ABSTRACT
Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.
Subject(s)
Allergens , Brain Stem , Bronchial Hyperreactivity , Dopamine beta-Hydroxylase , Lung , Neurons , Animals , Female , Male , Mice , Allergens/immunology , Asthma/immunology , Asthma/physiopathology , Brain Stem/cytology , Brain Stem/physiology , Bronchial Hyperreactivity/drug therapy , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/physiopathology , Interleukin-4/immunology , Lung/drug effects , Lung/immunology , Lung/innervation , Lung/physiopathology , Mast Cells/immunology , Neurons/enzymology , Neurons/physiology , Norepinephrine/antagonists & inhibitors , Norepinephrine/metabolism , Solitary Nucleus/cytology , Solitary Nucleus/physiology , Vagus Nerve/cytology , Vagus Nerve/physiology , Medulla Oblongata/cytology , Medulla Oblongata/drug effects , Ganglia, Autonomic/cytology , Dopamine beta-Hydroxylase/metabolismABSTRACT
Chronic exposure of the lung to irritants such as allergen is a primary cause of asthma characterized by exaggerated airway constriction, also called hyperreactivity, which can be life-threatening. Aside from immune cells, vagal sensory neurons are important for airway hyperreactivity 1-4 . However, the identity and signature of the downstream nodes of this adaptive circuit remains poorly understood. Here we show that a single population of Dbh + neurons in the nucleus of the solitary tract (nTS) of the brainstem, and downstream neurons in the nucleus ambiguous (NA), are both necessary and sufficient for chronic allergen-induced airway hyperreactivity. We found that repeated exposures of mice to inhaled allergen activates nTS neurons in a mast cell-, interleukin 4 (IL-4)-and vagal nerve-dependent manner. Single-nucleus RNA-seq of the nTS at baseline and following allergen challenges reveals that a Dbh + population is preferentially activated. Ablation or chemogenetic inactivation of Dbh + nTS neurons blunted, while chemogenetic activation promoted hyperreactivity. Viral tracing indicates that Dbh + nTS neurons, capable of producing norepinephrine, project to the NA, and NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that then directly drive airway constriction. Focusing on transmitters, delivery of norepinephrine antagonists to the NA blunted allergen-induced hyperreactivity. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. The knowledge opens the possibility of targeted neural modulation as an approach to control refractory allergen-induced airway constriction.
ABSTRACT
Real-time and accurate awareness of the grain situation proves beneficial for making targeted and dynamic adjustments to cleaning parameters and strategies, leading to efficient and effective removal of impurities with minimal losses. In this study, harvested maize was employed as the raw material, and a specialized object detection network focused on impurity-containing maize images was developed to determine the types and distribution of impurities during the cleaning operations. On the basis of the classic contribution Faster Region Convolutional Neural Network, EfficientNetB7 was introduced as the backbone of the feature learning network and a cross-stage feature integration mechanism was embedded to obtain the global features that contained multi-scale mappings. The spatial information and semantic descriptions of feature matrices from different hierarchies could be fused through continuous convolution and upsampling operations. At the same time, taking into account the geometric properties of the objects to be detected and combining the images' resolution, the adaptive region proposal network (ARPN) was designed and utilized to generate candidate boxes with appropriate sizes for the detectors, which was beneficial to the capture and localization of tiny objects. The effectiveness of the proposed tiny object detection model and each improved component were validated through ablation experiments on the constructed RGB impurity-containing image datasets.
ABSTRACT
Further investigation of material properties during the extrusion process is essential to achieve precise control of the quality of the extrudate. Whole oat flour was used to produce low moisture puffed samples by a twin-screw extruder. X-ray diffraction (XRD), Scanning electron microscopy (SEM), infrared spectroscopy (FTIR), thermal analysis, and rheological experiments were used to deeply characterize changes in the structure and cross-linking of oats in different extrusion zones. Results indicated that the melting region was the main region that changed oat starch, including the major transformation of oat starch crystal morphology and the significant decrease of enthalpy representing the starch pasting peak in the differential scanning calorimeter (DSC) pattern (p < 0.05). Moreover, the unstable structure of the protein increased in the barrel and then decreased significantly (p < 0.05) after being extruded through the die head. The viscosity of oats increased in the cooking zone but decreased after the melting zone. A transformation occurred from elastic-dominant behavior to viscoelastic-dominant behavior for oats in the melting zone and after being extruded. This study provides further theoretical support for the research of the change of materials during extrusion and the development of oat-based food.
