Significant Circadian Rhythm Research Articles

OP0086 Alterations of immune cellular circadian rhythms in rheumatoid arthritis

BackgroundThe circadian variation of clinical symptoms and the underlying variation of cytokine and hormone levels in rheumatoid arthritis (RA) are well described and have already led to the successful application...

Circadian variation of Valproic acid pharmacokinetics in mice

Valproic acid (VPA) is currently one of the most commonly used antiepileptic drugs. This study aims to investigate whether VPA pharmacokinetics varied according to circadian dosing-time. A single dose of VPA (350mgkg−1) was administered by intraperitonally (i.p.) route to a total of 132 mice synchronized for 3weeks to 12h light (rest span) and 12h dark (activity span). Four different circadian times (1, 7, 13 and 19 HALO) of drug injection were used (33 mice/circadian time). At each circadian time, blood samples were withdrawn at (0h) and at 0.083, 0.166, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2 and 3h after VPA injection. Plasma VPA concentrations were determined by an EMIT method.There were no significant differences in Tmax of VPA whatever the circadian-time of injections (Tmax=0.166h). However, there were relevant differences in Cmax between the four circadian groups (p<0.005), it varied between 386±30.86mgL−1 in mice treated at 7 HALO and 824±39.85mgL−1 in mice treated at 19 HALO. The AUC0–∞ was significantly two times higher when VPA was administered at 19 HALO as compared to the injection at 7 HALO. Drug dosing at 7 HALO resulted in highest ClT value: 0.405±0.006Lh−1kg−1, whereas ClT was significantly slower when VPA was administered at 19 HALO (0.157±0.009Lh−1kg−1) (p<0.0001). The AUC0–∞ was significantly 2-fold higher when VPA was administered at 19 HALO (2216.65±138.91mgh−1L−1) as compared to the injection at 7 HALO (864.09±16.82mgh−1L−1) (p<0.0001).Cosinor analysis showed circadian rhythm in different pharmacokinetic parameters. Cmax and AUC0–∞ have a significant circadian rhythm with an acrophase located at 20.16 HALO±0.16h (the middle of the active span) (p<0.001), whereas ClT and Vd showed a significant circadian rhythm with an acrophase located respectively at 7.86 HALO±0.57h and 6.13 HALO±0.07h (the middle of the rest span) (p<0.001).The large circadian variation of VPA pharmacokinetic processes might be involved in the mechanisms of circadian rhythm in murine toxicity since the optimal tolerance corresponded to the time which induces lowest Cmax and AUC values.

Circadian rhythm of TSH levels in subjects with Alzheimer’s disease (AD)

The circadian rhythm of serum thyroid stimulating hormone (TSH) levels in patients with Alzheimer's disease was measured by means of a case-control study. Serum samples from cases and controls were collected continuously for 2 days, and then once every 2 h (even number time-point during the first day and odd number time-point in the second). TSH was detected by radioimmunoassay. AD patients had no significant circadian rhythm in serum TSH levels, whereas normal controls did. In normal controls, serum TSH levels from 19:00 to 20:00 were the lowest (19:00, 3.89 ± 0.97 mIU/L; 20:00, 3.76 ± 0.84 mIU/L) and those in the period 2:00-4:00 were the highest (2:00, 6.15 ± 0.94 mIU/L; 3:00, 6.32 ± 1.04 mIU/L; 4:00, 6.39 ± 1.13 mIU/L; F = 6.762, df = 23, P = 0.002). However, in AD patients, 24-h serum TSH levels were 3.80-4.03 mIU/L (F = 0.897, df = 23, P = 0.996). At the 24 time-points, except for the four time-points from 16:00 to 19:00, TSH levels in AD patients were significantly lower than those in normal controls. The circadian rhythm of serum TSH levels in AD patients did not appear, and their serum TSH levels were significantly lower than those in normal controls. The circadian rhythm in serum TSH levels in AD patients differs greatly from that of the general population.

