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Corneal wound healing in diabetic patients is usually delayed and accompanied by excessive inflammation. However, the underlying cellular and molecular mechanisms remain poorly understood. Here, we found that somatostatin (SST), an immunosuppressive peptide produced by corneal nerve fibers, was significantly reduced in streptozotocin-induced diabetic mice. In addition, we discovered that topical administration of exogenous SST significantly improved re-epithelialization and nerve regeneration following diabetic corneal epithelial abrasion. Further analysis showed that topical SST significantly reduced the expression of injury inflammation-related genes, inhibited neutrophil infiltration, and shifted macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 in diabetic corneas' healing. Moreover, the application of L-817,818, an agonist of the SST receptor type 5 subtype, significantly reduced the inflammatory response following epithelial injury and markedly improved the process of re-epithelialization and nerve regeneration in mice. Taken together, these data suggest that activation of the SST-SST receptor type 5 pathway significantly ameliorates diabetes-induced abnormalities in corneal wound repair in mice. Targeting this pathway may provide a novel strategy to restore impaired corneal wound closure and nerve regeneration in diabetic patients.
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Sleep deprivation (SD) has a wide range of adverse health effects. However, the mechanisms by which SD influences corneal pathophysiology and its post-wound healing remain unclear. This study aimed to examine the basic physiological characteristics of the cornea in mice subjected to SD and determine the pathophysiological response to injury after corneal abrasion. Using a multi-platform water environment method as an SD model, we found that SD leads to disturbances of corneal proliferative, sensory, and immune homeostasis as well as excessive inflammatory response and delayed repair after corneal abrasion by inducing hyperactivation of the sympathetic nervous system and hypothalamic-pituitary-adrenal axis. Pathophysiological changes in the cornea mainly occurred through the activation of the IL-17 signaling pathway. Blocking both adrenergic and glucocorticoid synthesis and locally neutralizing IL-17A significantly improved corneal homeostasis and the excessive inflammatory response and delay in wound repair following corneal injury in SD-treated mice. These results indicate that optimal sleep quality is essential for the physiological homeostasis of the cornea and its well-established repair process after injury. Additionally, these observations provide potential therapeutic targets to ameliorate SD-induced delays in corneal wound repair by inhibiting or blocking the activation of the stress system and its associated IL-17 signaling pathway.
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Lesiones de la Cornea , Modelos Animales de Enfermedad , Interleucina-17 , Transducción de Señal , Privación de Sueño , Cicatrización de Heridas , Animales , Ratones , Interleucina-17/metabolismo , Privación de Sueño/inmunología , Lesiones de la Cornea/metabolismo , Lesiones de la Cornea/etiología , Masculino , Córnea/metabolismo , Córnea/inmunología , Córnea/patología , Inflamación/inmunología , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Ratones Endogámicos C57BL , Estrés FisiológicoRESUMEN
Allergic conjunctivitis (AC), an allergen-induced ocular inflammatory disease, primarily involves mast cells (MCs) and eosinophils. The role of neuroimmune mechanisms in AC, however, remains to be elucidated. We investigated the effects of transient receptor potential vanilloid 1 (TRPV1)-positive sensory nerve ablation (using resiniferatoxin) and TRPV1 blockade (using Acetamide, N-[4-[[6-[4-(trifluoromethyl)phenyl]-4-pyrimidinyl]oxy]-2-benzothiazolyl] (AMG-517)) on ovalbumin-induced conjunctival allergic inflammation in mice. The results showed an exacerbation of allergic inflammation as evidenced by increased inflammatory gene expression, MC degranulation, tumor necrosis factor-α production by MCs, eosinophil infiltration and activation, and C-C motif chemokine 11 (CCL11) (eotaxin-1) expression in fibroblasts. Subsequent findings demonstrated that TRPV1+ sensory nerves secrete somatostatin (SST), which binds to SST receptor 5 (SSTR5) on MCs and conjunctival fibroblasts. SST effectively inhibited tumor necrosis factor-α production in MCs and CCL11 expression in fibroblasts, thereby reducing eosinophil infiltration and alleviating AC symptoms, including eyelid swelling, lacrimation, conjunctival chemosis, and redness. These findings suggest that targeting TRPV1+ sensory nerve-mediated SST-SSTR5 signaling could be a promising therapeutic strategy for AC, offering insights into neuroimmune mechanisms and potential targeted treatments.