ABSTRACT
Camalexin, an indolic antimicrobial metabolite, is the major phytoalexin in Arabidopsis thaliana, and plays a crucial role in pathogen resistance. Our previous studies revealed that the Arabidopsis mitogen-activated protein kinases MPK3 and MPK6 positively regulate pathogen-induced camalexin biosynthesis via phosphoactivating the transcription factor WRKY33. Here, we report that the ethylene and jasmonate (JA) pathways act synergistically with the MPK3/MPK6-WRKY33 module at multiple levels to induce camalexin biosynthesis in Arabidopsis upon pathogen infection. The ETHYLENE RESPONSE FACTOR1 (ERF1) transcription factor integrates the ethylene and JA pathways to induce camalexin biosynthesis via directly upregulating camalexin biosynthetic genes. ERF1 also interacts with and depends on WRKY33 to upregulate camalexin biosynthetic genes, indicating that ERF1 and WRKY33 form transcriptional complexes to cooperatively activate camalexin biosynthetic genes, thereby mediating the synergy of ethylene/JA and MPK3/MPK6 signaling pathways to induce camalexin biosynthesis. Moreover, as an integrator of the ethylene and JA pathways, ERF1 also acts as a substrate of MPK3/MPK6, which phosphorylate ERF1 to increase its transactivation activity and therefore further cooperate with the ethylene/JA pathways to induce camalexin biosynthesis. Taken together, our data reveal the multilayered synergistic regulation of camalexin biosynthesis by ethylene, JA, and MPK3/MPK6 signaling pathways via ERF1 and WRKY33 transcription factors in Arabidopsis.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cyclopentanes , Ethylenes/metabolism , Gene Expression Regulation, Plant , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Oxylipins , Sesquiterpenes , Transcription Factors/genetics , Transcription Factors/metabolism , PhytoalexinsABSTRACT
Liquid organic electrolytes commonly employed in commercial Li-ion batteries suffer from safety issues such as flammability and explosions. Replacing liquid electrolytes with nonflammable electrolytes has become increasingly attractive in the development of safe, high-energy Li-metal batteries (LMBs). In this work, nonflammable, robust, and flexible composite polymer-polymer electrolytes (PPEs) were successfully fabricated by flame-retardant solution casting with polyimide (PI) and polyvinylidene fluoride (PVDF). The optimized nonflammable PPEs (e.g., PPE-50) demonstrate not only good mechanical properties (i.e., a high tensile strength of 29.6 MPa with an elongation at break of 87.2%), but also high Li salts dissolubility, the former of which ensures the suppression of Li dendrites, while the latter further improves the ionic conductivity (â¼1.86 × 10-4 S cm-1 at 30 °C). The resulting symmetric cells (Li|PPE-50|Li) offer excellent Li stripping and plating stability for 1000 h at 0.5 mA cm-2/0.25 mAh cm-2 and 600 h at 2.0 mA cm-2/1.0 mAh cm-2. In addition, the LiFePO4|PPE-50|Li half cells show high cycling performance (e.g., a reversible discharge capacity of 135.9 mAh g-1 after 300 cycles at 1C) and rate capability (e.g., 117.2 mAh g-1 at 4C). The PPE-50 is also compatible with a high-voltage cathode (e.g., LiNi0.5Mn0.3Co0.2O2), and the resulting batteries demonstrate long-term cycling stability with a high cut-off voltage of 4.5 V vs. Li/Li+. Because of the incorporation of a mechanically robust and thermally stable PI, a polar PVDF, and flame-retardant trimethyl phosphate (TMP) within PPEs, as well as the coordination between Li salts and TMP, and the interaction between Li salts and polymers (especially between Li bis(oxalato)borate) and PI, as well as the bis(oxalato)borate anion and PI), PPEs show great potential for practical and high-energy LMBs without safety concerns.
