Effects of Circadian Rhythm Disorders on Wound Healing and Strength of Bowel Anastomosis in Rats

Introduction. Many body systems, especially the immune system, are affected by circadian rhythm disorders (CRDs). This study investigated the effects of shift lag on wound healing and bowel anastomosis in rats. Materials and Methods. Forty-five rats were randomly divided into 3 groups: placebo, control, and experimental. Circadian rhythm was disturbed with altered light/dark cycles. Colon anastomosis was performed in rats with and without disturbed circadian rhythms. Serum melatonin levels and bowel anastomosis bursting pressures were measured. Tissue samples were used to measure tissue hydroxyproline levels and for histological assessment. Results. In the groups with an altered circadian rhythm, the bowel bursting pressures, tissue hydroxyproline levels, and fibrosis were greater than the control group. Conclusion. This study found that a CRD, simulated by altered light/dark cycles, positively affected the anastomotic bursting pressure. This suggests that cytokines such as interleukin (IL)-1, IL-6, transforming growth factor α, and stress hormones affect fibroblasts and thereby increase collagen synthesis in the proliferative phase. More studies are necessary to understand the effects of CRDs.

Factors in modern lifestyles, such as working conditions, social activities, and widespread transmeridianal flights, are common causes of circadian rhythm disorders (CRDs). Circadian rhythem disorder after a long-distance flight is often called jet lag, whereas that experienced by people working unusual shifts is called shift lag. Malaise, ailments, fatigue, lassitude, insomnia, constipation, diarrhea, nausea, swelling, nervousness, perception disorders, and reduction in working performance are the main biological symptoms of CRDs.^1-4

  Wound healing takes place in 3 steps: the inflammatory phase, the proliferative phase, and the remodeling phase.⁵ Neutrophils, T cells, macrophages, platelets, and fibroblasts are important cellular elements of wound healing.⁶ Immune system cells and their products stimulate wound healing during the proliferative phase and angiogenesis.⁷

  In humans, melatonin is produced by the pineal gland. The signal from melatonin forms part of the system that regulates the sleep/wake cycle by chemically inducing drowsiness and lowering body temperature.⁸ Aside from its function as a biological clock synchronizer, melatonin is a powerful free-radical scavenger and wide-spectrum antioxidant.⁹ Endogenous melatonin in human lymphocytes is related to interleukin (IL)-2 production and the expression of IL-2 receptors.¹⁰

  In 2007, the World Health Organization identified late-night shift work as a probable cancer-causing agent. Melatonin is an antioxidant and suppressor of tumor development that is produced at night. When an individual works in artificial light, they generally have lower melatonin levels, and may be more likely to develop cancer.¹¹

  Sleep deprivation promotes several changes in body chemistry, including a marked increase in the production of stress hormones, such as catecholamines and cortisol; a reduction in cognitive capacity; and a reduction in alertness.¹²

  Levels of IL-1 in cerebrospinal fluid and plasma as well as mRNA expression in the brain vary with circadian rhythms. In humans, plasma levels of IL-1 peak at the onset of sleep. Another proinflammatory cytokine involved in sleep regulation is tumor necrosis factor α (TNFα). There is a diurnal rhythm to TNFα levels in plasma, and the ability of monocytes to produce TNFα is related to sleep/wake behavior and increases during sleep deprivation.¹³

  Immunogens, including sleep deprivation, are stressors that activate the hypothalamo-pituitary-adrenal axis and the sympathetic nervous system. The effects are mediated by the release of cytokines from activated cells of the immune system. Activation of these stress pathways regulates the immune response to restore and protect internal homeostasis. Stress mediators act on immune cells to modulate the production of key regulatory cytokines.^14,15 The plasma concentration of IL-6 displays a diurnal rhythm, with peak levels during sleep and nadirs during wakefulness.¹⁶ Also, IL-6 and TNFα plasma concentrations are increased in humans with sleep disorders such as sleep apnea, narcolepsy, and obesity.¹⁷

  Studies have shown that prolonged sleep deprivation leads to a reduction in body mass, elevated energy metabolism, changes in circulating hormones, and loss of immune system integrity.¹⁸