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Antineoplásicos , Conjuntivitis Alérgica , Ratones , Animales , Factor de Necrosis Tumoral alfa/metabolismo , Conjuntiva/metabolismo , Conjuntiva/patología , Eosinófilos , Antineoplásicos/efectos adversos , Inflamación/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The lacrimal gland is responsible for maintaining the health of the ocular surface through the production of tears. However, our understanding of the immune system within the lacrimal gland is currently limited. Therefore, in this study, we utilized single-cell RNA sequencing and bioinformatic analysis to identify and analyze immune cells and molecules present in the lacrimal glands of normal mice. A total of 34,891 cells were obtained from the lacrimal glands of mice and classified into 18 distinct cell clusters using Seurat clustering. Within these cell populations, 26 different immune cell subpopulations were identified, including T cells, innate lymphocytes, macrophages, mast cells, dendritic cells, and B cells. Network analysis revealed complex cell-cell interactions between these immune cells, with particularly significant interactions observed among T cells, macrophages, plasma cells, and dendritic cells. Interestingly, T cells were found to be the main source of ligands for the Thy1 signaling pathway, while M2 macrophages were identified as the primary target of this pathway. Moreover, some of these immune cells were validated using immunohistological techniques. Collectively, these findings highlight the abundance and interactions of immune cells and provide valuable insights into the complexity of the lacrimal gland immune system and its relevance to associated diseases.
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Aparato Lagrimal , Aparato Lagrimal/patología , Lágrimas/metabolismo , Linfocitos T , Linfocitos , ARN/metabolismoRESUMEN
Purpose: This study aims to investigate the potential involvement of spleen-derived monocytes in the repair process following corneal epithelial abrasion. Methods: A corneal epithelial abrasion model was established in male C57BL/6J mice, and the dynamic changes of monocyte subpopulations in the injured cornea were analyzed using flow cytometry. The effects of Ly6Chi monocyte depletion and local adoptive transfer of purified Ly6Chi monocytes on wound closure and neutrophil recruitment to the injured cornea were observed. The effect of sympathetic nerves on the recruitment of spleen-derived Ly6Chi monocytes to the injured cornea was also investigated using multiple methods. The emigration of fluorescence-labeled monocytes to the injured cornea was validated through intravital microscopy. Finally, differential genes between different groups were identified through high-throughput RNA sequencing and analyzed for functional enrichment, followed by verification by quantitative PCR. Results: Ly6Chi monocytes were present in large numbers in the injured cornea prior to neutrophil recruitment. Predepletion of Ly6Chi monocytes significantly inhibited neutrophil recruitment to the injured cornea. Furthermore, surgical removal of the spleen significantly reduced the number of Ly6Chi monocytes in the injured cornea. Further observations revealed that sympathetic blockade significantly reduced the number of Ly6Chi monocytes recruited to the injured cornea. In contrast, administration of the ß2-adrenergic receptor agonist significantly increased the number of Ly6Chi monocytes recruited to the injured cornea in animals treated with sympathectomy and catecholamine synthesis inhibition. Conclusions: Our results suggest that spleen-derived Ly6Chi monocytes, under the control of the sympathetic nervous system, play a critical role in the inflammatory response following corneal injury.
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Lesiones de la Cornea , Bazo , Masculino , Ratones , Animales , Ratones Endogámicos C57BL , Monocitos , Córnea , Sistema Nervioso Simpático , Cicatrización de HeridasRESUMEN
Hernia and life-threatening intestinal obstruction often result from abdominal wall injuries, and the regeneration of abdominal wall defects is limited due to the lack of biocompatible, antibacterial and angiogenic scaffolding materials for treating injured tissues. Taking inspiration from the facile preparation of dopamine polymerization and its surface modification technology, in this study, multi-therapeutic copper element was introduced into porcine small intestinal submucosa (SIS) bio-patches through polydopamine (PDA) deposition, in order to regenerate abdominal wall injury. In both in vitro antibacterial assays, cytocompatibility assays and in vivo abdominal wall repair experiments, the SIS/PDA/Cu bio-patches exhibited robust antibacterial efficiency (ï¼99%), excellent biocompatibility to cells (ï¼90%), and enhanced neovascularization and improved collagen maturity compared to other commercially available patches (3.0-fold higher than the PP mesh), due to their activation of VEGF pathway. These findings indicated the bio-patch was a promising application for preventing visceral adhesion, bacterial infection, and promoting soft tissue regeneration.
