Chronic sleep deprivation impairs retinal circadian transcriptome and visual function.

Sleep loss is common in modern society and is increasingly associated with eye diseases. However, the precise effects of sleep loss on retinal structure and function, particularly on the retinal circadian system, remain largely unexplored. This study investigates these effects using a chronic sleep deprivation (CSD) model in mice. Our investigation reveals that CSD significantly alters the retinal circadian transcriptome, leading to remarkable changes in the temporal patterns of enriched pathways. This perturbation extends to metabolic and immune-related transcriptomes, coupled with an accumulation of reactive oxygen species in the retina. Notably, CSD rhythmically affects the thickness of the ganglion cell complex, along with diurnal shifts in microglial migration and morphology within the retina. Most critically, we observe a marked decrease in both scotopic and photopic retinal function under CSD conditions. These findings underscore the broad impact of sleep deprivation on retinal health, highlighting its role in altering circadian gene expression, metabolism, immune response, and structural integrity. Our study provides new insights into the broader impact of sleep loss on retinal health.

Long-term high fructose intake promotes lacrimal gland dysfunction by inducing gut dysbiosis in mice.

The lacrimal gland is essential for maintaining ocular surface health through the secretion of the aqueous layer of the tear film. It is therefore important to explore the intrinsic and extrinsic factors that affect the structure and function of the lacrimal gland and the mechanisms underlying them. With the prevalence of Westernized diets characterized by high sugar and fat content, the susceptibility to many diseases, including ocular diseases, is increased by inducing dysbiosis of the gut microbiome. Here, we found that the composition, abundance, and diversity of the gut microbiome was significantly altered in mice by drinking 15% high fructose water for one month, as determined by 16S rRNA sequencing. This was accompanied by a significant increase in lipid deposition and inflammatory cell infiltration in the extraorbital lacrimal glands (ELGs) of mice. Transcriptome analysis based on bulk RNA-sequencing revealed abnormal activation of some of several metabolic and immune-related pathways. In addition, the secretory response to stimulation with the cholinergic receptor agonist pilocarpine was significantly reduced. However, when the composition and diversity of the gut microbiome of high fructose intake (HFI)-treated mice were improved by transplanting feces from normal young healthy mice, the pathological alterations in ELG structure, inflammatory cell infiltration, secretory function and transcriptome analysis described above were significantly reversed compared to age-matched control mice. In conclusion, our data suggest that prolonged HFI may cause pathological damage to the structure and function of the ELG through the induction of gut dysbiosis. Restoration of intestinal dysbiosis in HFI-treated mice by fecal transplantation has a potential role in ameliorating these pathological impairments.

Microbial Reconstitution Improves Aging-Driven Lacrimal Gland Circadian Dysfunction.

Lacrimal glands are highly susceptible to aging and exhibit age-related structural and functional alterations. However, the mechanisms by which aging affects the lacrimal glands are not well-established. The current study explores the crosstalk between the aging process, gut microbiota, and circadian rhythm in age-associated lacrimal gland dysfunction. C57BL/6J mice were divided into young, old, and fecal microbiota transplant (FMT)-treated old groups. The gut bacterial community diversity was analyzed by 16S rRNA sequencing. Exorbital lacrimal glands (ELGs) were collected at 3-hour intervals over a 24-hour circadian cycle, and total RNA was subjected to high-throughput sequencing. Rhythmic transcriptional data were analyzed using the Jonckheere-Terpstra-Kendall algorithm and bioinformatics analysis technology. Immunostaining was used to identify lymphocytic infiltration, lipid deposition, and nerve innervation in the ELGs. Compared with young mice, old mice underwent a significant gut microbial community shift. The rhythmically transcriptomic profile was significantly reprogrammed over a 24-hour cycle in the old ELG group. Intervention with serial FMT from young donors for 1 month rejuvinated the gut microbial community of the old mice. Most alterations in rhythmic transcriptomic profiling were improved. Furthermore, chronic inflammation, lipid deposition, and aberrant neural response of the aging lacrimal glands were significantly reduced. Thus, the study shows that reconstitution of age-associated gut dysbiosis with FMTs from young donors improves aging-driven lacrimal gland circadian dysfunction.

Recent advances in modulators of circadian rhythms: an update and perspective.

