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Keywords:

  • circadian rhythm;
  • colorectal distension;
  • cortisol;
  • visceral hyperalgesia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Most patients with functional bowel disorders complain of daytime symptoms while they remain asymptomatic at night. As symptoms are associated with heightened visceral sensitivity, we hypothesized that circadian fluctuations of the visceral sensory function occur. At four different timepoints (06.00, 12.00, 18.00 and 24.00 h), colorectal distensions (CRD) were performed in fasting conscious male Lewis rats using a balloon catheter and a barostat device. The abdominal wall contractions (behavioural pain response) were assessed during colorectal distension by abdominal wall electromyography (EMG). Plasma levels for endogenous cortisol were determined simultaneously at these timepoints. EMG responses to CRD were significantly (P < 0.05) higher at midnight and in the early morning. Plasma cortisol levels peaked in the evening. In night-active Lewis rats, the behavioural pain response to noxious visceral stimulation is augmented at night and fluctuations of visceral sensitivity are accompanied by circadian changes of plasma concentrations of endogenous cortisol. We conclude that there are marked circadian fluctuations in visceral sensory functions. Thresholds are low during time periods of normal behavioural activity. These findings suggest that fluctuation of the sensory functions may be linked to the circadian variability of symptoms in patients with functional GI disorders.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Functional bowel disorders are characterized by a more or less typical pattern of symptoms and the absence of structural lesions or obvious biochemical abnormalities to explain these symptoms.1 It is now widely accepted that an abnormal visceral sensory function plays an important role in the development of symptoms in these patients.2–5 The majority of patients with functional gastrointestinal (GI) disorders reports an improvement in, or even a complete absence of, symptoms at night. Indeed, the absence of symptoms at night has been considered a positive indicator for functional bowel disorder rather than an organic disease. Martinez and coworkers6 have described changes in pain thresholds in rats with oestrus and time of day using the tail-flick test. In cell physiological studies, circadian oscillations in N-methyl-D-aspartate (NMDA)-evoked Ca2+ transients have been observed in neurones of the suprachiasmatic nucleus.7 Similarly, nitric oxide synthase in the rat pineal gland is known to exhibit a circadian rhythm.8 Thus, a potential explanation for the obvious diurnal fluctuations of symptoms associated with visceral pain are differences in the processing of visceral afferent information.

In the current study, we aimed to analyse diurnal variations in the behavioural pain response to a standardized noxious visceral (colonic) stimulation. We hypothesized that the behavioural pain response to a standardized visceral stimulus peaks during time periods of normal behavioural activity.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Subjects and stimuli

Male Lewis rats (Harlan, Borchen, Germany), weighing 340–390 g, were used. As a visceral stimulus, mechanical distensions of the descending colon and rectum were performed by pressure-controlled air inflation of a 4.5-cm flexible plastic balloon connected to an electronic distension device (Distender Series IITM barostat; G & J Electronics, Willowdale, Ontario, Canada). The balloon was lubricated (Vaseline; University of Essen, Pharmacy, Essen, Germany), inserted intra-anally and positioned such that the end of the balloon was 1 cm proximal to the anus. The balloon was secured in place by taping the catheter to the tail. The visceromotor response to colorectal distension (CRD) in the rat has been intensively characterized by Ness and Gebhart9 and consists of a contraction of the abdominal and hindlimb musculature. In this study, the visceromotor response to CRD was quantified by electromyographic (EMG) recordings of abdominal wall muscle activity.

Surgical preparation

Under combined anaesthesia with ketamine hydrochloride (Ketamin® 10%; Sanofi-Ceva, Dusseldorf, Germany; 1 mL kg−1 bodyweight) intraperitoneally (i.p.) and xylazin hydrochloride (Xylazin® 2%; Sanofi-Ceva, Dusseldorf, Germany; 0.05 mL per animal i.p.), a midline incision was performed under sterile conditions to open the abdomen. For the EMG recordings, Teflon-insulated silver wires (Cooner Wire Co., Cathsworth, CA, USA) were used as bipolar electrodes and implanted into the musculus obliquus externus above the ligamentum inguinale. The EMG electrodes were externalized at the dorsal neck of the animals. Animals were housed individually after surgery with food and water ad libitum. After surgery, rats were allowed to recover for 4 days before experimental use. The animals were kept on a 12-h light/dark cycle.

