Epigenetic signals associated with delirium replicated across four independent cohorts.

Delirium is risky and indicates poor outcomes for patients. Therefore, it is crucial to create an effective delirium detection method. However, the epigenetic pathophysiology of delirium remains largely unknown. We aimed to discover reliable and replicable epigenetic (DNA methylation: DNAm) markers that are associated with delirium including post-operative delirium (POD) in blood obtained from patients among four independent cohorts. Blood DNA from four independent cohorts (two inpatient cohorts and two surgery cohorts; 16 to 88 patients each) were analyzed using the Illumina EPIC array platform for genome-wide DNAm analysis. We examined DNAm differences in blood between patients with and without delirium including POD. When we compared top CpG sites previously identified from the initial inpatient cohort with three additional cohorts (one inpatient and two surgery cohorts), 11 of the top 13 CpG sites showed statistically significant differences in DNAm values between the delirium group and non-delirium group in the same directions as found in the initial cohort. This study demonstrated the potential value of epigenetic biomarkers as future diagnostic tools. Furthermore, our findings provide additional evidence of the potential role of epigenetics in the pathophysiology of delirium including POD.

NSAIDs use history: impact on the genome-wide DNA methylation profile and possible mechanisms of action.

BACKGROUND AND OBJECTIVE: NSAIDs inhibit cyclooxygenase, but their role in aging and other diseases is not well understood. Our group previously showed the potential benefit of NSAIDs in decreasing the risk of delirium and mortality. Concurrently, epigenetics signals have also been associated with delirium. Therefore, we sought to find differentially methylated genes and biological pathways related to exposure with NSAIDs by comparing the genome-wide DNA methylation profiles of patients with and without a history of NSAIDs use. METHODS: Whole blood samples were collected from 171 patients at the University of Iowa Hospital and Clinics from November 2017 to March 2020. History of NSAIDs use was assessed through a word-search function in the subjects' electronic medical records. DNA was extracted from the blood samples, processed with bisulfite conversion, and analyzed using Illumina's EPIC array. The analysis of top differentially methylated CpG sites and subsequent enrichment analysis were conducted using an established pipeline using R statistical software. RESULTS: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) showed several biological pathways relevant to NSAIDs' function. The identified GO terms included "arachidonic acid metabolic process," while KEGG results included "linoleic acid metabolism," "cellular senescence," and "circadian rhythm." Nonetheless, none of the top GO and KEGG pathways and the top differentially methylated CpG sites reached statistical significance. CONCLUSION: Our results suggest a potential role of epigenetics in the mechanisms of the action of NSAIDs. However, the results should be viewed with caution as exploratory and hypothesis-generating given the lack of statistically significant findings.

Bispectral EEG (BSEEG) Algorithm Captures High Mortality Risk Among 1,077 Patients: Its Relationship to Delirium Motor Subtype.

OBJECTIVE: Delirium is dangerous and a predictor of poor patient outcomes. We have previously reported the utility of the bispectral EEG (BSEEG) with a novel algorithm for the detection of delirium and prediction of patient outcomes including mortality. The present study employed a normalized BSEEG (nBSEEG) score to integrate the previous cohorts to combine their data to investigate the prediction of patient outcomes. We also aimed to test if the BSEEG method can be applicable regardless of age, and independent of delirium motor subtypes. METHODS: We calculated nBSEEG score from raw BSEEG data in each cohort and classified patients into BSEEG-positive and BSEEG-negative groups. We used log-rank test and Cox proportional hazards models to predict 90-day and 1-year outcomes for the BSEEG-positive and -negative groups in all subjects and motor subgroups. RESULTS: A total of 1,077 subjects, the BSEEG-positive group showed significantly higher 90-day (hazard ratio 1.33 [95% CI 1.16-1.52] and 1-year (hazard ratio 1.22 [95% CI 1.06-1.40] mortality rates than the negative group after adjustment for covariates such as age, sex, CCI, and delirium status. Among patients with different motor subtypes of delirium, the hypoactive group showed significantly higher 90-day (hazard ratio 1.41 [95% CI 1.12-1.76] and 1-year mortality rates (hazard ratio 1.32 [95% CI 1.05-1.67], which remained significant after adjustment for the same covariates. CONCLUSION: We found that the BSEEG method is capable of capturing patients at high mortality risk.