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Conventional tissue adhesives face challenges for hemostasis and tissue regeneration in large-scaled hemorrhage and capillary hypobaric bleeding due to weak adhesion, and inability to degrade at specific sites. Herein, convenient and injectable poly(ethylene glycol) (PEG)-based adhesives are developed to address the issues for liver hemostasis. The PEG-bioadhesives are composed of tetra-armed PEG succinimide glutarate (PEG-SG), tetra-armed PEG amine (PEG-NH2 ), and tri-lysine. By mixing the components, the PEG-bioadhesives can be rapidly formulated for use of liver bleeding closure in hepatectomy. The PEG-bioadhesives also possess mechanical compliance to native tissues (elastic modulus ≈40 kPa) and tough tissue adhesion (≈28 kPa), which enables sufficient adhering to the injured tissues and promotes liver regeneration with the PEG-bioadhesive degradation. In both rats of liver injury and pigs of large-scaled hepatic hemorrhage, the PEG-bioadhesives show effective hemostasis with superior blood loss than conventional tissue adhesives. Due to biocompatibility and degradability, the PEG-bioadhesive is advantageous for liver regeneration, while commercial adhesives (e.g., N-octyl cyanoacrylate) display adhesion failure and limited liver reconstructions. These PEG-bioadhesive components are FDA-approved, and demonstrate excellent adhesion to various tissues not only for liver hemostasis, it is a promising candidate in biomedical translations and clinical applications.
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Polietilenglicoles , Adhesivos Tisulares , Ratas , Animales , Porcinos , Polietilenglicoles/farmacología , Adhesivos Tisulares/farmacología , Adhesivos Tisulares/uso terapéutico , Adhesivos , Hemostasis , Hígado , Hemorragia/tratamiento farmacológicoRESUMEN
We generated an induced pluripotent stem (iPS) cell line by reprogramming peripheral blood mononuclear cells of a patient with Usher syndrome type II carrying USH2A gene mutation (c.8559-2A > G). The iPS cell line with confirmed patient-specific point mutation exhibited typical iPS cell characteristics and maintained a normal karyotype. It can be used as 2D and 3D models to investigate the underlying pathogenic mechanism and lay a solid foundation for future personalized therapy.
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Células Madre Pluripotentes Inducidas , Síndromes de Usher , Humanos , Síndromes de Usher/genética , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación/genética , Línea Celular , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
Timely initiation and termination of inflammatory response after corneal epithelial abrasion is critical for the recovery of vision. The cornea is innervated with rich sensory nerves with highly dense TRPV1 nociceptors. However, the roles of TRPV1+ sensory neurons in corneal inflammation after epithelial abrasion are not completely understood. Here, we found that depletion of TRPV1+ sensory nerves using resiniferatoxin (RTX) and blockade of TRPV1 using AMG-517 delayed corneal wound closure and enhanced the infiltration of neutrophils and γδ T cells to the wounded cornea after epithelial abrasion. Furthermore, depletion of TRPV1+ sensory nerves increased the number and TNF-α production of corneal CCR2+ macrophages and decreased the number of corneal CCR2- macrophages and IL-10 production. In addition, the TRPV1+ sensory nerves inhibited the recruitment of neutrophils and γδ T cells to the cornea via RAMP1 and SSTR5 signaling, decreased the responses of CCR2+ macrophages via RAMP1 signaling, and increased the responses of CCR2- macrophages via SSTR5 signaling. Collectively, our results suggest that the TRPV1+ sensory nerves suppress inflammation to support corneal wound healing via RAMP1 and SSTR5 signaling, revealing potential approaches for improving defective corneal wound healing in patients with sensory neuropathy.
