.

Substances that can efficiently enhance skin penetration while exerting no adverse effect are useful for drug and cosmetics formulation. To investigate the safety and enhance skin penetration efficacy of Putocrin®, a combination containing 2% isosorbide dimethyl ether, 1% pentanediol, and 0.5% inositol. An invitro keratinocyte cell assay using 3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT), and an invitro EpiKutis® skin study adopted hematoxylin and eosin staining, immunostaining, and liquid chromatography-mass spectrometry (LC-MS) analysis were carried out to investigate the safety of Putocrin®. A pigskin-Franz cell system experiment applied high-performance liquid chromatography (HPLC) to compare the skin penetration efficiency of fluorescein isothiocyanate (Fitc)-labeled tranexamic acid with or without the assistance of Putocrin®. The safety and efficacy of Putocrin® was further evaluated on zebrafish embryos. The MTT assay showed that Putocrin® at concentration ≤2.5% did not significantly affect cell viability. The invitro EpiKutis® skin study revealed that 2.5% Putocrin® did not affect skin morphology, filaggrin content, ceramide/protein, or fatty acid/protein ratios, but significantly increased loricrin content by 86.00% (p < 0.001). The pigskin-Franz cell penetration experiment demonstrated that Fitc-labeled tranexamic acid could barely penetrate the skin (with penetration rate of 1.121%), while Putocrin® significantly enhanced the penetration rate up to 83.983%, which was close to unlabeled tranexamic acid (90.013%). The zebrafish embryo study showed that 2.5% Putocrin® did not exert observable toxicity and obviously assisted the skin penetration of Fitc-labeled tranexamic acid into fish embryos. These results indicate the strong enhancing skin penetration potency of Putocrin®. This study demonstrated the safety as well as the strong enhancing skin penetration potency of Putocrin® for cosmetics formulation use.