Circadian rhythm is a universal life phenomenon that plays an important role in maintaining the multiple physiological functions and regulating the adaptability to internal and external environments of flora and fauna. Circadian alignment in humans has the greatest effect on human health, and circadian misalignment is closely associated with increased risk for metabolic syndrome, cardiovascular diseases, neurological diseases, immune diseases, cancer, sleep disorders, and ophthalmic diseases. The recent description of clock proteins and related post-modification targets was involved in several diseases, and numerous lines of evidence are emerging that small molecule modulators of circadian rhythms can be used to rectify circadian disorder. Herein, we attempt to update the disclosures about the modulators targeting core clock proteins and related post-modification targets, as well as the relationship between circadian rhythm disorders and human health as well as the therapeutic role and prospect of these small molecule modulators in circadian rhythm related disease.

Analysis of the heterogeneity and complexity of murine extraorbital lacrimal gland via single-cell RNA sequencing.

PURPOSE: The lacrimal gland is essential for maintaining ocular surface health and avoiding external damage by secreting an aqueous layer of the tear film. However, a healthy lacrimal gland's inventory of cell types and heterogeneity remains understudied. METHODS: Here, 10X Genome-based single-cell RNA sequencing was used to generate an unbiased classification of cellular diversity in the extraorbital lacrimal gland (ELG) of C57BL/6J mice. From 43,850 high-quality cells, we produced an atlas of cell heterogeneity and defined cell types using classic marker genes. The possible functions of these cells were analyzed through bioinformatics analysis. Additionally, the CellChat was employed for a preliminary analysis of the cell-cell communication network in the ELG. RESULTS: Over 37 subclasses of cells were identified, including seven types of glandular epithelial cells, three types of fibroblasts, ten types of myeloid-derived immune cells, at least eleven types of lymphoid-derived immune cells, and five types of vascular-associated cell subsets. The cell-cell communication network analysis revealed that fibroblasts and immune cells play a pivotal role in the dense intercellular communication network within the mouse ELG. CONCLUSIONS: This study provides a comprehensive transcriptome atlas and related database of the mouse ELG.

Screening marker genes of type 2 diabetes mellitus in mouse lacrimal gland by LASSO regression.

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and a relative deficiency of insulin. This study aims to screen T2DM-related maker genes in the mouse extraorbital lacrimal gland (ELG) by LASSO regression.C57BLKS/J strain with leptin db/db homozygous mice (T2DM, n = 20) and wild-type mice (WT, n = 20) were used to collect data. The ELGs were collected for RNA sequencing. LASSO regression was conducted to screen marker genes with the training set. Five genes were selected from 689 differentially expressed genes by LASSO regression, including Synm, Elovl6, Glcci1, Tnks and Ptprt. Expression of Synm was downregulated in ELGs of T2DM mice. Elovl6, Glcci1, Tnks, and Ptprt were upregulated in T2DM mice. Area under receiver operating curve of the LASSO model was 1.000(1.000-1.000) and 0.980(0.929-1.000) in the training set and the test set, respectively. The C-index and the robust C-index of the LASSO model were 1.000 and 0.999, respectively, in the training set, and 1.000 and 0.978, respectively, in the test set. In the lacrimal gland of db/db mice, Synm, Elovl6, Glcci1, Tnks and Ptprt can be used as marker genes of T2DM. Abnormal expression of marker genes is related to lacrimal gland atrophy and dry eye in mice.

Mechanisms of Extraorbital Lacrimal Gland Aging in Mice: An Integrative Analysis of the Temporal Transcriptome.

Purpose: This study used high-throughput RNA sequencing (RNA-Seq) and bioinformatics analysis to investigate the altered transcriptome profile of aging lacrimal glands in mice that occurs over the course of a 24-hour cycle. Methods: Male C57BL/6J mice aged 12 weeks (young) and 20 months (aging) were housed in a pathogen-free setting with a 12-hour light/12-hour dark cycle. Throughout a 24-hour cycle, mouse extraorbital lacrimal glands (ELGs) were collected at eight time points at three-hour intervals. To prepare for the high-throughput RNA-Seq, whole mRNA was extracted. Differentially expressed genes (DEGs) in the young and aging groups were subjected to bioinformatic analysis based on diurnal patterns. Furthermore, the cell populations in which significant DEGs express and signaling pathways occur were validated at the single-cell RNA sequencing (scRNA-seq) level. Results: The total transcriptome composition was significantly altered in aging ELGs compared with that in young mouse ELGs at eight time points during the 24-hour cycle, with 864 upregulated and 228 downregulated DEGs, which were primarily enriched in inflammatory pathways. Further comparative analysis of the point-to-point transcriptome revealed that aging ELGs underwent alterations in the temporal transcriptome profile in several pathways, including the inflammation-related, metabolism-related, mitochondrial bioenergetic function-associated, synaptome neural activity-associated, cell processes-associated, DNA processing-associated and fibrosis-associated pathways. Most of these pathways occurred separately in distinct cell populations. Conclusions: Transcriptome profiles of aging lacrimal glands undergo considerable diurnal time-dependent changes; this finding offers a comprehensive source of information to better understand the pathophysiology of lacrimal gland aging and its underlying mechanisms.