ABSTRACT
The human lung plays vital roles in respiration, host defense, and basic physiology. Recent technological advancements such as single-cell RNA sequencing and genetic lineage tracing have revealed novel cell types and enriched functional properties of existing cell types in lung. The time has come to take a new census. Initiated by members of the NHLBI-funded LungMAP Consortium and aided by experts in the lung biology community, we synthesized current data into a comprehensive and practical cellular census of the lung. Identities of cell types in the normal lung are captured in individual cell cards with delineation of function, markers, developmental lineages, heterogeneity, regenerative potential, disease links, and key experimental tools. This publication will serve as the starting point of a live, up-to-date guide for lung research at https://www.lungmap.net/cell-cards/. We hope that Lung CellCards will promote the community-wide effort to establish, maintain, and restore respiratory health.
Subject(s)
Lung/cytology , Lung/physiology , Cell Differentiation/genetics , Databases as Topic , Humans , Lung/metabolism , Regeneration/genetics , Single-Cell Analysis/methodsABSTRACT
Photodynamic therapy (PDT) is a non-invasive treatment method for tumors by exciting photosensitizers (PS) upon light irradiation to generate cytotoxic reactive oxygen species (ROS). However, the low oxygen concentration near the tumor tissue limits the therapeutic effect of PDT. Herein, we synthesized six chlorin e6 derivatives containing NO-donors to enhance their antitumor activity by synergistic effect of ROS and NO. The results revealed that the new NO-donor containing photosensitizers (PS-NO) exhibited more potent photodynamic activity than chlorin e6, and the introduction of NO donor moieties to chlorin e6 increased the level of NO and ROS in cells. The addition of Ferrostatin-1, a ferroptosis inhibitor, markedly reduced the photodynamic activity of PS-NO as well as the level of NO and ROS in cells. Mechanism studies further showed that PS-NO could reduce intracellular GSH level, inhibit GPX4 activity and promote malondialdehyde (MDA) accumulation upon light irradiation, which suggested the ferroptosis mechanism underlying the PDT effect of PS-NO.
Subject(s)
Cyclohexylamines/pharmacology , Phenylenediamines/pharmacology , Photosensitizing Agents/pharmacology , Cyclohexylamines/chemical synthesis , Cyclohexylamines/chemistry , Dose-Response Relationship, Drug , Ferroptosis/drug effects , HeLa Cells , Humans , Molecular Structure , Nitric Oxide/analysis , Nitric Oxide/metabolism , Phenylenediamines/chemical synthesis , Phenylenediamines/chemistry , Photosensitizing Agents/chemical synthesis , Photosensitizing Agents/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type-specific enhancers remain poorly understood. Here, we show that the transcriptional "master regulator" ATOH1-which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis-is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Hair Cells, Auditory/metabolism , Homeodomain Proteins/metabolism , Mechanoreceptors/metabolism , Transcription Factor Brn-3C/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation , Cochlea/metabolism , Enhancer Elements, Genetic , Epigenesis, Genetic , Hair Cells, Auditory/cytology , Homeodomain Proteins/genetics , Humans , Merkel Cells/metabolism , Mice , Transcription Factor Brn-3C/geneticsABSTRACT
Adult mammalian tissues such as heart, brain, retina, and the sensory structures of the inner ear do not effectively regenerate, although a latent capacity for regeneration exists at embryonic and perinatal times. We explored the epigenetic basis for this latent regenerative potential in the mouse inner ear and its rapid loss during maturation. In perinatal supporting cells, whose fate is maintained by Notch-mediated lateral inhibition, the hair cell enhancer network is epigenetically primed (H3K4me1) but silenced (active H3K27 de-acetylation and trimethylation). Blocking Notch signaling during the perinatal period of plasticity rapidly eliminates epigenetic silencing and allows supporting cells to transdifferentiate into hair cells. Importantly, H3K4me1 priming of the hair cell enhancers in supporting cells is removed during the first post-natal week, coinciding with the loss of transdifferentiation potential. We hypothesize that enhancer decommissioning during cochlear maturation contributes to the failure of hair cell regeneration in the mature organ of Corti.