Anti-inflammatory medication use associated with reduced delirium risk and all-cause mortality: A retrospective cohort study.

OBJECTIVE: To investigate the relationship between history of anti-inflammatory medication use and delirium risk, as well as long-term mortality. METHODS: In this retrospective cohort study, subjects recruited between January 2016 and March 2020 were analyzed. Information about anti-inflammatory medication use history including aspirin, NSAIDs, glucosamine, and other anti-inflammatory drugs, was collected. Logistic regression analysis investigated the relationship between anti-inflammatory medications and delirium. Log-rank analysis and cox proportional hazards model investigated the relationship between anti-inflammatory medications and one-year mortality. RESULTS: The data from 1274 subjects were analyzed. The prevalence of delirium was significantly lower in subjects with NSAIDs usage (23.0%) than in those without NSAIDs usage (35.0%) (p < 0.001). Logistic regression analysis controlling for age, sex, dementia status, and hospitalization department showed that the risk of delirium tended to be reduced by a history of NSAIDs use (OR, 0.76 [95% CI, 0.55 to 1.03]). The one-year mortality in the subjects with NSAIDs (survival rate, 0.879 [95% CI, 0.845 to 0.906]) was significantly lower than in the subjects without NSAIDs (survival rate, 0.776 [95% CI, 0.746 to 0.803]) (p < 0.001). A history of NSAIDs use associated with the decreased risk of one-year mortality even after adjustment for age, sex, Charlson Comorbidity Index, delirium status, and hospitalization department (HR, 0.70 [95% CI, 0.51 to 0.96]). CONCLUSION: This study suggested that NSAIDs usage was associated with decreased delirium prevalence and lower one-year mortality. The potential benefit of NSAIDs on delirium risk and mortality were shown.

Metformin use history and genome-wide DNA methylation profile: potential molecular mechanism for aging and longevity.

BACKGROUND: Metformin, a commonly prescribed anti-diabetic medication, has repeatedly been shown to hinder aging in pre-clinical models and to be associated with lower mortality for humans. It is, however, not well understood how metformin can potentially prolong lifespan from a biological standpoint. We hypothesized that metformin's potential mechanism of action for longevity is through its epigenetic modifications. METHODS: To test our hypothesis, we conducted a post-hoc analysis of available genome-wide DNA methylation (DNAm) data obtained from whole blood collected from inpatients with and without a history of metformin use. We assessed the methylation profile of 171 patients (first run) and only among 63 diabetic patients (second run) and compared the DNAm rates between metformin users and nonusers. RESULTS: Enrichment analysis from the Kyoto Encyclopedia of Genes and Genome (KEGG) showed pathways relevant to metformin's mechanism of action, such as longevity, AMPK, and inflammatory pathways. We also identified several pathways related to delirium whose risk factor is aging. Moreover, top hits from the Gene Ontology (GO) included HIF-1α pathways. However, no individual CpG site showed genome-wide statistical significance (p < 5E-08). CONCLUSION: This study may elucidate metformin's potential role in longevity through epigenetic modifications and other possible mechanisms of action.

The genome-wide DNA methylation profiles among neurosurgery patients with and without post-operative delirium.

AIMS: There is no previous study demonstrating the differences of genome-wide DNA methylation (DNAm) profiles between patients with and without postoperative delirium (POD). We aimed to discover epigenetic (DNAm) markers that are associated with POD in blood obtained from patients before and after neurosurgery. METHODS: Pre- and post-surgical blood DNA samples from 37 patients, including 10 POD cases, were analyzed using the Illumina EPIC array genome-wide platform. We examined DNAm differences in blood from patients with and without POD. Enrichment analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes terms were also conducted. RESULTS: When POD cases were tested for DNAm change before and after surgery, enrichment analyses showed many relevant signals with statistical significance in immune response related-pathways and inflammatory cytokine related-pathways such as "cellular response to cytokine stimulus", "regulation of immune system process", "regulation of cell activation", and "regulation of cytokine production". Furthermore, after excluding the potential effect of common factors related to surgery and anesthesia between POD cases and non-POD controls, the enrichment analyses showed significant signals such as "immune response" and "T cell activation", which are same pathways previously identified from an independent non-surgical inpatient cohort. CONCLUSIONS: Our first genome-wide DNAm investigation of POD showed promising signals related to immune response, inflammatory response and other relevant signals considered to be associated with delirium pathophysiology. Our data supports the hypothesis that epigenetics play an important role in the pathophysiological mechanism of delirium and suggest the potential usefulness of an epigenetics-based biomarker of POD.