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Lesiones de la Cornea , Proteína 1 Modificadora de la Actividad de Receptores , Receptores de Somatostatina , Canales Catiónicos TRPV , Animales , Córnea , Inflamación , Ratones , Ratones Endogámicos C57BL , Proteína 1 Modificadora de la Actividad de Receptores/metabolismo , Receptores de Somatostatina/metabolismo , Canales Catiónicos TRPV/metabolismo , Cicatrización de HeridasRESUMEN
PURPOSE: Thyroid hormones have a critical role in maintaining metabolic and physiological homeostasis. However, understanding of the possible effects of thyroid dysfunction on corneal homeostasis and the wound healing process is quite limited. To explore the influence of hypothyroidism on corneal homeostasis and the post-wound repair processes of the murine cornea. METHODS: A hypothyroidism model was established by total thyroidectomy (TThy) in C57BL/6J mice. On day 10 after TThy, hypothyroidism was confirmed via thyronine (T3 and T4) and thyroid-stimulating hormone serum levels. We further assessed changes in corneal thickness, corneal sensitivity, sub-basal nerve density, and the corneal expression of thyroid hormone receptors. A corneal epithelial abrasion model was established via mechanical removal of a central epithelium 2 mm in diameter. Wound closure and recruitment of inflammatory cells (neutrophils and γδ T-cells) were evaluated. RNA-sequencing and gene set enrichment analysis were performed in injured corneas after abrasion. The effect of local T3 administration on corneal wound healing in thyroidectomized mice was also observed. RESULTS: Compared with sham-operated control mice, the TThy-treated mice showed the following: (1) a significant decrease in corneal epithelial thickness, sensitivity to external stimuli, and sub-basal nerve density, as well as an alteration in thyroid hormone receptor expression in the steady state; (2) delayed corneal wound repair and enhanced inflammatory response after corneal abrasion; (3) down-regulation of actin-skeleton and DNA replication pathways and up-regulation of inflammation-associated pathways in abraded corneas; and (4) significant restoration of delayed corneal wound repair and inhibition of excessive inflammation following topical T3 administration. CONCLUSIONS: We conclude that deficient thyroid hormone secretion significantly affects corneal homeostasis and post-wound repair processes. Topical T3 administration might have a potential reversal effect on delayed corneal wound repair among hypothyroid individuals.
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Lesiones de la Cornea , Epitelio Corneal , Hipotiroidismo , Animales , Córnea/metabolismo , Lesiones de la Cornea/metabolismo , Epitelio Corneal/metabolismo , Homeostasis , Hipotiroidismo/metabolismo , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Cicatrización de Heridas/fisiologíaRESUMEN
Mast cells (MCs) regulate wound healing and are influenced by the autonomic nervous system (ANS). However, the underlying mechanisms affecting wound healing outcomes remain elusive. Here, we explored the specific role of the ANS by regulating MC degranulation following corneal epithelium abrasion. A mouse model of corneal abrasion was established by mechanically removing a 2-mm central epithelium. Wound closure, neutrophil infiltration, and transcription of injured corneas were investigated using whole-mount immunostaining, flow cytometry, and RNA-sequencing analysis, respectively. Inhibition of MC degranulation by the MC stabilizers cromolyn sodium and lodoxamide tromethamine increased the infiltration of neutrophils and delayed healing of abraded corneas. Moreover, transcriptomic profiling analysis showed that purified MCs from the limbus expressed adrenergic and cholinergic receptors. Pharmacological manipulation and sympathectomy with 6-hydroxydopamine confirmed that sympathetic nervous system signaling inhibited MC degranulation after corneal abrasion, whereas parasympathetic nervous system signaling enhanced MC degranulation. We conclude that normal degranulation of MCs in the corneal limbus and crosstalk between the ANS and MCs are crucial for the appropriate control of inflammation and the repair progress of wounded corneas. This suggests a potential approach for improving defective corneal wound healing by the administration of clinically available autonomic activity-modulating agents.
