The distribution and morphology of neuronal degeneration were observed and analyzed in each sector of the zona incerta in a lithium‐pilocarpine (LiCl) Wistar rat model of status epilepticus in 12, 15, 18, 21, and 25‐day‐old rats and survival intervals of 4, 8, 12, 24, and 48 hours. Status epilepticus was induced via intraperitoneal (IP) injection of LiCl (3 mmol/kg) 24 hours before an injection of pilocarpine (40 mg/kg, IP). Motor seizures were suppressed by paraldehyde (0.3‐0.6 ml/kg, IP) two hours after status epilepticus onset. Animals were anesthetized using urethane and perfused with phosphate‐buffered saline followed by 4% paraformaldehyde. Brains were sectioned and Nissl stained for map guidance, with fluoro‐Jade B fluorescence used to detect degenerated neurons. Fluoro‐jade B‐positive neurons were plotted to a standard stereotaxic atlas, their distribution was quantified, and their long‐axis diameter was measured. Fluoro‐jade B‐positive neurons were found in pups aged 15 days and older 24 hours after status epilepticus, in which their numbers increased, and their perikaryon size decreased with advancing age. Thus, neuronal damage severity was dependent on age and survival interval. Neuronal damage was only found in the rostral sector of the zona incerta, a region that exhibits a small number of inhibitory neurons and is reciprocally connected to the limbic cortex. This system of hyperactivity, coupled with inhibitory neurons, may be the underlying cause of the neuronal degeneration and explain why it was confined to the rostral sector of the zona incerta.
- MeSH
- Lithium Chloride toxicity MeSH
- Nerve Degeneration * pathology etiology MeSH
- Fluoresceins MeSH
- Rats MeSH
- Disease Models, Animal MeSH
- Neurons * pathology MeSH
- Animals, Newborn MeSH
- Pilocarpine toxicity MeSH
- Rats, Wistar MeSH
- Status Epilepticus * pathology chemically induced complications MeSH
- Age Factors MeSH
- Zona Incerta * pathology MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Úvod: Flexibilní endoskopické vyšetření polykání (FEES) je jednou ze základních metod k objektivní diagnostice poruch polykání. Principem je polykání bolusů různých konzistencí pod endoskopickou kontrolou. Z dostupných údajů i našich zkušeností vyplývá, že kvalita a výtěžnost vyšetření závisí na viditelnosti testovaného bolusu. Cíl práce: Ověřit, zda použití fluoresceinu zlepšuje citlivost FEES vyšetření v porovnání se standardním potravinářským barvivem. Metodika: Ve studii jsme u 40 pacientů provedli FEES s polknutím vody obarvené zeleným potravinářským barvivem a vody obarvené fluoresceinem. Hodnocena byla přítomnost predeglutivního leaku, stagnace bolusu v hltanu (valekulách, na stěnách hltanu, piriformních recesech), průnik bolusu do dýchacích cest na základě Penetračně-aspirační škály podle Rosenbeka (PAS), tendence k penetraci přes zadní komisuru a také subjektivní porovnání obou metod ve sledovaných parametrech. Výsledky: Výsledky prokazují statisticky významně vyšší záchyt stagnace bolusu na stěnách hltanu (p < 0,001) a ve valekulách (p = 0,038) v případě tekutiny obarvené fluoresceinem. Při hodnocení průniku bolusu do dýchacích cest dosáhla hodnota reliability (spolehlivosti) statistické významnosti (k = 0,438; p < 0,001) mezi testovanými metodami (zelené barvivo oproti fluoresceinu), což svědčí pro dobrou citlivost obou metod. Na bodové škále podle Rosenbeka (1–8 bodů) se ale metody statisticky významně lišily v hodnocení závažnosti penetrace/aspirace (p = 0,001). Při použití fluoresceinu byla detekována statisticky významně větší hloubka průniku do dýchacích cest (PAS 3,13) než při použití zeleného barviva (PAS 2,10) (p = 0,001). Při subjektivním porovnání obou metod vyšetřujícím lékařem je statisticky významně lepší viditelnost fluoresceinu ve všech hodnocených parametrech. Závěr: Studie prokázala lepší senzitivitu FEES při použití fluoresceinu v porovnání s běžně používaným potravinářským barvivem. Fluorescein se jeví jako vhodné barvivo k vyšetřování poruch polykání.