The potential benefit of metformin to reduce delirium risk and mortality: a retrospective cohort study.

PURPOSE: Metformin has been reported to improve age-related disorders, including dementia, and to lower mortality. This study was conducted to investigate whether metformin use lower delirium risk, as well as long-term mortality. METHODS: In this retrospective cohort study, previously recruited 1,404 subjects were analyzed. The relationship between metformin use and delirium, and the relationship between metformin use and 3-year mortality were investigated. MAIN FINDINGS: 242 subjects were categorized into a type 2 diabetes mellitus (DM)-without-metformin group, and 264 subjects were categorized into a DM-with-metformin group. Prevalence of delirium was 36.0% in the DM-without-metformin group, and 29.2% in the DM-with-metformin group. A history of metformin use reduced the risk of delirium in patients with DM (OR, 0.50 [95% CI, 0.32 to 0.79]) after controlling for confounding factors. The 3-year mortality in the DM-without-metformin group (survival rate, 0.595 [95% CI, 0.512 to 0.669]) was higher than in the DM-with-metformin group (survival rate, 0.695 [95% CI, 0.604 to 0.770]) (p=0.035). A history of metformin use decreased the risk of 3-year mortality after adjustment for confounding factors (HR, 0.69 [95% CI, 0.48 to 0.98]). CONCLUSIONS: Metformin use may lower the risk of delirium and mortality in DM patients.

Genome-wide DNA methylation analysis of post-operative delirium with brain, blood, saliva, and buccal samples from neurosurgery patients.

OBJECTIVE: No previous study demonstrates the difference in the genome-wide DNA methylation status of post-operative delirium (POD) using human brain tissue obtained from neurosurgery and multiple peripheral tissues such as blood, saliva, and buccal samples from the same individuals. We aimed to identify epigenetic marks of DNA methylation in the brain and peripheral tissues to elucidate the potential pathophysiological mechanism of POD. METHODS: The four tissue types (brain, blood, saliva, buccal) of DNA samples from up to 40 patients, including 11 POD cases, were analyzed using Illumina EPIC array. DNAm differences between patients with and without POD were examined. We also conducted enrichment analysis based on the top DNAm signals. RESULTS: The most different CpG site between control and POD was found at cg16526133 near the ADAMTS9 gene from the brain tissue(p = 8.66E-08). However, there are no CpG sites to reach the genome-wide significant level. The enrichment analysis based on the 1000 top hit CpG site (p < 0.05) on the four tissues showed several intriguing pathways. In the brain, there are pathways including "positive regulation of glial cell differentiation". Blood samples showed also pathways related to immune function. Besides, both saliva and the buccal sample showed pathways related to circadian rhythm, although these findings were not FDR significant. CONCLUSION: Enrichment analysis found several intriguing pathways related to potential delirium pathophysiology. Present data may further support the role of epigenetics, especially DNA methylation, in the molecular mechanisms of delirium pathogenesis.

DNA methylation in the inflammatory genes after neurosurgery and diagnostic ability of post-operative delirium.

The pathophysiological mechanisms of postoperative delirium (POD) are still not clear, and no reliable biomarker is available to differentiate those with and without POD. Pre- and post-surgery blood from epilepsy subjects undergoing neurosurgery were collected. DNA methylation (DNAm) levels of the TNF gene, IL1B gene, and IL6 gene by the Illumina EPIC array method, and DNAm levels of the TNF gene by pyrosequencing, were analyzed. Blood from 37 subjects were analyzed by the EPIC array method, and blood from 27 subjects were analyzed by pyrosequencing. Several CpGs in the TNF gene in preoperative blood showed a negative correlation between their DNAm and age both in the POD group and in the non-POD group. However, these negative correlations were observed only in the POD group after neurosurgery. Neurosurgery significantly altered DNAm levels at 17 out of 24 CpG sites on the TNF gene, 8 out of 14 CpG sites on the IL1B gene, and 4 out of 14 CpG sites on the IL6 gene. Furthermore, it was found that the Inflammatory Methylation Index (IMI), which was based on the post-surgery DNAm levels at the selected five CpG sites, can be a potential detection tool for delirium with moderate accuracy; area under the curve (AUC) value was 0.84. The moderate accuracy of this IMI was replicated using another cohort from our previous study, in which the AUC was 0.79. Our findings provide further evidence of the potential role of epigenetics and inflammation in the pathophysiology of delirium.