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Lesiones de la Cornea , Epitelio Corneal , Animales , Sistema Nervioso Autónomo , Degranulación de la Célula , Epitelio Corneal/fisiología , Inflamación , Mastocitos , Ratones , Ratones Endogámicos C57BL , Cicatrización de Heridas/fisiologíaRESUMEN
USH type 2 (USH2) is an autosomal recessive disorder that is characterized by inherited retinopathies and sensorineural hearing loss. USH type 2 (USH2) is frequently caused by USH2A mutations, which account for 74-90% of USH2 cases. We used peripheral blood mononuclear cells (PBMCs) from a USH2 patient with a USH2A gene mutation (c.8559-2A > G) to create an induced pluripotent stem (iPS) cell line. The patient-specific iPS cell line with the specific point mutation exhibited typical iPS cell characteristics, and it can be used as a model to investigate the pathogenic mechanisms underlying USH2A-associated retinal degeneration and sensorineural hearing loss.
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Células Madre Pluripotentes Inducidas , Síndromes de Usher , Línea Celular , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación/genética , Síndromes de Usher/genéticaRESUMEN
Purpose: Endogenous and exogenous stressors, including nutritional challenges, may alter circadian rhythms in the cornea. This study aimed to determine the effects of high fructose intake (HFI) on circadian homeostasis in murine cornea. Methods: Corneas of male C57BL/6J mice subjected to 10 days of HFI (15% fructose in drinking water) were collected at 3-hour intervals over a 24-hour circadian cycle. Total extracted RNA was subjected to high-throughput RNA sequencing. Rhythmic transcriptional data were analyzed to determine the phase, rhythmicity, unique signature, metabolic pathways, and cell signaling pathways of transcripts with temporally coordinated expression. Corneas of HFI mice were collected for whole-mounted techniques after immunofluorescent staining to quantify mitotic cell number in the epithelium and trafficking of neutrophils and γδ-T cells to the limbal region over a circadian cycle. Results: HFI significantly reprogrammed the circadian transcriptomic profiles of the normal cornea and reorganized unique temporal and clustering enrichment pathways, but did not affect core-clock machinery. HFI altered the distribution pattern and number of corneal epithelial mitotic cells and enhanced recruitment of neutrophils and γδ-T cell immune cells to the limbus across a circadian cycle. Cell cycle, immune function, metabolic processes, and neuronal-related transcription and associated pathways were altered in the corneas of HFI mice. Conclusions: HFI significantly reprograms diurnal oscillations in the cornea based on temporal and spatial distributions of epithelial mitosis, immune cell trafficking, and cell signaling pathways. Our findings reveal novel molecular targets for treating pathologic alterations in the cornea after HFI.
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Ritmo Circadiano/genética , Epitelio Corneal/efectos de los fármacos , Proteínas del Ojo/genética , Fructosa/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , ARN/genética , Administración Oral , Animales , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epitelio Corneal/citología , Proteínas del Ojo/biosíntesis , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Animales , ARN/metabolismo , Edulcorantes/administración & dosificación , TranscriptomaRESUMEN
BACKGROUND: Cervical cancer (CC) is a common gynecological tumor that affects women's health. Circular RNA hsa_circ_0084927 (hsa_circ_0084927) has been reported to be upregulated in CC. However, the role and regulatory mechanism of hsa_circ_0084927 in CC are unclear. METHODS: Expression of hsa_circ_0084927, microRNA (miR)-634, and tumor protein D52 (TPD52) mRNA in CC tissues and cells was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, colony formation, cell cycle progression, apoptosis, migration, and invasion of CC cells were determined with cell counting kit-8 (CCK-8), plate clone, flow cytometry, or transwell assays. The levels of cyclin D1, cleaved-caspase-3 (c-caspase 3), matrix metalloproteinase (MMP)-2, MMP-9, and TPD52 protein were evaluated with Western blotting. The targeting relationship between hsa_circ_0084927 or TPD52 and miR-634 was verified via dual-luciferase reporter and/or RNA immunoprecipitation (RIP) assays. Xenograft assay was conducted to confirm the role of hsa_circ_0084927 in vivo. RESULTS: Hsa_circ_0084927 and TPD52 were upregulated while miR-634 was downregulated in CC tissues and cells. Hsa_circ_0084927 silencing reduced tumor growth in vivo and induced cell cycle arrest, apoptosis, and curbed proliferation, colony formation, migration, and invasion of CC cells in vitro. Hsa_circ_0084927 regulated TPD52 expression through sponging miR-634. MiR-634 inhibitor reversed hsa_circ_0084927 knockdown-mediated impact on the malignancy of CC cells. TPD52 elevation abolished the repressive influence of miR-634 mimics on the malignancy of CC cells. CONCLUSION: Hsa_circ_0084927 accelerated CC advancement via upregulating TPD52 via sponging miR-634, offering a new evidence to support hsa_circ_0084927 as a promising target for CC treatment.