Temporal profiles of physical activity and energy expenditure in cancer in-patients

Fifty-three cancer in-patients (37 males and 16 females) were randomly selected from the Regional Cancer Center, Pt. J.N.M. College, Dr. B.R. Ambedkar Hospital, Raipur, India. Rhythms in physical activity (PA) and energy expenditure (EE) were studied non-invasively using Actical (Mini Mitter Co. Inc., USA) and compared with 24 apparently healthy subjects (11 males and 13 females). Data were gathered at 1-min epoch length for at least three to four consecutive days and were analyzed using several statistical techniques, such as Cosinor rhythmometry, ANOVA, Duncan's multiple-range test, and t-test. Most of the cancer in-patients and all control subjects exhibited a statistically significant circadian rhythm in PA and EE. However, the rhythm detection ratio was low among cancer in-patients. Patients had significantly lower 24-h average, lower amplitude, and an earlier acrophase in PA and EE rhythms. Further, significant effect of factor disease was discerned on total activity count (TAC) and total energy expenditure (TEE). TAC and TEE were significantly lower in cancer in-patients as compared to control subjects, irrespective of gender. In addition, a gradual decrement in PA intensity levels from sedentary to vigorous was validated in patients. From the present findings, it can be concluded that the factor disease might alter the temporal profiles of the PA and EE. However, further intensive studies involving more patients are required to reinforce the above conclusion.

The internal circadian clock increases hunger and appetite in the evening independent of food intake and other behaviors

ObjectiveDespite the extended overnight fast, paradoxically, people are typically not ravenous in the morning and breakfast is typically the smallest meal of the day. Here we assessed whether this paradox could be explained by an endogenous circadian influence on appetite with a morning trough, while controlling for sleep/wake and fasting/feeding effects.Design and MethodsWe studied 12 healthy non-obese adults (6 male; age, 20–42 year) throughout a 13-day laboratory protocol that balanced all behaviors, including eucaloric meals and sleep periods, evenly across the endogenous circadian cycle. Participants rated their appetite and food preferences by visual analog scales.ResultsThere was a large endogenous circadian rhythm in hunger, with the trough in the biological morning (8 AM) and peak in the biological evening (8 PM; peak-to-trough amplitude=17%; P=0.004). Similarly phased significant endogenous circadian rhythms were present in appetite for sweets, salty and starchy foods, fruits, meats/poultry, food overall, and for estimates of how much food participants could eat (amplitudes 14–25%; all P < 0.05).ConclusionsIn people who sleep at night, the intrinsic circadian evening peak in appetite may promote larger meals before the fasting period necessitated by sleep. Furthermore, the circadian decline in hunger across the night would theoretically counteract the fasting-induced hunger increase that could otherwise disrupt sleep.

Ventricular arrhythmias in children with attention deficit disorder – a symptom of autonomic imbalance?

Potential side effects of stimulants for attention deficit disorder are in the focus of scientific discussions, intensified by the higher number of prescriptions. Children with known arrhythmias or other severe cardiac problems should not receive stimulants because of their sympathomimetic effects. This is a retrospective analysis of 24-hour Holter electrocardiograms from 100 consecutive children with attention deficit disorder from January, 2006 to April, 2012. In all, nine children had significant ventricular arrhythmia (mean age 11.4 ± 3.1 years, 77% male, 77% received methylphenidate). All these children had ventricular parasystole - four of them with an accelerated idioventricular rhythm. A significant circadian rhythm of premature ventricular contractions in seven children and the effect of standing and exercise clearly indicate the influence of the autonomic nervous system. In these children, hourly analysis of circadian rhythm within a 24-hour period showed a highly significant correlation between premature ventricular contractions and the vagal tone indicated by the heart rate variability parameter RMSSD (r = -0.83; p < 0.001). Ventricular arrhythmia was unaffected in seven children who received methylphenidate before diagnosis and decreased during metoprolol treatment in two children. By Holter electrocardiogram analysis, we observed a remarkably high incidence of ventricular parasystole and accelerated idioventricular rhythm in nine of 100 children with attention deficit disorder, which depends on autonomic imbalance and not on stimulant treatment.