Experimental protocol

After a fasting period (18–24 h), the balloon was inserted into the descending colon and rectum under light halothane (Halothan Eurim®; Eurim-Pharm Arzneimittel, Piding, Germany) anaesthesia. EMG electrodes were connected with an EMG recording system (ISOLAB/IDAA Intestinal Data Acquisition and Analysis; Fa Standard Instruments, Karlsruhe, Germany). Rats were placed in Boreman cages. In pilot experiments we tested various colonic distensions. Distending pressures of 20 and 40 mmHg did not result in a significant behavioural pain response in all animals. A distending pressure of 60 mmHg induced a visible behavioural pain response in all animals tested. Increasing the distending to 80 mmHg further increased the pain response with no further increase for a pressure of 100 mmHg. Thus we used distensions at a pressure of 60 mmHg and 3-min duration as a noxious colonic stimulus.

EMG was recorded until 60 s after the distension. The EMG signal was amplified by the factor 100.000 on the ISOLAB system and filtered by a bandpass filter with high selectivity at a centre frequency of 120 Hz. The integrated EMG response was used as the response parameter. The measurement of the visceromotor reflex to tonic distension was repeated three times and the experiments were performed every other day. There were four different timepoints: 06.00, 12.00, 18.00 and 24.00 h. Each rat was distended once at every timepoint. On a given study day, only one time period was assessed and all measurements completed within 45 min (i.e. measurements done between 06.00 and 06.45 h). To avoid an artificial bias in the data by a day-to-day desensitization,10 four groups of rats were started randomly at these four different times.

Measurement of cortisol levels in the serum

Blood samples were drawn by intracardiac exsanguination, 30 min after the last CRD. The blood was collected into plastic tubes with EDTA. After centrifugation, plasma was stored at −20 °C until the assays were started. To measure the plasma concentrations of cortisol, an automated immunoassay with chemiluminescence technology (ACS:180 Cortisol; Chiron Diagnostics Corp., CA, USA) was used.

Statistical analysis

To take into account the different starting points of the four groups of animals, a split-plot design was used. Data are expressed as mean ± standard error throughout the manuscript. In order to determine differences in sensory function, ANOVA was performed to identify differences in the visceral motor response for different timepoints and to assess the influence of the repetition of the measurement. P < 0.05 was considered significant. In addition, Spearman rank correlations were assessed to determine the association between the plasma cortisol level and the EMG response to CRD.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Diurnal visceromotor response to distension stimulus

The EMG responses showed marked diurnal fluctuation. While abdominal EMG responses were lowest during daytime (345 ± 74 μV × 100 at 12.00 h), responses peaked at midnight and during the early morning (757 ± 174 μV × 100 at 24.00 h) (Fig. 1).

image

Figure 1.  Circadian variability of pain response (abdominal EMG activity) to colorectal distension (60 mmHg, 3 min) in male Lewis rats (n=8). Electrical recording of abdominal musculature activity during distension period. In contrast to the daytime recordings, EMG response to CRD was significantly increased at night (*P < 0.01 vs. 24.00 and 06.00 h).

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Hormone concentrations in the serum

Fluctuations of cortisol plasma levels also demonstrated a biphasic pattern. However, the peak of cortisol concentrations was measured in the evening: 94.8 ± 5.3 nmol mL−1 at 18.00 h vs. 69.3 ± 3.3 nmol mL−1 at 12.00 h (Fig. 2).

image

Figure 2.  Plasma cortisol levels at 06.00, 12.00, 18.00 and 24.00 h (male Lewis rats, n=8). Following a maximum concentration in the evening, the cortisol levels consecutively decreased to a minimum at 12.00 h (*P < 0.05 vs. 06.00 h and **P < 0.01 vs. 12.00 h).