  The risk of coronary heart disease, diabetes mellitus, breast cancer, and colorectal cancer may also be higher in shift workers,^2–4,19 where one study reported that Parkinson’s disease was less prevalent among nurses working in nightshifts.²⁰ Circadian rhythm disorders affect the levels of IL-1, IL-2, IL-6, TNFα, natural killer cells, adrenocorticotropic hormone (ACTH), cortisol, growth hormone, and melatonin, all of which are important in wound healing.^21–24

  The effects of CRDs on wound healing and bowel anastomosis are unknown. In the present study, the effects of shift lag on wound healing and the strength of bowel anastomosis in rats is examined.

 Animals and preoperative preparation. The study was approved by the Kocaeli University Local Ethics Committee for Animal Experiments (number: 2008/039).

  Forty-five Wistar albino male rats (200-300 g) were used. They were housed under standard laboratory conditions, with the room temperature at 22ºC, and were acclimated for 2 weeks to a 12 hour light/dark cycle to induce CRD. They received normal rodent food and water ad libitum. The rats were divided into 3 groups: placebo (unaltered circadian rhythm without bowel anastomosis), control (unaltered circadian rhythm with bowel anastomosis), and experimental (disturbed circadian rhythm by altering the light/dark cycle and with bowel anastomosis) groups.

  Special cages were used that were impermeable to light and had a timed lamp. The light/dark cycles of the experimental group were changed, and rats were subjected to twice-weekly 12-hour shifts of the daily light/dark cycle with respect to the protocol of Tsai’s model, and placed in the special cages for 2 weeks.²⁵ The other groups remained on the 12 hour light/dark cycle. The same light/dark cycle was continued after the operation.

  Surgical technique. Investigators were blinded to the groups during the surgical procedures. The same anesthetic protocol was used with all animals: 50 mg/kg ketamine (Ketalar, Pfizer, Istanbul, Turkey) with 10 mg/kg xylazine HCl (Rompun, Bayer, Istanbul, Turkey). Preoperative weights were evaluated and noted. Bowel anastomosis was performed on the control and experimental groups. The abdominal skin was shaved and painted with iodine povidone solution under general anesthesia. All surgical procedures were performed under sterile conditions. The colon was cut 5 cm distal to the ileocecal valve. Anastomosis was performed with a single layer of 6-8 sutures with 6/0 prolene. Midline incisions were closed with 3/0 silk sutures. All rats were returned to their cages after operations and kept at an ambient temperature of 22ºC. They were fed a standard rat diet after surgery. Weights were evaluated and noted on postoperative day 7, and rats were anesthetized using the same protocol as above. All groups underwent the surgical procedure. Three cm of colon centered on the anastomosis were resected. The distal parts of the resected segments were closed with 3/0 silk sutures. The proximal parts were attached to an intraluminal pressure manometer (monitoring kit L978- A07; Abbott, Slingo, Ireland). The segments were filled with isotonic sodium chloride solution with a continuous infusion (4 mL/min). The rising intraluminal pressures were monitored, and the bursting pressures were noted. Then 4 mL blood was sampled from the right ventricle. All surgical procedures were performed in the afternoon so more elevated serum melatonin levels were expected.

  Histopathologic evaluation. For histopathological examination, 1 cm of tissue from the anastomosis site was fixed in 10% buffered formaldehyde solution and stained with Masson’s trichrome. Inflammatory cells and collagen levels were evaluated.

  Biochemical evaluation. The ELISA method was used to measure serum melatonin levels using Melatonin ELISA (EIA–1431, DRG International, Inc, Springfield, NJ). Serum was separated by centrifugation, and then stored at -80ºC. Tests were manually evaluated, and results are noted in pg/mL. For the determination of total hydroxyproline expressed per mg of tissue, the Switzer method was used, which consists of hydrolysis of the tissue homogenate and determination of free hydroxyproline in hydrolyzates. A portion of the anastomosis was resected, washed with saline solution, and dried. Dry tissue weight was measured, and samples were stored at -80ºC.