Circadian rhythm characteristics of oral squamous cell carcinoma growth in an orthotopic xenograft model

BackgroundRecent studies show that circadian rhythm changes are closely related to the occurrence and development of various tumors, such as breast, liver, and prostate. However, there are significant differences in circadian rhythm between different tumors. At present, the circadian rhythm characteristics of oral cancer remain unknown. The purpose of this study is to investigate the circadian rhythm characteristics of the in vivo growth of oral squamous cell carcinoma (OSCC).Materials and methodsThirty-two nude mice were placed under 12-hour light/12-hour dark cycles. The human OSCC cell line BcaCD885 was inoculated in the cheek of nude mice. After 3 weeks, eight mice were sacrificed at four time points, including 4 hours after light onset (HALO), 10 HALO, 16 HALO, and 22 HALO, during a period of 24 hours. The volume of excised tumors was measured and the proliferative index (PI) and apoptotic index (AI) of tumor cells were determined by flow cytometry. A cosine analysis method was used to determine whether the tumor volume, PI, and AI obeyed a circadian rhythm.ResultsThere was a significant circadian rhythm in the tumor volume and PI of OSCC cells. For the tumor volume, there were significant differences between the four time points. The peak and trough values of the tumor volume appeared at 3.23 HALO and 15.23 HALO, whereas the peak and trough values of PI appeared at 6.60 HALO and 18.16 HALO, respectively. However, there was no circadian rhythm in the AI of tumor cells, despite significant differences between the four time points.ConclusionThis study demonstrates, for the first time, that the tumor volume and PI of in vivo growing OSCC undergo circadian rhythms. These results support the assertion that time factor should be considered in the occurrence, development, treatment, efficacy evaluation and pathophysiology of OSCC.

Assessing circadian rhythms during prolonged midazolam infusion in the pediatric intensive care unit (PICU) children

BackgroundThis study evaluates possible circadian rhythms during prolonged midazolam infusion in 27 pediatric intensive care unit (PICU) children under mechanical ventilation. MethodsBlood samples for midazolam and 1-OH-midazolam assay were collected throughout the infusion at different times of the day. The blood pressure, heart rate and body temperature were recorded every hour for the rhythms analysis. Population nonlinear mixed-effect modeling with NONMEM was used for data analysis. ResultsA two-compartment model for midazolam pharmacokinetics and a one-compartment model for midazolam metabolite adequately described the data. The 24h profiles of all monitored physiological parameters were greatly disturbed/abolished in comparison with the well-known 24h rhythmic patterns in healthy subjects. There was no significant circadian rhythm detected with respect to midazolam pharmacokinetics, its active metabolite pharmacokinetics and all monitored parameters. ConclusionsWe concluded that the light-dark cycle did not influence midazolam pharmacokinetics in intensive care units children. Also, endogenous rhythms in critically ill and sedated children are severely disturbed and desynchronized. Our results confirmed that it is necessary to adjust the dose of midazolam to the patient's body weight. The low value of midazolam clearances observed in our study was probably caused by mechanical ventilation, which was shown to decrease the cardiac output.

Sleep-Wake Behavior in the Rat

Ultradian rhythms are a prominent but little-studied feature of mammalian sleep-wake and rest-activity patterns. They are especially evident in long-term records of behavioral state in polyphasic animals such as rodents. However, few attempts have been made to incorporate ultradian rhythmicity into models of sleep-wake dynamics, and little is known about the physiological mechanisms that give rise to ultradian rhythms in sleep-wake state. This study investigated ultradian dynamics in sleep and wakefulness in rats entrained to a 12-h:12-h light-dark cycle (LD) and in rats whose circadian rhythms were suppressed and free-running following long-term exposure to uninterrupted bright light (LL). We recorded sleep-wake state continuously for 7 to 12 consecutive days and used time-series analysis to quantify the dynamics of net cumulative time in each state (wakefulness [WAKE], rapid eye movement sleep [REM], and non-REM sleep [NREM]) in each animal individually. Form estimates and autocorrelation confirmed the presence of significant ultradian and circadian rhythms; maximum entropy spectral analysis allowed high-resolution evaluation of multiple periods within the signal, and wave-by-wave analysis enabled a statistical evaluation of the instantaneous period, peak-trough range, and phase of each ultradian wave in the time series. Significant ultradian periodicities were present in all 3 states in all animals. In LD, ultradian range was approximately 28% of circadian range. In LL, ultradian range was slightly reduced relative to LD, and circadian range was strongly attenuated. Ultradian rhythms were found to be quasiperiodic in both LD and LL. That is, ultradian period varied randomly around a mean of approximately 4 h, with no relationship between ultradian period and time of day.