Subject(s)
Cell Differentiation/physiology , Hair Cells, Auditory/metabolism , Receptors, Notch/metabolism , Regeneration/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Transdifferentiation/genetics , Cell Transdifferentiation/physiology , Epigenesis, Genetic/genetics , Epigenesis, Genetic/physiology , Hair Cells, Auditory/cytology , Mice, Transgenic , Regulatory Sequences, Nucleic Acid/geneticsABSTRACT
The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 and other factors gain access to cartilage-specific cis-regulatory regions during skeletal development was unknown. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. These results show that Foxc1 promotes chondrogenesis in the face by establishing chromatin accessibility at a number of cartilage-associated gene enhancers.
Animals with backbones (or vertebrates) have body shape determined, in part, by their skeletons. These emerge in the embryo in the form of cartilage structures that will then get replaced by bone during development. The neural crest is a group of embryonic cells that can become different tissues. In the head, it forms the cartilage scaffold for some of the facial bones and the base of the skull. During this process, a protein called Sox9 is required for neural crest cells to morph into cartilage. This transcription factor binds to regulatory sequences in the genome to turn cartilage genes on. But Sox9 is also required to form non-cartilage tissues in organs such as the liver, lung, and kidneys. How, then, does Sox9 only turn on the genes required for cartilage formation in the embryonic face? This specificity can be controlled by which regulatory sequences Sox9 can physically access in a cell: controlling which regulatory sequences Sox9 can access determines which genes it can activate, and which type of tissue a cell will become. Xu, Yu et al. wanted to understand exactly how Sox9 switches on the genes that turn neural crest cells into facial cartilage. They studied the genomes of zebrafish embryos, which have a cartilaginous skeleton similar to other vertebrates, and found out which areas were accessible to transcription factors in the neural crest cells that became facial cartilage. Analyzing these regions suggested that sites where Sox9 could bind were often close to binding sites for another protein, called Foxc1. When zebrafish embryos were genetically modified to inactivate Foxc1 proteins, many of the regulatory sequences in cartilage failed to become accessible, and the cartilaginous skeleton did not form properly. These results support a model in which Foxc1 opens up the genomic regions that Sox9 needs to bind for cartilage to form, as opposed to the regions that Sox9 would bind to make different organ cell types. The findings of Xu, Yu et al. uncover the stepwise process by which cartilage cells are made during development. Further research based on these results could allow scientists to develop new ways of replacing cartilage in degenerative conditions such as arthritis.
Subject(s)
Chondrogenesis , Forkhead Transcription Factors/genetics , Skull/embryology , Zebrafish Proteins/genetics , Zebrafish/embryology , Animals , Cartilage/embryology , Cell Differentiation , Chondrocytes/metabolism , Embryo, Nonmammalian/embryology , Forkhead Transcription Factors/metabolism , Neural Crest/embryology , Zebrafish Proteins/metabolismABSTRACT
Pulmonary neuroendocrine cells (PNECs) are rare airway epithelial cells that also uniquely harbor neuronal and endocrine characteristics. In vitro data indicate that these cells respond to chemical or mechanical stimuli by releasing neuropeptides and neurotransmitters, implicating them as airway sensors. Emerging in vivo data corroborate this role and demonstrate that PNECs are important for lung response to signals, such as allergens. With close proximity to steady-state immune cells and innervating nerves, PNECs, as prototype tissue-resident neuroendocrine cells, are at the center of a neuro-immune module that enables the fundamental ability of an organ to sense and respond to the environment.
Subject(s)
Epithelial Cells/metabolism , Epithelium/metabolism , Lung/metabolism , Neuroendocrine Cells/metabolism , Animals , Epithelial Cells/immunology , Epithelium/immunology , Humans , Lung/immunology , Neuroendocrine Cells/immunology , Neuropeptides/metabolismABSTRACT
Whereas the gill chambers of jawless vertebrates open directly into the environment, jawed vertebrates evolved skeletal appendages that drive oxygenated water unidirectionally over the gills. A major anatomical difference between the two jawed vertebrate lineages is the presence of a single large gill cover in bony fishes versus separate covers for each gill chamber in cartilaginous fishes. Here, we find that these divergent patterns correlate with the pharyngeal arch expression of Pou3f3 orthologs. We identify a deeply conserved Pou3f3 arch enhancer present in humans through sharks but undetectable in jawless fish. Minor differences between the bony and cartilaginous fish enhancers account for their restricted versus pan-arch expression patterns. In zebrafish, mutation of Pou3f3 or the conserved enhancer disrupts gill cover formation, whereas ectopic pan-arch Pou3f3b expression generates ectopic skeletal elements resembling the multimeric covers of cartilaginous fishes. Emergence of this Pou3f3 arch enhancer >430 Mya and subsequent modifications may thus have contributed to the acquisition and diversification of gill covers and respiratory strategies during gnathostome evolution.