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Stem cell therapy holds promises for treating corneal scarring. Here, we use multilineage-differentiating stress-enduring (Muse) cells to study their differentiation and therapeutic potential for treating corneal injury. Muse cells were isolated from lipoaspirate, which presented biphenotype properties of both pluripotent stem cells and some mesenchymal stem cells. Muse cells expanded by about 100-fold from the initial seeding cell number to Muse spheroids with the maintenance of the Muse cell phenotype and high cell viability at 33 days by static spheroid culture. We revealed that Muse spheroids were activated by the dynamic rotary cell culture system (RCCS), as characterized by increased stemness, improved activity, and enhanced adherence. Gene and protein expression of the pluripotent markers OCT3/4, SOX2, and NANOG and of the proliferation marker KI67 in Muse spheroids cultured under RCCS were higher than those in the static group. These activated Muse spheroids enabled ready differentiation into corneal stromal cells (CSCs) expressing characteristic marker genes and proteins. Furthermore, implantation of Muse cells-differentiated CSCs (Muse-CSCs) laden assembled with two orthogonally stacked stretched compressed collagen (cell-SCC) in mouse and tree shrew wounded corneas prevented the formation of corneal scarring, increased corneal re-epithelialization and nerve regrowth, and reduced the severity of corneal inflammation and neovascularization. cell-SCC retained the capacity to suppress corneal scarring after long-distance cryopreserved transport. Thus, Muse cell therapy is a promising avenue for developing therapeutics for treating corneal scarring.
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Lesiones de la Cornea , Células Madre Pluripotentes , Alprostadil , Animales , Diferenciación Celular , Cicatriz , Lesiones de la Cornea/terapia , Ratones , TupaiidaeRESUMEN
Eosinophils are a major cause of tissue injury in allergic conjunctivitis. The biological nature of eosinophils in the conjunctiva and the mechanisms that control eosinophils' responses in allergic conjunctivitis are currently not completely understood. This study reports that conjunctival eosinophils comprise two populations-Siglec-Fint and Siglec-Fhi-in different life stages. Siglec-Fint eosinophils partly expressed CD34 and were in the immature (or steady) state. Siglec-Fhi eosinophils did not express CD34, sharply increased in number after short ragweed (SRW) pollen challenge, and were in the mature (or activated) state. Moreover, chemical sympathectomy by 6-hydroxydopamine reduced the recruitment and activation of eosinophils, whereas the activation of the sympathetic nerve system (SNS) with restraint stress accelerated the recruitment and activation of eosinophils in SRW-induced conjunctivitis. It was also found that two eosinophil populations expressed alpha-1a-adrenergic receptors (α1a-ARs); in SRW-induced conjunctivitis, treatment with an α1a-AR antagonist decreased eosinophil responses, whereas treatment with an α1a-AR agonist aggravated eosinophil responses. Thus, eosinophil responses in conjunctivitis are regulated by the SNS via α1a-AR signaling. SNS inputs or α1a-AR function may be potential targets for the treatment of allergic conjunctivitis.