Differential Patterns in the Periodicity and Dynamics of Clock Gene Expression in Mouse Liver and Stomach

The rhythmic recurrence of biological processes is driven by the functioning of cellular circadian clocks, operated by a set of genes and proteins that generate self-sustaining transcriptional-translational feedback loops with a free-running period of about 24 h. In the gastrointestinal apparatus, the functioning of the biological clocks shows distinct patterns in the different organs. The aim of this study was to evaluate the time-related variation of clock gene expression in mouse liver and stomach, two components of the digestive system sharing vascular and autonomic supply, but performing completely different functions. The authors analyzed the periodicity by cosinor analysis and the dynamics of variation by computing the fractional variation to assess the rate of change in gene expression. Five-week-old male Balb/c mice were exposed to 2 wks of 12-h light/12-h dark cycles, then kept in complete darkness for 3 d as a continuation of the dark span of the last light-dark cycle. The authors evaluated the expression of Bmal1, Clock, Cry1, Cry2, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, Timeless, Dbp, Csnk1d, and Csnk1e by using real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) in mouse liver and stomach. A significant 24-h rhythmic component was found for 10 genes in the liver (Bmal1, Clock, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp), and for 9 genes in the stomach (Bmal1, Cry1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp). In particular, Clock showed marked rhythm differences between liver and stomach, putatively due to some compensation by Npas2. The acrophase of the original values of Bmal1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 14.30 ± 7.94 degrees (57.20 ± 31.78 minutes). A statistically significant difference was found in the acrophases of Bmal1 (p = .015) and Npas2 (p = .011). Fractional variations provided significant circadian rhythms for nine genes in the liver (Bmal1, Per1, Per2, Per3, Rev-erbα, Rev-erbβ, Npas2, Timeless, and Dbp), and for seven genes in the stomach (Bmal1, Clock, Per2, Rev-erbα, Npas2, Dbp, and Csnk1e). The acrophase of the fractional variations of Bmal1, Per2, Per3, Rev-erbα, Rev-erbβ, and Dbp expression was delayed in the stomach, and the average delay expressed as mean ± SD was 19.10 ± 9.39 degrees (76.40 ± 37.59 minutes). A significantly greater fractional variation was found in the liver for Clock at 06:00 h (p = .034), Per1 at 02:00 h (p = .037), and Per3 at 02:00 h (p = .029), whereas the fractional variation was greater in the stomach for Clock at 10:00 h (p = .016), and for Npas2 at 02:00 h (p = .029) and at 06:00 h (p = .044). In conclusion, liver and stomach show different phasing and dynamics of clock gene expression, which are probably related to prevailing control by different driving cues, and allow them to keep going the various metabolic pathways and diverse functional processes that they manage. (Author correspondence: g.mazzoccoli@operapadrepio.it)

Circadian variability and nocturnal dipping pattern in blood pressure in young normotensive subjects

Blood pressure variability (BPV) is one of the recognized risk factors for different types of cardiovascular diseases (CVDs). Several physiological, behavioral and ethnic factors are known to modulate blood pressure (BP). Such studies on the population of southeast India are altogether absent. It is worthwhile, therefore, to examine the circadian variability in systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) and nocturnal dipping pattern in BP in apparently healthy human subjects as a function of gender. Sixty females and 40 males voluntarily participated in the study. All subjects wore an Ambulatory Blood Pressure Monitor (ABPM, TM 2430) for two to four consecutive days. SBP, DBP and HR was measured every 15 and 30 minutes between 07:00–22:00 and 22:00–07:00 hrs, respectively. In addition, double product (DP) and mean arterial pressure (MAP) were also computed. Data were analyzed using A&D, Cosinor and Spectre software. Prevalence of extreme dipper, dipper and non-dipper was 13%, 63% and 24%, respectively. A statistically significant circadian rhythm was validated in all studied variables, irrespective of gender and dipping pattern in BP. However, the rhythm detection ratio was low among non-dippers. Chi-square test revealed a statistically significant association of the frequency of prominent periods of SBP and MAP with nocturnal dipping categories. Further, significant differences for the circadian Mesors of SBP, DBP, HR, DP and MAP and acrophase of DBP and MAP were noticed between males and females. Dipping pattern produced a significant effect on the rhythm characteristics of all the studied variables. On the basis of our findings we can conclude that variability in BP may be associated with factor gender to some extent, whereas nocturnal dipping in BP is independent of gender. Interestingly in the present study about 24% subjects are non-dippers, which may be an indication of higher risk of CVDs among individuals belonging to younger generation of this region. However, further extensive study is desirable to strengthen the above conjecture.

Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.

A Circadian Rhythm in Heart Rate Variability Contributes to the Increased Cardiac Sympathovagal Response to Awakening in the Morning

Morning hours are associated with a heightened risk of adverse cardiovascular events. Recent evidence suggests that the sleep-wake cycle and endogenous circadian system modulate cardiac function in humans and may contribute to these epidemiological findings. The aim of the present study was to investigate the interaction between circadian and sleep-wake–dependent processes on heart rate variability (HRV). Fifteen diurnally active healthy young adults underwent a 72-h ultradian sleep-wake cycle (USW) procedure (alternating 60-min wake episodes in dim light and 60-min nap opportunities in total darkness) in time isolation. The present study revealed a significant main effect of sleep-wake–dependent and circadian processes on cardiac rhythmicity, as well as a significant interaction between these processes. Turning the lights off was associated with a rapid increase in mean RR interval and cardiac parasympathetic modulation (high-frequency [HF] power), whereas low-frequency (LF) power and sympathovagal balance (LF:HF ratio) were reduced (p ≤ .001). A significant circadian rhythm in mean RR interval and HRV components was observed throughout the wake and nap episodes (p ≤ .001). Sleep-to-wake transitions occurring in the morning were associated with maximal shifts towards sympathetic autonomic activation as compared to those occurring during the rest of the day. Namely, peak LF:HF ratio was observed in the morning, coincidental with peak salivary cortisol levels. These results contribute to our understanding of the observed increase in cardiovascular vulnerability after awakening in the morning. (Author correspondence: diane.boivin@douglas.mcgill.ca)

Chronotolerance study of the antiepileptic drug valproic acid in mice

BackgroundValproic acid (VPA) is an antiepileptic drug widely used for the treatment of absence seizures and generalized tonic-clonic seizures. The present work aims to study whether VPA-induced toxicity varies according to the dosing-time in the 24 hour-scale.MethodsThe influence of dosing-time on tolerance to VPA was investigated in 120 male Swiss mice synchronized under a light-dark cycle (12:12). The mean VPA lethal dose was first determined to be 850 ± 0.2 mg/kg, i.p.. Such a dose was administered by i.p. route to a total of 90 mice divided in six circadian stages [1, 5, 9, 13, 17 and 21 Hours After Light Onset (HALO)] (15 mice/circadian time); 30 mice were used as control (5 mice / circadian time).ResultsThe surviving treated mice exhibited a significant circadian variation in rectal temperature and body weight loss (p < 0.001). The least rectal temperature change and body weight loss occurred when VPA was injected at 9 HALO. Drug dosing at 9 HALO resulted in -9 % weight loss whereas drug dosing at 17 HALO was -15 % (Ø = 20.3 HALO ± 1.1 h, p ≤ 0.0001). Lethal toxicity also varied according to circadian dosing-time (χ2 = 42.1, p < 0.0001). The highest (60 %) and the lowest (6.67 %) survival rates were observed at 9 HALO and 17 HALO respectively. Cosinor analyses validated a significant circadian rhythm in survival duration with an acrophase at 8.4 HALO ± 0.75 h (p < 0.001).ConclusionsWith regards to these data the optimal tolerance to VPA occurred when the drug was administered in the second half of the light-rest span of mice which is physiologically analogous to the second half of the night for human patients.

Leptin-sensitive neurons in the arcuate nuclei contribute to endogenous feeding rhythms