Background: Flexible endoscopic evaluation of swallowing (FEES) is one of the basic methods for objective diagnostics of swallowing disorders. The principle is to swallow boluses of various consistencies under endoscopic control. According to available data and our experience, the quality and accuracy of the examination depends on the visibility of the tested bolus. Aim of the study: Verify that the use of fluorescein improves the sensitivity of the FEES compared to a standard food colouring. Methods: In the study, we performed FEES on 40 patients using green food colouring and fluorescein dyed water. The presence of pre-deglutive leak, bolus stagnation in the pharynx (valleculas, pharyngeal walls, piriform sinuses), bolus penetration into the airways based on the Rosenbek Penetration-Aspiration Scale (PAS), tendency to penetrate through the posterior commissure and subjective comparison of both methods in parameters mentioned above were evaluated. Results: The results show a statistically significantly higher detection of bolus stagnation on the pharyngeal walls (P <0.001) and in the epiglottic valleculas (P = 0.038) with fluorescein-dyed water. When assessing airway bolus penetration, the reliability value reached statistical significance (k = 0.438; P <0.001) between the tested methods (green food colouring vs. fluorescein), which indicates good sensitivity of both methods. However, on the Rosenbek score scale (1–8 points), the methods differed statistically significantly in the assessment of penetration/aspiration severity (P = 0.001). A statistically significantly greater depth of airway penetration was detected with fluorescein (PAS 3.13) compared to green food colouring (PAS 2.10) (P = 0.001). In a subjective comparison of both methods by the examining physician, the visibility of fluorescein is statistically significantly better in all evaluated parameters. Conclusions: The study showed a better sensitivity of FEES when using fluorescein compared to conventional food colouring. Fluorescein appears to be a good colouring for diagnostics of swallowing disorders.
- MeSH
- Staining and Labeling methods MeSH
- Diagnostic Imaging methods MeSH
- Adult MeSH
- Endoscopy classification methods MeSH
- Fluorescein * pharmacology metabolism MeSH
- Humans MeSH
- Oropharynx diagnostic imaging pathology MeSH
- Deglutition Disorders diagnostic imaging diagnosis etiology MeSH
- Prospective Studies MeSH
- Pyriform Sinus diagnostic imaging pathology MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Fluorescein, eosin Y, and rose bengal are dyes used in clinical medicine and considered (photo-)chemically stable. Upon extensive irradiation with visible light in aqueous solutions, we found that these compounds release carbon monoxide (CO) - a bioactive gasotransmitter - in 40-100% yields along with the production of low-mass secondary photoproducts, such as phthalic and formic acids, in a multistep degradation process. Such photochemistry should be considered in applications of these dyes, and they could also be utilized as visible-light activatable CO-releasing molecules (photoCORMs) with biological implications.
- MeSH
- Adult MeSH
- Fluorescein MeSH
- Contact Lenses * MeSH
- Humans MeSH
- Myopia * therapy MeSH
- Tomography, Optical methods MeSH
- Corneal Topography methods instrumentation MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
The diagnosis of solid tumors of epithelial origin (carcinomas) represents a major part of the workload in clinical histopathology. Carcinomas consist of malignant epithelial cells arranged in more or less cohesive clusters of variable size and shape, together with stromal cells, extracellular matrix, and blood vessels. Distinguishing stroma from epithelium is a critical component of artificial intelligence (AI) methods developed to detect and analyze carcinomas. In this paper, we propose a novel automated workflow that enables large-scale guidance of AI methods to identify the epithelial component. The workflow is based on re-staining existing hematoxylin and eosin (H&E) formalin-fixed paraffin-embedded sections by immunohistochemistry for cytokeratins, cytoskeletal components specific to epithelial cells. Compared to existing methods, clinically available H&E sections are reused and no additional material, such as consecutive slides, is needed. We developed a simple and reliable method for automatic alignment to generate masks denoting cytokeratin-rich regions, using cell nuclei positions that are visible in both the original and the re-stained slide. The registration method has been compared to state-of-the-art methods for alignment of consecutive slides and shows that, despite being simpler, it provides similar accuracy and is more robust. We also demonstrate how the automatically generated masks can be used to train modern AI image segmentation based on U-Net, resulting in reliable detection of epithelial regions in previously unseen H&E slides. Through training on real-world material available in clinical laboratories, this approach therefore has widespread applications toward achieving AI-assisted tumor assessment directly from scanned H&E sections. In addition, the re-staining method will facilitate additional automated quantitative studies of tumor cell and stromal cell phenotypes.
The preocular tear film is critically important for maintaining healthy ocular surface. In lagophthalmos, increased evaporation and tear film instability can occur. The level of tear matrix metalloproteinase 9 (MMP-9) is considered as a possible marker of ocular surface damage and inflammation. The aim of this study was to evaluate the possible usefulness of measuring tear film levels of MMP-9 in patients with lagophthalmos. Sixteen adult patients with unilateral lagophthalmos due to cerebellopontine angle mass surgery were included. Basic clinical examination including tear film osmolarity, degree of lagophthalmos, ocular surface sensitivity testing, corneal fluorescein staining, and tear break-up time (TBUT) were performed. Furthermore, tear MMP-9 quantification was performed and the values from lagophthalmic and contralateral healthy eye were compared. Possible correlations between tear MMP-9 levels and other parameters were analyzed. The Oxford score was higher in lagophthalmic eyes in comparison to healthy eyes. TBUT and corneal sensitivity were lower in lagophthalmic eyes. There was no difference in osmolarity between the two groups. Tear MMP-9 values were higher in lagophthalmic eyes. A higher MMP-9 value was associated with an increase in ocular surface fluorescein staining and a decrease of TBUT in lagophthalmic eyes. Tear MMP-9 may be used for monitoring ocular surface damage, contribute to early detection of inflammation progression and facilitate treatment adjustments.