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Conjuntivitis Alérgica/metabolismo , Eosinófilos/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Sistema Nervioso Simpático/metabolismo , Animales , Conjuntiva/inmunología , Conjuntiva/metabolismo , Conjuntivitis Alérgica/inmunología , Modelos Animales de Enfermedad , Eosinófilos/inmunología , Ratones , Transducción de Señal/fisiología , Sistema Nervioso Simpático/inmunologíaRESUMEN
Antibiotics are extremely useful, but they can cause adverse impacts on host bodies. We found that antibiotic treatment altered the composition of the gut microbiota and the gene expression profile in the corneal tissues of postnatal mice and decreased the corneal size and thickness, the angiogenesis of limbal blood vessels, and the neurogenesis of corneal nerve fibers. The reconstitution of the gut microbiota with fecal transplants in antibiotic-treated mice largely reversed these impairments in corneal development. Furthermore, C-C chemokine receptor type 2 negative (CCR2-) macrophages were confirmed to participate in corneal development, and their distribution in the cornea was regulated by the gut microbiota. We propose that the CCR2- macrophage population is a crucial mediator through which gut microbiota affect corneal development in postnatal mice. In addition, probiotics were shown to have the potential effect of restoring corneal development in antibiotic-treated mice. Abx-induced gut dysbiosis has significant, long-term effects on the development of the cornea, and reversal of these suppressive effects takes a long time.
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Antibacterianos/efectos adversos , Córnea/fisiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/inmunología , Disbiosis/inmunología , Microbioma Gastrointestinal/genética , Macrófagos/inmunología , ARN Ribosómico 16S/genética , Animales , Animales Recién Nacidos , Antibacterianos/uso terapéutico , Movimiento Celular , Células Cultivadas , Disbiosis/etiología , Trasplante de Microbiota Fecal , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Atención Posnatal , Receptores CCR2/metabolismoRESUMEN
Retinitis pigmentosa (RP) represents a group of inherited retinopathies with early-onset nyctalopia followed by progressive photoreceptor degeneration causing irreversible vision loss. Mutations in USH2A are the most common cause of non-syndromic RP. Here, we reprogrammed induced pluripotent stem cells (iPSCs) from a RP patient with a mutation in USH2A (c.8559-2A > G/c.9127_9129delTCC). Then, multilayer retinal organoids including neural retina (NR) and retinal pigment epithelium (RPE) were generated by three-step "induction-reversal culture." The early retinal organoids derived from the RP patient with the USH2A mutation exhibited significant defects in terms of morphology, immunofluorescence staining and transcriptional profiling. To the best of our knowledge, the pathogenic mutation (c.9127_9129delTCC) in USH2A has not been reported previously among RP patients. Notably, the expression of laminin in the USH2A mutation organoids was significantly lower than in the iPSCs derived from healthy, age- and sex-matched controls during the retinal organogenesis. We also observed that abnormal retinal neuroepithelium differentiation and polarization caused defective retinal progenitor cell development and retinal layer formation, disordered organization of NRs in the presence of the USH2A mutation. Furthermore, the USH2A mutation bearing RPE cells presented abnormal morphology, lacking pigmented foci and showing an apoptotic trend and reduced expression of specific makers, such as MITF, PEDF, and RPE65. In addition, the USH2A mutation organoids had lower expression of cilium-associated (especially CFAP43, PIFO) and dopaminergic synapse-related genes (including DLGAP1, GRIK1, SLC17A7, and SLC17A8), while there was higher expression of neuron apoptotic process-related genes (especially HIF1A, ADARB1, and CASP3). This study may provide essential assistance in the molecular diagnosis and screening of RP. This work recapitulates the pathogenesis of USH2A using patient-specific organoids and demonstrated that alterations in USH2A function due to mutations may lead to cellular and molecular abnormalities.
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Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na+-K+-ATPase. The gene expression levels of NIFK, Na+-K+-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.
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Exposure to tobacco smoke is a major public health concern that can also affect ophthalmic health. Based on previous work demonstrating the important role of the sympathetic nervous system (SNS) in corneal wound repair, we postulated that acute tobacco smoke exposure (ATSE) may act through the SNS in the impairment of corneal wound repair. Here we find that ATSE rapidly increases the markers of inflammatory response in normal corneal limbi. After an abrasion injury, ATSE exaggerates inflammation, impairs wound repair, and enhances the expression of nuclear factor-κB (NF-κB) and inflammatory molecules such as interleukin-6 (IL-6) and IL-17. We find that chemical SNS sympathectomy, local adrenergic receptor antagonism, NF-κB1 inactivation, and IL-6/IL-17A neutralization can all independently attenuate ATSE-induced excessive inflammatory responses and alleviate their impairment of the healing process. These findings highlight that the SNS may represent a major molecular sensor and mediator of ATSE-induced inflammation.