Neural sites that interact with the suprachiasmatic nuclei (SCN) to generate rhythms of unrestricted feeding remain unknown. We used the targeted toxin, leptin conjugated to saporin (Lep-SAP), to examine the importance of leptin receptor-B (LepR-B)-expressing neurons in the arcuate nucleus (Arc) for generation of circadian feeding rhythms. Rats given Arc Lep-SAP injections were initially hyperphagic and rapidly became obese (the "dynamic phase" of weight gain). During this phase, Lep-SAP rats were arrhythmic under 12:12-h light-dark (LD) conditions, consuming 59% of their total daily intake during the daytime, compared with 36% in blank-SAP (B-SAP) controls. Lep-SAP rats were also arrhythmic in continuous dark (DD), while significant circadian feeding rhythms were detected in all B-SAP controls. Approximately 8 wk after injection, Lep-SAP rats remained obese but transitioned into a "static phase" of weight gain marked by attenuation of their hyperphagia and rate of weight gain. In this phase, Arc Lep-SAP rats exhibited circadian feeding rhythms under LD conditions, but were arrhythmic in continuous light (LL) and DD. Lep-SAP injections into the ventromedial hypothalamic nucleus did not cause hyperphagia, obesity, or arrhythmic feeding in either LD or DD. Electrolytic lesion of the SCN produced feeding arrhythmia in DD but not hyperphagia or obesity. Results suggest that both Arc Lep-SAP neurons and SCN are required for generation of feeding rhythms entrained to photic cues, while also revealing an essential role for the Arc in maintaining circadian rhythms of ad libitum feeding independent of light entrainment.

Evolution of time-control mechanisms in subterranean organisms: cave fishes under light-dark cycles (Teleostei: Siluriformes, Characiformes)

Subterranean organisms are excellent models for chronobiological studies, yet relatively few taxa have been investigated with this focus. Former results were interpreted as a pattern of regression of circadian locomotor activity rhythms in troglobitic (exclusively subterranean) species. In this paper we report results of experiments with cave fishes showing variable degrees of troglomorphism (reduction of eyes, melanic pigmentation and other specializations related to the hypogean life) submitted to light-dark cycles, preceded and followed by several days in constant darkness. Samples from seven species have been monitored in our laboratory for the detection of significant circadian rhythms in locomotor activity: S. typhlops, an extremely troglomophic species, presented the lowest number of significant components in the circadian range (only one individual out of eight in DD1 and three other fish in LD), all weak (low values of spectral power). Higher incidence of circadian components was observed for P. kronei – only one among six studied catfish without significant circadian rhythms under DD1 and DD2; spectral powers were generally high. Intermediate situations were observed for the remaining species, however all of them presented relatively strong significant rhythms under LD. Residual oscillations (circadian rhythms in DD2) were detected in at least part of the studied individuals of all species but S. typhlops, without a correlation with spectral powers of LD rhythms, i.e., individuals exhibiting residual oscillations were not necessarily those with the strongest LD rhythms. In conclusion, the accumulated evidence for troglobitic fishes strongly supports the hypothesis of external, environmental selection for circadian locomotor rhythms.

Spinal astrocytes contribute to the circadian oscillation of glutamine synthase, cyclooxygenase-1 and clock genes in the lumbar spinal cord of mice

Spinal astrocytes have key roles in the regulation of pain transmission. However, the relationship between astrocytes and the circadian system in the spinal cord remains poorly defined. In the current study, the circadian variations in the expression of several clock genes in the lumbar spinal cord of mice were examined by using real-time PCR. The expression of Period1, Period2 and Cryptochrome1 showed significant circadian oscillations, each gene peaking in the early evening. The expression of Bmal1 mRNA also exhibited a circadian pattern, peaking from around midnight to early morning. The mRNA levels of Cryptochrome2 were slightly, but not significantly altered. Molecules related to pain transmission were also investigated. The mRNA expression of glutamine synthase (GS), and cyclooxygenases (COXs), known to be involved in various spinal sensory functions, showed rhythmicity with a peak in the early evening, although the expression of the neurokinin-1 receptor, subunits of the N-methyl-d-aspartate receptor, and glutamate transporters did not change. In addition, we found that protein levels of GS and COX-1 were also high at midnight compared with midday. Furthermore, we examined the effect of intrathecal fluorocitrate (100pmol), an inhibitor of astrocytic metabolism, on the expression of oscillating genes in lumbar spinal cord. Fluorocitrate significantly suppressed astrocyte function. Furthermore, the circadian oscillation of clock gene expression and GS and COX-1 expression were suppressed. Together, these results suggest that a significant circadian rhythmicity of the expression of clock genes is present in the spinal cord and that the components of the circadian clock timed by astrocytes might contribute to spinal functions, including nociceptive processes.