- MeSH
- Adult MeSH
- Fluorescein MeSH
- Humans MeSH
- Matrix Metalloproteinase 9 MeSH
- Eyelid Diseases * MeSH
- Osmolar Concentration MeSH
- Eye Injuries * MeSH
- Tears MeSH
- Dry Eye Syndromes * diagnosis MeSH
- Inflammation MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Fluorescein is a fluorescent dye used as a diagnostic tool in various fields of medicine. Although fluorescein itself possesses low toxicity, after photoactivation, it releases potentially toxic molecules, such as singlet oxygen (1O2) and, as we demonstrate in this work, also carbon monoxide (CO). As both of these molecules can affect physiological processes, the main aim of this study was to explore the potential biological impacts of fluorescein photochemistry. In our in vitro study in a human hepatoblastoma HepG2 cell line, we explored the possible effects on cell viability, cellular energy metabolism, and the cell cycle. We observed markedly lowered cell viability (≈30%, 75-2400 μM) upon irradiation of intracellular fluorescein and proved that this decrease in viability was dependent on the cellular oxygen concentration. We also detected a significantly decreased concentration of Krebs cycle metabolites (lactate and citrate < 30%; 2-hydroxyglutarate and 2-oxoglutarate < 10%) as well as cell cycle arrest (decrease in the G2 phase of 18%). These observations suggest that this photochemical reaction could have important biological consequences and may account for some adverse reactions observed in fluorescein-treated patients. Additionally, the biological activities of both 1O2 and CO might have considerable therapeutic potential, particularly in the treatment of cancer.
- MeSH
- Angiography MeSH
- Hep G2 Cells MeSH
- Citric Acid Cycle drug effects radiation effects MeSH
- Fluorescein chemistry pharmacology MeSH
- Photochemical Processes MeSH
- Cell Cycle Checkpoints drug effects radiation effects MeSH
- Humans MeSH
- Carbon Monoxide analysis MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Antineoplastic Agents chemistry pharmacology MeSH
- Singlet Oxygen analysis MeSH
- Light MeSH
- Cell Survival drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Rose bengal and erythrosin B are xanthene dyes mainly known and used as antimicrobial agents, but due to their photodynamic activity they are also potential photosensitizers for cancer photodynamic therapy. The aim of this work is to study a photodynamic efficacy of rose bengal and erythrosin B against human skin melanoma and mouse fibroblast cell lines, compare them with each other and find out their photodynamic properties induced by light emitting diodes with total light dose of 5 J/cm2. To fully identify and understand photodynamic properties of both potentially effective photo-sensitizers, a set of complex in vitro tests such as cell cytotoxic assay, measurement of reactive oxygen species production, mitochondrial membrane potential change assay, mode of cell death determination or comet assay were made. Although both photosensitizers proved to have similar properties such as increasing production of reactive oxygen species with the higher concentration, predominance of necrotic mode of death or genotoxicity, the more effective photosensitizer was rose bengal because its EC50 was over 20 times lower for both cell lines than in case of erythrosine B.
- MeSH
- Rose Bengal therapeutic use MeSH
- Erythrosine MeSH
- Cancer-Associated Fibroblasts drug effects radiation effects MeSH
- Photochemotherapy * methods MeSH
- Humans MeSH
- Melanoma therapy MeSH
- Disease Models, Animal MeSH
- Mice MeSH
- Neoplasms therapy MeSH
- Reactive Oxygen Species MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.
- MeSH
- Eosine Yellowish-(YS) chemistry MeSH
- Hematoxylin chemistry MeSH
- Hormones genetics metabolism MeSH
- In Vitro Oocyte Maturation Techniques * MeSH
- Cells, Cultured MeSH
- Lipids analysis MeSH
- Oocytes growth & development metabolism MeSH
- Oxazines chemistry MeSH
- Swine MeSH
- Signal Transduction MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.
- MeSH
- Alkaline Phosphatase chemistry MeSH
- Biosensing Techniques methods MeSH
- DNA Probes chemistry genetics MeSH
- DNA analysis chemistry genetics MeSH
- Electrochemical Techniques methods MeSH
- Fluoresceins chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- Glucuronidase genetics MeSH
- Nucleic Acid Hybridization MeSH
- Immobilized Nucleic Acids chemistry genetics MeSH
- Humans MeSH
- Limit of Detection MeSH
- Naphthalenes chemistry MeSH
- Organophosphorus Compounds chemistry MeSH
- Sulfhydryl Compounds chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