Mortality among patients with sepsis associated with a bispectral electroencephalography (BSEEG) score.

We have previously developed a bispectral electroencephalography (BSEEG) device, which was shown to be effective in detecting delirium and predicting patient outcomes. In this study we aimed to apply the BSEEG approach for a sepsis. This was a retrospective cohort study conducted at a single center. Sepsis-positive cases were identified based on retrospective chart review. EEG raw data and calculated BSEEG scores were obtained in the previous studies. The relationship between BSEEG scores and sepsis was analyzed, as well as the relationship among sepsis, BSEEG score, and mortality. Data were analyzed from 628 patients. The BSEEG score from the first encounter (1st BSEEG) showed a significant difference between patients with and without sepsis (p = 0.0062), although AUC was very small indicating that it is not suitable for detection purpose. Sepsis patients with high BSEEG scores showed the highest mortality, and non-sepsis patients with low BSEEG scores showed the lowest mortality. Mortality of non-sepsis patients with high BSEEG scores was as bad as that of sepsis patients with low BSEEG scores. Even adjusting for age, gender, comorbidity, and sepsis status, BSEEG remained a significant predictor of mortality (p = 0.008). These data are demonstrating its usefulness as a potential tool for identification of patients at high risk and management of sepsis.

DNA methylation in the TNF-alpha gene decreases along with aging among delirium inpatients.

It has been suggested that aging and inflammation play key roles in the development of delirium. In the present study, we investigated the differences of the DNAm patterns in the TNF gene between patients with delirium and without. The data and samples derived from previous and ongoing cohort studies were analyzed. DNAm levels of the TNF gene were analyzed using the Illumina EPIC array genome-wide method and pyrosequencing method. Correlations between age and DNAm levels of each CpG were calculated. Several CpG in the TNF gene in blood showed negative correlation between their DNAm and age in delirium cases both with the EPIC array and by the pyrosequencing method. However, there was no CpG that had significant correlation between their DNAm and age regardless of delirium status among buccal samples. On the other hand, among peripheral blood mononuclear cells samples, it was found that several CpG showed negative correlation between their DNAm and age in delirium cases. The evidence of DNAm change in the TNF gene among delirious subjects was demonstrated.

Evaluation of point-of-care thumb-size bispectral electroencephalography device to quantify delirium severity and predict mortality.

BACKGROUND: We have developed the bispectral electroencephalography (BSEEG) method for detection of delirium and prediction of poor outcomes. AIMS: To improve the BSEEG method by introducing a new EEG device. METHOD: In a prospective cohort study, EEG data were obtained and BSEEG scores were calculated. BSEEG scores were filtered on the basis of standard deviation (s.d.) values to exclude signals with high noise. Both non-filtered and s.d.-filtered BSEEG scores were analysed. BSEEG scores were compared with the results of three delirium screening scales: the Confusion Assessment Method for the Intensive Care Unit (CAM-ICU), the Delirium Rating Scale-Revised-98 (DRS) and the Delirium Observation Screening Scale (DOSS). Additionally, the 365-day mortalities and the length of stay (LOS) in the hospital were analysed. RESULTS: We enrolled 279 elderly participants and obtained 620 BSEEG recordings; 142 participants were categorised as BSEEG-positive, reflecting slower EEG activity. BSEEG scores were higher in the CAM-ICU-positive group than in the CAM-ICU-negative group. There were significant correlations between BSEEG scores and scores on the DRS and the DOSS. The mortality rate of the BSEEG-positive group was significantly higher than that of the BSEEG-negative group. The LOS of the BSEEG-positive group was longer compared with that of the BSEEG-negative group. BSEEG scores after s.d. filtering showed stronger correlations with delirium screening scores and more significant prediction of mortality. CONCLUSIONS: We confirmed the usefulness of the BSEEG method for detection of delirium and of delirium severity, and prediction of patient outcomes with a new EEG device.