High-fat intake reshapes the circadian transcriptome profile and metabolism in murine meibomian glands.

Background: Nutritional and food components reshape the peripheral clock and metabolism. However, whether food challenges affect the circadian clock and metabolism of meibomian glands (MGs) has not been fully explored. This study was designed to analyze alterations in the rhythmic transcriptome and metabolism of MGs of murine fed a balanced diet or a high-fat diet (HFD). Methods: Male C57BL/6J mice were maintained on a 12/12 h light/dark cycle and fed ad libitum on normal chow (NC) or HFD for 4 weeks. MGs were collected from sacrificed animals at 3-h intervals throughout a 24-h circadian cycle. The circadian transcriptome of MGs was analyzed via bioinformatics approaches using high-throughput RNA sequencing (RNA-seq). In addition, circadian oscillations of lipid components in MGs were analyzed. Results: Meibomian glands displayed robust transcriptome rhythmicity. HFD feeding significantly altered the circadian transcriptome profile of MGs-including composition and phase-and spatiotemporally affected the enriched signaling pathways. In addition, HFD feeding significantly altered the normal rhythmic oscillations of lipid components in MGs. Conclusion: Our data show that HFD significantly affects MGs' rhythmicity, which reveals a high sensitivity of MGs' clocks to lipid composition in food.

High-Fat Nutritional Challenge Reshapes Circadian Signatures in Murine Extraorbital Lacrimal Glands.

Purpose: A high-fat diet (HFD) increases the risk of developing many systemic diseases; however, the effects of high fat intake on lacrimal gland functions and the molecular mechanisms underlying these effects are unknown. We explored the effects of an HFD on the circadian rhythms of the extraorbital lacrimal glands (ELGs). Methods: Male C57BL/6J mice maintained on a 12/12-hour light/dark cycle were fed an ad libitum HFD or normal chow (NC) for 2 weeks. The ELGs were collected from euthanized animals every 3 hours throughout the circadian cycle (24 hours). Using high-throughput RNA-sequencing (RNA-Seq), we studied the circadian transcriptomic profile of the ELGs. Circadian oscillations in cell size, secretion response, lipid deposition, and immune cell trafficking of the ELGs were also analyzed. Results: An HFD modulated the circadian transcriptomic profile of the ELGs, including the composition, phase, and amplitude of cyclical transcript oscillations, and affected the associated signaling pathways at spatiotemporal levels. HFD feeding significantly altered the normal rhythmic oscillations of ELG cell size, immune cell trafficking, secretion response, and lipid deposition. After dietary reversal in HFD-fed animals, the activity, core temperature, and lipid accumulation in lacrimal glands recovered partially to the level of NC-fed animals. However, the average cell size of the ELGs, the recruitment of immune cells, and the rhythm of lacrimal secretion did not return to the levels of the NC-fed group. Conclusions: HFD perturbation interferes with the cyclical transcriptomic profile, cell size, immune cell trafficking, and secretion function of the ELGs with a strikingly high sensitivity.

Sleep Loss Causes Dysfunction in Murine Extraorbital Lacrimal Glands.