Subject(s)
Enhancer Elements, Genetic , Evolution, Molecular , Gills/growth & development , POU Domain Factors/genetics , Vertebrates/genetics , Animals , Fishes/classification , Fishes/genetics , Fishes/growth & development , Mutation , Phylogeny , Sharks/classification , Sharks/genetics , Sharks/growth & development , Vertebrates/classification , Vertebrates/growth & developmentABSTRACT
The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.
Worldwide, hearing loss is the most common loss of sensation. Most cases of hearing loss are due to the death of specialized hair cells found deep inside the ear. These hair cells convert sounds into nerve impulses which can be understood by the brain. Hair cells naturally degrade as part of aging and can be damaged by other factors including loud noises, and otherwise therapeutic drugs, such as those used in chemotherapy for cancer. In humans and other mammals, once hair cells are lost they cannot be replaced. Hair cells have often been studied using mice, but the small number of hair cells in their ears, and their location deep inside the skull, makes it particularly difficult to study them in this way. Scientists are seeking ways to grow hair cells in the laboratory to make it easier to understand how they work and the factors that contribute to their damage and loss. Different cell types in the body are formed in response to specific combinations of biological signals. Currently, scientists do not have an efficient way to grow hair cells in the laboratory, because the correct signals needed to create them are not known. Menendez et al. have now identified four proteins which, when activated, convert fibroblasts, a common type of cell, into hair cells similar to those in the ear. These proteins are called Six1, Atoh1, Pou4f3 and Gfi1. Menendez et al. termed the resulting cells induced hair cells, or iHCs for short, and analyzed these cells to identify those characteristics that are similar to normal hair cells, as well as their differences. Importantly, the iHCs were found to be damaged by the same chemicals that specifically harm normal hair cells, suggesting they are useful test subjects. The ability to create hair cells in the laboratory using more easily available cells has many uses. These cells can help to understand the normal function of hair cells and how they become damaged. They can also be used to test new drugs to assess their success in preventing or reversing hearing loss. These findings may also lead to genetic solutions to curing hearing loss.
Subject(s)
Cell Lineage , Fibroblasts/physiology , Hair Cells, Auditory, Inner/physiology , Labyrinth Supporting Cells/physiology , Mice/physiology , Animals , Mice, Transgenic , Tail , Transcription Factors/metabolismABSTRACT
The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Epigenesis, Genetic , Gene Expression Regulation , Hair Cells, Auditory/metabolism , Regeneration/physiology , Saccule and Utricle/metabolism , Transcriptome , Animals , Cell Cycle , Cell Death , Cochlea , Female , Male , Mice , Transcription Factors , Transduction, GeneticABSTRACT
Lidocaine has been reported to induce apoptosis on rabbit corneal endothelial cells. However, the apoptotic effect and exact mechanism involved in cytotoxicity of lidocaine are not well-established in human corneal endothelial (HCE) cells. In this study, we investigated the apoptosis-inducing effect of lidocaine on HCE cells in vitro. After HCE cells were treated with lidocaine at concentrations of 0.15625-10.0 g/l, the morphology and ultrastructure of the cells were observed by inverted light microscope and transmission electron microscope (TEM). Cell viability was measured by MTT assay, and the apoptotic ratio was evaluated with flow cytometry and fluorescent microscopic counting after FITC-Annexin V/PI and AO/EB staining. DNA fragmentation was detected by electrophoresis, and the activation of caspases was evaluated by ELISA. In addition, changes in mitochondrial membrane potential were determined by JC-1 staining. Results suggest that lidocaine above 1.25 g/l reduced cellular viability and triggered apoptosis in HCE cells in a time- and dose-dependent manner. Diminishment of ΔΨm and the activation of caspases indicate that lidocaine-induced apoptosis was caspase dependent and may be related to mitochondrial pathway.