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Correlation between EMG response and cortisol plasma levels

Cortisol levels and the EMG responses to CRD were significantly correlated (r=0.62; P < 0.02).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Intensity of symptoms in patients with functional GI disorders are characterized by a marked diurnal variability and most patients are free of symptoms at night.11 Although the precise mechanisms for the development of symptoms in these patients are still unknown, longitudinal studies suggest a link between sensory thresholds and symptom severity.12 Thus, the diurnal variability of symptoms may be linked to diurnal variations of sensory thresholds. It is now widely believed that the intensity of the behavioural response to a noxious stimulation of the colon reflects sensory function. For this reason we assessed diurnal variations of the behavioural pain response to a standardized colonic stimulation. Although eating very probably affects GI sensory function, our animals were restrained from eating for 18–24 h prior to the study, thus it is very unlikely that their eating habits or the direct influence of nutrients affected sensory thresholds. The aim of our study was to identify the role of daytime changes rather than reversing the cycle. Indeed, we observed distinct diurnal fluctuation of the behavioural pain response to a standardized noxious visceral stimulus. At night, the time period of normal behavioural activity in these rats, responses were significantly higher compared to the daytime response, when the rats usually rest. Thus, our data for the first time demonstrate a distinct diurnal variability in the sensory function during noxious colorectal stimulation in rats.

A previous study examined the behavioural response to cyclophosphamide-induced cystitis. In this study,13 the behavioural response 2 h after the injection was augmented if the noxious stimulus was administered during the time period of normal rat activity. Thus, these data mirror the results of our study. However, it is noteworthy that there is a distinct circadian susceptibility to cyclophosphamide toxicity, which is believed to be linked to a diurnal variation of metabolism of cyclophosphamide. Thus it has been argued that the diurnal variations in the behavioural response to a noxious cyclophosphamide application simply reflect differences in the severity of mucosal damage.

The methodology of rectal distensions for the assessment of mechanosensory function has been introduced by Ness and Gebhart14 and is now widely accepted as a reliable and valid tool to apply a noxious visceral stimulus to conscious and unanaesthetized animals. In our study, barostat technology15 was used for the distension instead of a volume-controlled distension. This technology accounted for potential diurnal variations of colonic tone, thus the stimulus was highly standardized and was not influenced by potential diurnal fluctuations of the colonic tone.

Colorectal stimulation is an acute mechanical stimulation of the colon. At high intensities, it can cause nociception in rats and may cause pain in humans. Thus, the colorectal stimulation can be considered a highly standardized mechanical colonic stimulus that induces acute colonic pain.14

Colonic balloon distensions have been extensively tested and characterized. They are associated with locus coeruleus activation, triggering of the central stress response and activation of endogenous pain inhibition systems involving γ-amino butyric acid, noradrenaline, 5-hydroxytryptamine and opioids.14

Circadian fluctuations in CNS activity and the release of peptides that are associated with sensory functions most likely play a role.16 Thus, diurnal fluctuations of brain activity may directly influence the behavioural response or modify the transmission of afferences. Besides the direct link between brain activity and the intensity of a behavioural response, other mechanisms may also play a role for the diurnal fluctuation in the behavioural pain response. In our study, the behavioural pain response to visceral stimulation was higher following the daily cortisol peak. This observation is in line with a pronociceptive effect of corticotropin-releasing factor (CRF) or other peptides that are released by this peptide. In one study, CRH administration resulted in an augmented adrenocorticotropic hormone release in irritable bowel syndrome (IBS) patients compared to healthy controls. In addition, abdominal symptoms that occurred after CRH administration lasted 10 times longer in IBS patients (21 vs. 2 min) compared to controls.17 These data suggest that: (i) the hypothalamic–pituitary axis may play a role in the development of symptoms in patients with functional GI disorders; and (ii) patients with functional GI disorders exert an augmented response of this endocrine axis.

However, multiple alterations in body chemistry occur with diurnal variation.18,19 While we observed a statistically significant correlation between the cortisol level and the behavioural pain response, we believe that the cycling of cortisol that mirrors ‘cycling’ in the behavioural response to a standardized noxious colorectal distension does not prove a causal relation between fluctuations of mechanosensory function and cortisol. However, based upon our data there is no doubt that diurnal fluctuations of visceral sensory function occur. The precise mechanisms, however, remain to be established.

In summary, the present study demonstrates a marked diurnal fluctuation in the behavioural response to a noxious colonic stimulation. While the diurnal variability may be due to altered visceral afferent or efferent function, it is a likely explanation for the striking diurnal variability of symptom intensities in patients with functional GI disorders. Further studies are needed to identify the precise mechanisms that cause diurnal fluctuations in the visceral sensory function.

ACKNOWLEDGMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

The skilful assistance of Ms. A. Pietsch is greatly appreciated. The study was supported by a grant from the IFORES Program of the University of Essen.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References
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