Purpose: Sleep loss markedly affects the structure and function of the lacrimal gland and may cause ocular surface disease as a common public health problem. This study aims to investigate the circadian disturbance caused by sleep loss leading to dysfunction of extraorbital lacrimal glands (ELGs). Methods: A mouse sleep deprivation (SD) model for sleep loss studies was built in C57BL/6J male mice. After four weeks, the ELGs were collected at three-hour intervals during a 24-hour period. The Jonckheere-Terpstra-Kendall algorithm was used to determine the composition, phase, and rhythmicity of transcriptomic profiles in ELGs. Furthermore, we compared the non-sleep-deprived and SD-treated mouse ELG (i) reactive oxygen species (ROS) by fluorescein staining, (ii) DNA damage by immunostaining for γ-H2Ax, and (iii) circadian migration of immune cells by immunostaining for CD4, CD8, γδ-TCR, CD64, and CX3CR1. Finally, we also evaluated (i) the locomotor activity and core body temperature rhythm of mice and (ii) the mass, cell size, and tear secretion of the ELGs. Results: SD dramatically altered the composition and phase-associated functional enrichment of the circadian transcriptome, immune cell trafficking, metabolism, cell differentiation, and neural secretory activities of mouse ELGs. Additionally, SD caused the ROS accumulation and consequent DNA damage in the ELGs, and the ELG dysfunction caused by SD was irreversible. Conclusions: SD damages the structure, function, and diurnal oscillations of ELGs. These results highlight comprehensive characterization of insufficient sleep-affected ELG circadian transcriptome that may provide a new therapeutic approach to counteract the effects of SD on ELG function.

Light cycle phase advance as a model for jet lag reprograms the circadian rhythms of murine extraorbital lacrimal glands.

PURPOSE: Jet lag causes a disruption in physiological rhythms in humans. This study aims to explore the extent to which jet lag affects the circadian rhythmicity in the lacrimal glands. METHODS: C57BL/6J mice were subjected to a 12-h light/12-h dark (LD) cycle and an 8-h advanced LD schedule as a model for jet lag. On day 5 after the LD advance, the extraorbital lacrimal glands (ELGs) were collected at 3-h intervals during a 24-h cycle. Total mRNA was extracted from normal and advanced LD-treated ELGs and assayed using high-throughput RNA sequencing. The rhythmic transcripts were identified, analyzed, and visualized by bioinformatics techniques. Finally, (i) animal behavior; (ii) the mass, cell size, and secretion response of ELGs; and (iii) circadian migration of immune cells to ELGs were also assayed. RESULTS: Jet lag treatment drastically altered the phase and composition of the rhythmic transcripts compared to that of normal ELGs. The key biological processes, signaling pathways, and protein-protein association networks were also dramatically altered in a spatiotemporal pattern. Furthermore, the circadian migration of neutrophils, T cells, B cells, and macrophages to the ELGs increased and shifted later by 6-h. Finally, the circadian rhythms of the ELGs with respect to mass, cell size, and secretion response were also impaired in jet lag-treated animals. CONCLUSIONS: Jet lag impairs the circadian rhythm of the transcriptomic profile, structure, and secretion function of the lacrimal glands. This information provides novel insight into the negative effects of jet lag on ELGs.

Type 1 diabetes mellitus impairs diurnal oscillations in murine extraorbital lacrimal glands.

PURPOSE: People with diabetes are at high risk of lacrimal gland dysfunction, but the underlying mechanism is not well understood. In this study, we determined how type 1 diabetes mellitus (T1DM) influences circadian homeostasis of the murine extraorbital lacrimal glands (ELGs). METHODS: A T1DM animal model was established by systemic streptozotocin injection in C57BL/6J mice. After 5 weeks, ELGs were collected at 3-h intervals over a 24-h circadian cycle. Total extracted RNA was subjected to high-throughput RNA sequencing, and rhythmic transcriptional data were evaluated using the Jonckheere-Terpstra-Kendall algorithm, Kyoto Encyclopedia of Genes and Genomes pathway analysis, Phase Set Enrichment Analysis, and time series cluster analysis to determine the phase, rhythmicity, and unique signature of the transcripts over temporally coordinated expression. Additionally, mass, cell size, histology, and tear secretion of the ELGs were evaluated. RESULTS: T1DM globally altered the composition of the ELG transcriptome. Specifically, T1DM significantly reprogrammed the circadian transcriptomic profiles of normal ELGs and reorganized core clock machinery. Unique temporal and clustering enrichment pathways were also rewired by T1DM. Finally, normal daily rhythms of mass, cell size, and tear secretion of mouse ELGs were significantly impaired by streptozotocin-induced diabetes. CONCLUSIONS: T1DM significantly reprograms the diurnal oscillations of the lacrimal glands and impairs their structure and tear secretion. This information may reveal potential targets for improving lacrimal gland dysfunction in patients with diabetes.