Blunted Heart Rate Circadian Rhythms in Small for Gestational Age Infants during the Early Neonatal Period

Infants born with intrauterine growth restriction are at increased risk for adverse cardiovascular outcomes in neonatal and later life. Although circadian rhythm is a prognostic marker of cardiovascular health, the concern over the circadian rhythm of these infants is rarely observed. To determine the influence of intrauterine growth retardation on the pattern of circadian rhythm, heart rate (HR) circadian rhythmicity was analyzed in 39 small for gestational age (SGA; birth weight and height below <-2.0 standard deviation score [SDS]) and 117 appropriate for gestational age (AGA; >-1.5 to <1.5 SDS) infants within 72 hours of birth using spectral analysis and cosinor analysis. Amplitude, midline estimating statistic of rhythm, and acrophase calculated from circadian rhythm were analyzed with clinical variables. A significant HR circadian rhythm was observed in 23.1% of the SGA and 24.8% of the AGA group without significant differences; however, SGA infants exhibited remarkable smaller amplitudes compared with AGA in all gestational age (GA) groups (p < 0.001). Amplitudes in AGA infants were positively correlated with the GA or body composition relevant variables (p < 0.001, respectively), but not SGA infants. The blunted HR circadian rhythmicity in SGA infants showed in this study might indicate the vulnerability to pathophysiological condition and could potentially refer to cardiovascular disease in later life.

Mice Show Circadian Rhythms of Blood Pressure During Each Wake-Sleep State

A daily rhythm of blood pressure (BP), with maximum values in the activity period, carries important prognostic information. The extent to which this rhythm depends on behavioral factors remains debated. Mice are the species of choice for functional genomics. In mice, episodes of wakefulness and sleep are not restricted to particular daily periods, allowing BP in each wake-sleep state to be measured at each time of day. The aim of this study was to investigate whether a circadian rhythm of BP is manifest in each wake-sleep state in mice. Mice with B6 genetic background (n = 26) were implanted with a telemetric BP transducer and electrodes to discriminate wake-sleep states and recorded while housed under a 12:12 h light-dark period. For each mouse, 8 values of BP were obtained in each wake-sleep state (wakefulness, non-rapid-eye-movement sleep, and rapid-eye-movement sleep) by averaging over successive 3-h time bins. Analysis of variance evidenced a significant time effect in each wake-sleep state as well as a significant wake-sleep state × time interaction effect. In an additional group of mice (n = 3) recorded in constant darkness, the Lomb-Scargle periodogram also revealed a significant circadian rhythm of BP in each wake-sleep state. These findings demonstrate that during each wake-sleep state, mice show daily and circadian rhythms of BP in conditions of entrainment to the light-dark cycle and in free-running conditions of constant darkness, respectively. (Author correspondence: giovanna.zoccoli@unibo.it)

Alteration of the Circadian Clock in Children with Smith-Magenis Syndrome

Smith-Magenis syndrome (SMS) is associated with sleep disturbances and disrupted melatonin production. The study aimed to ascertain whether the sleep and melatonin production anomalies in SMS patients may be due to an alteration of the molecular mechanism of the circadian clock. Five SMS patients (3-17 yr old) and five healthy age-matched control subjects were involved in the study. Saliva and buccal scrub samples were collected every 4 h during a 24-h period. Daily profiles of melatonin were determined in saliva using a direct double-antibody radioimmunoassay. Daily profiles of clock gene mRNA levels (Per1, Per2, and Rev-erbα) were determined in buccal scrub samples by RT-PCR. In controls, melatonin levels were elevated during the nighttime and very low during the daytime. Daily profiles of clock genes, Per1, Per2, and Rev-erbα, mRNA levels in buccal mucosa exhibited significant and mutually synchronized circadian variations (Per1 and Rev-erbα: P < 0.001; Per2: P < 0.05); the mRNA levels were elevated during the daytime and decreased during the nighttime. In SMS patients, melatonin profiles were significantly altered compared with controls, being phase reversed, phase advanced, depressed, or abolished. Only Per1 and Rev-erbα mRNA profiles exhibited significant circadian rhythms (P < 0.05); the Per2 expression exhibited high variability, and the profile was out of phase with the other clock genes. Our findings suggest that the anomalies in melatonin profiles of SMS patients might be due to a disturbance of the molecular circadian clockwork.