Subject(s)
Cornea/drug effects , Endothelial Cells/drug effects , Lidocaine/toxicity , Apoptosis/drug effects , Benzimidazoles/metabolism , Carbocyanines/metabolism , Cell Survival/drug effects , Cells, Cultured , Cornea/cytology , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , Microscopy, Electron, Transmission , Mitochondria/drug effectsABSTRACT
PURPOSE: To evaluate the performance of reconstructed tissue-engineered human corneal endothelium (TE-HCE) by corneal transplantation in cat models. METHODS: TE-HCE reconstruction was performed by culturing 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled monoclonal HCE cells on denuded amniotic membranes (dAMs) in 20% fetal bovine serum-containing Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (1:1) medium and 5% CO(2) at 37 ° C on a 24-well culture plate. The reconstructed TE-HCE was transplanted into cat corneas via lamellar keratoplasty with all of the endothelium and part of Descemet's membrane stripped. Postsurgical corneas were monitored daily with their histological properties examined during a period of 104 days after transplantation. RESULTS: The reconstructed TE-HCE at a density of 3,413.33 ± 111.23 cells/mm(2) in average established intense cell-cell and cell-dAM junctions. After lamellar keratoplasty surgery, no obvious edema was found in TE-HCE-transplanted cat corneas, which were transparent throughout the monitoring period. In contrast, intense corneal edema developed in dAM-transplanted cat corneas, which were turbid. The corneal thickness gradually decreased to 751.33 ± 11.37 µm on day 104 after TE-HCE transplantation, while that of dAM eye was over 1,000 µm in thickness during the monitoring period. A monolayer of endothelium consisting of TE-HCE-originated cells at a density of 2,573.33 ± 0.59 cells/mm(2) attached tightly to the surface of remnant Descemet's membrane over 104 days; this was similar to the normal eye control in cell density. CONCLUSIONS: The reconstructed TE-HCE was able to function as a corneal endothelium equivalent and restore corneal function in cat models.
Subject(s)
Endothelium, Corneal/transplantation , Tissue Engineering/methods , Animals , Cats , Cattle , Cells, Cultured , Endothelium, Corneal/cytology , Endothelium, Corneal/physiology , Humans , Male , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Models, Animal , Tissue Scaffolds , Transplantation, HeterologousABSTRACT
AIM: To demonstrate the apoptosis-inducing effect of lidocaine on human corneal stromal (HCS) cells in vitro, and provide experimental basis for safety anesthetic usage in clinic of ophthalmology. METHODS: In vitro cultured HCS cells were treated with lidocaine at different doses and times, and their morphology was monitored successively with inverted phase contrast microscopy. The membrane permeability of them was detected by acridine orange/ethidium bromide (AO/EB) double staining. The DNA fragmentation of them was examined by agarose gel electrophoresis, and their ultrastructure was observed by transmission electron microscopy (TEM), respectively. RESULTS: Exposure to lidocaine at doses from 0.3125g/L to 20g/L induced morphological changes of HCS cells such as cytoplasmic vacuolation, cellular shrinkage, and turning round, and elevated membrane permeability of these cells in AO/EB staining. The change of morphology and membrane permeability was dose- and time-dependent, while lidocaine at dose below 0.15625g/L could not induce these changes. Furthermore, lidocaine induced DNA fragmentation and ultrastructural changes such as cytoplasmic vacuolation, structural disorganization, chromatin condensation, and apoptotic body appearance of the cells. CONCLUSION: Lidocaine has significant cytotoxicity on human corneal stromal cells in vitro in a dose- and time-dependent manner by inducing apoptosis of these cells. The established experimental model and findings based on this model here help provide new insight into the apoptosis-inducing effect of local anesthetics in eye clinic.
ABSTRACT
AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575µm in thickness during the monitoring period. A 4-5 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.