Transcriptional Profiling of Daily Patterns of mRNA Expression in the C57BL/6J Mouse Cornea.

Purpose: The purpose of this study was to determine how the transcriptome of the murine cornea adapts to diurnal changes in physiology. Methods: C57BL/6J mice were maintained under a 12-h light/12-h dark (LD) cycle for two weeks. Corneas were collected from euthanized mice at Zeitgeber time (ZT) 0, 3, 6, 9, 12, 15, 18, and 21. Total RNA was extracted and subjected to RNA sequencing (RNA-Seq). A JTK_CYCLE algoithm and other software tools were used to analyze the transcriptional data to determine the periodicity, rhythmicity, and amplitude of the transcripts. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the enrichment of cycling transcripts. Results: Approximately 24% of the total transcripts from the murine corneal genome were rhythmically expressed over an LD cycle. GO analysis showed that these cycling genes are primarily involved in cellular and metabolic processes. A KEGG pathway analysis identified 6 branches and 44 pathways that encode the gene outputs necessary for basic cellular functions and processes. More importantly, most of the rhythmic genes between the day and night are enriched in their own unique pathways in addition to some common pathways. Furthermore, most of the rhythmic gene expression was concentrated in the 12-h and 24-h periods. A comparative analysis of GO and KEGG showed large differences in metabolic processes, but not cellular processes. Finally, the murine cornea also rhythmically expressed 11 canonical components of circadian clock genes over an LD cycle at the transcriptional level. Conclusions: One fourth of the corneal transcriptome follows a rhythmic expression pattern involved in basic molecular and cellular mechanisms. This implies that the time of day contributes significantly to the overall temporal organization of the corneal transcriptome.

Short-term High Fructose Intake Reprograms the Transcriptional Clock Rhythm of the Murine Extraorbital Lacrimal Gland.

Purpose: To determine whether high fructose intake (HFI) influences the daily transcriptional clock rhythms of murine extraorbital lacrimal glands (ELGs). Methods: Timed ELGs were collected from two groups of C57BL/6J mice subjected to a 12-hour light/12-hour dark (LD) cycle for 10 days; the first group received water-only feeding and the second received water with 15% fructose. Total RNA was extracted and subjected to RNA sequencing. A JTK_CYCLE algorithm and computational software were used to determine the periodicity, rhythmicity, and amplitude of the cycling transcripts. The rhythmic transcripts from different conditions were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results: HFI feeding caused massive remodeling of the preexisting rhythmic genes in the normal control (NC)-fed ELGs. The induced transcripts in HFI-fed mice resulted in a profound reorganization of the coordinated transcriptional oscillations and KEGG pathways. Moreover, HFI feeding significantly altered the distribution of the KEGG pathways over an LD in the ELGs. Finally, we found that the ELGs have a robust core clock machinery and HFI feeding altered amplitude and the peak phase of clock gene transcriptional oscillation in ELGs. Conclusions: Short-term HFI reprograms the daily transcriptomic oscillation of murine ELGs. This information may deepen our understanding of the outcomes of lacrimal glands altered by nutritional challenge.

Acute tobacco smoke exposure exacerbates the inflammatory response to corneal wounds in mice via the sympathetic nervous system.

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.

Epothilone B Speeds Corneal Nerve Regrowth and Functional Recovery through Microtubule Stabilization and Increased Nerve Beading.

The successful restoration of corneal innervation and function after a corneal injury is a clinically challenging issue. Structural and functional recovery after a nerve injury involves a complex series of steps in which microtubules play a key role. The aim of the current study was to investigate the effects of epothilone B (EpoB), a microtubule-stabilizing agent, on corneal innervation and the functional recovery of the corneal nerve in mice after corneal epithelial abrasion. The pretreatment of mice with EpoB has a remarkable effect on the stabilization of beta-III tubulin, as demonstrated by substantial increases in the visualization of beta-III tubulin, nerve beading, corneal reinnervation, and reaction to stimuli. Furthermore, a pharmacokinetic analysis showed that EpoB remains at a high concentration in the cornea and the trigeminal ganglion for at least 6 days after administration. In addition, the administration of EpoB at 24 hours after corneal abrasion has a marked therapeutic effect on nerve regrowth and functional recovery. In conclusion, EpoB treatment may have therapeutic utility for improving corneal reinnervation and restoring sensitivity following corneal injury.