Circadian Rhythms of Melatonin, Cortisol, and Clock Gene Expression in the Hyperacute Phase of Wake-Up Stroke: Study Design and Measurement
Wake-up stroke (WUS) refers to an ischemic stroke that occurs during nighttime sleep, accounting for approximately 20% of all acute ischemic stroke (AIS) cases. Epidemiological studies have shown that AIS exhibits a circadian distribution, with a higher frequency in the morning and a lower frequency during sleep. However, it remains unclear whether WUS is a random event or if it is driven by a distinct pathophysiological process, such as circadian rhythm disorder, which differentiates it from daytime stroke (non-WUS).
The circadian distribution of AIS may be attributed to the rhythmicity of cardiovascular parameters, such as blood pressure (BP), fibrinolytic activity, and platelet aggregation. These rhythms are regulated by the biological clock, a complex network of endogenous clocks that govern circadian rhythms. Researchers have proposed that WUS may result from the decompensation of endogenous circadian protective mechanisms in the brain. Clinical findings support this hypothesis. For example, Lundholm et al. compared BP variability in WUS and non-WUS patients and suggested that nocturnal autonomic nerve instability may play a role in WUS. Additionally, obstructive sleep apnea (OSA), a recognized risk factor for WUS, has been linked to abnormal circadian rhythms, which may contribute to vascular disease risk factors.
This study hypothesizes that in some WUS patients, nighttime rhythmicity may be attenuated or abolished. Circadian rhythms are driven by the suprachiasmatic nuclei (SCN) in the anterior hypothalamus, which regulate the expression of core clock genes, including CLOCK, BMAL1, CRY1, CRY2, PER1, PER2, and PER3. These genes form auto-regulatory feedback loops that maintain accurate circadian rhythms, even in the absence of external cues. Peripheral blood cells also contain a circadian clock similar to the SCN. Melatonin and cortisol are traditional circadian biomarkers. Melatonin, considered a reliable output of the endogenous clock, is minimally influenced by environmental factors. Cortisol secretion follows the light/dark cycle and exhibits robust circadian oscillations.
To explore these mechanisms, this study aims to evaluate circadian phase variations in WUS patients by measuring plasma concentrations of cortisol and melatonin, as well as clock gene expression in peripheral blood cells. These measurements will be compared with those of non-WUS patients and healthy volunteers. The study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University and registered in the Chinese Clinical Trial Registry.
This single-center, cross-sectional, observational study includes three groups: the WUS group, the non-WUS group, and the healthy control group. The WUS group consists of individuals who go to sleep healthy and wake up with stroke symptoms. The non-WUS group includes patients with a definite daytime stroke onset. The healthy control group comprises age- and sex-matched volunteers. All AIS patients arriving at the Emergency Department are screened for eligibility based on strict inclusion and exclusion criteria to minimize confounding factors. Inclusion criteria include age 40 to 80 years, lesions located in the anterior circulation, clear consciousness, and hospital arrival time between 6 AM and 6 PM. Exclusion criteria include thalamic lesions, mechanical thrombectomy treatment, consumption of caffeinated beverages within 24 hours, confirmed depression or psychiatric disorders, history of stroke or neurological diseases, sleep disorders, blindness, severe liver or renal dysfunction, malignant diseases, shift work, travel across time zones within six weeks, and use of medications such as beta-receptor blockers, steroids, benzodiazepines, opioids, and immunosuppressants.
All enrolled patients undergo basic examinations within 24 hours of admission, including physical and neurological examinations, laboratory tests, electrocardiograms, cerebral computed tomography (CT), CT angiography, and perfusion scanning. Participants are instructed to maintain their normal nocturnal sleep patterns, avoid daytime naps, and are exposed to natural and conventional fluorescent light during awake hours. Lights are turned off between 8 PM and 10 PM, depending on individual sleeping habits. Participants wear activity bracelet monitors and eye masks while sleeping. During overnight sampling, a dim flashlight is used to avoid light-induced suppression of melatonin secretion. Blood samples are collected at 6-hour intervals starting at a fixed time point after enrollment. Each sample is tested for melatonin and cortisol concentrations, as well as the expression levels of seven clock genes: CLOCK, BMAL1, PER1, PER2, PER3, CRY1, and CRY2. Heart rate, body temperature, and BP are recorded hourly over 24 hours. Sleep parameters, including total sleep time, number of awakenings, rapid eye movement sleep percentage, light sleep percentage, and deep sleep percentage, are obtained after participants wake up.
The study aims to recruit 15 subjects per group, with an anticipated dropout rate of 25%, resulting in approximately 11 subjects per group. Dropout reasons may include reduced sleep quality during the study or the observation of thalamic or posterior circulation lesions on magnetic resonance imaging at a later stage.
The primary endpoints of the study are the circadian rhythmicity of plasma melatonin and cortisol concentrations, clock gene expression, heart rate, body temperature, and BP in the WUS group, and the differences in circadian rhythms among the three groups. Secondary endpoints include the potential impact of the biological clock on WUS occurrence.
Data analysis will compare study endpoints among the three groups using the MetaCycle R package, with multiple comparison testing corrected using the Fisher method. Continuous variables will be presented as mean ± standard deviation or median (interquartile range), depending on the normality of distribution. Analysis of variance (ANOVA) or Welch ANOVA will be used to compare variables among groups, and categorical variables will be compared using the Chi-square test. A P-value of 0.05 will be considered statistically significant.
This study is among the first to investigate circadian rhythms in the hyperacute phase of WUS. However, as a cross-sectional study, it can only evaluate associations, not causation. If positive results are found, they will suggest an association between circadian rhythm and WUS, providing novel insights for further exploration of WUS pathogenesis.
This study is supported by grants from the National Key Research & Development Program of China and the Social Development Project of Jiangsu. The authors declare no conflicts of interest.
doi.org/10.1097/CM9.0000000000001111
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