Statistical Analysis. Data were analyzed using the commercially available PASW Statistics for Windows (version 18.0, SPSS Inc, Chicago, IL). A P value < 0.05 was considered significant. The variable distribution of the results did not have a normal distribution. Therefore the data were analyzed with nonparametric tests (Wilcoxon, Kruskal-Wallis, Mann-Whitney U, and correlation).

  The mean preoperative weights of the placebo, control, and experimental groups were 236 g, 214 g, and 229 g, respectively. On postoperative day 7, mean weights were 231 g, 218 g, and 240 g, respectively. The differences among groups were not significant (P = 0.505).

  The mean serum melatonin levels were 41 pg/mL in the placebo group, 52 pg/mL in the control group, and 99 pg/mL in the experimental group. Despite the melatonin level of the experimental group being twice as high as the other groups, the result was not significant (P = 0.262).

  The mean hydroxyproline levels in tissue were 1053 µg/g in the placebo group, 150 µg/g in the control group, and 1205 µg/g in the experimental group. The levels in the placebo and experimental groups were significantly higher than in the control group (P < 0.001). There was not a significant difference between the placebo and experimental groups (P = 0.543).

  The mean bursting pressures were 278 mm Hg in the placebo group, 212 mm Hg in the control group, and 270 mm Hg in the experimental group. The pressures were significantly higher in the experimental group and placebo group than in control group (P = 0.004, P = 0.003). There was not a significant difference between the experimental and placebo groups (P = 0.861). These results are summarized in Table 1.

Circadian rhythm disorders due to shift work and jet lag are common. For the purposes of this study, CRD was induced in rats by altering their light/dark cycles; the effects on wound healing and bowel anastomosis were evaluated. The main hormones altered in CRDs are ACTH, cortisol, growth hormone, and melatonin. Many studies have investigated the effects of these molecules on wound healing.^26-30

  In a previous study that assessed bowel bursting pressure and tissue hydroxyproline levels after colon anastomosis, the effect of melatonin was not determined to be significant.²⁷ Another study reported positive effects of pinealectomy and negative effects of melatonin on wound healing.²⁹ Yet another study found that melatonin improved wound healing when given in concert with a normal circadian rhythm.³⁰ This variability in results is likely caused by differences in applied melatonin levels, application time, and the pathophysiologies of the study subjects.

  The effects of circadian rhythm on the immune system and wound healing are not yet clearly identified. These results help clarify the relationship between CRDs and wound healing. Melatonin reaches a maximum plasma concentration between 3 and 4 in the morning. Melatonin secretion continues at a baseline level during the day. Pharmacologically suppressing melatonin synthesis at night and increasing its levels during the day can lead to CRD.³¹ Melatonin has potential effects on the immune system, as inhibition of pineal melatonin synthesis with propranolol results in immunosupression.³²

  During the intestinal healing process, fibroblasts and smooth muscle cells in the submucosa activate collagen synthesis. Interleukin-1 accelerates smooth muscle proliferation in this phase. In the proliferative phase of healing, collagen is both synthesized and degraded. Collagen degradation is the greatest in the early phase, and its synthesis becomes dominant with the help of TGF-β, which affects migrating cells for this purpose. The strength of the anastomosis depends on the submucosa. Collagen condenses in the submucosa and provides strength for increased intraluminal pressures. Until postoperative day 4, collagen degradation leads the process, but at postoperative day 7 collagen synthesis predominates.³³

  Although there was no anastomosis performed in the placebo group, the bursting pressure in the experimental group was almost as high as in the placebo group. Also, tissue hydroxyproline levels were higher in the experimental and placebo groups. These results may be due to the effects of cytokines IL-1, IL-6, and TNFα on increasing collagen synthesis via their effects on fibroblasts.

  The most important steps of the proliferative phase are epithelization, angiogenesis, granulation, and collagen accumulation. In the present study, investigations were done on postoperative day 7 when the proliferative phase was dominant. Collagen accumulation increases tissue durability; however, this does not explain why these measurements were lower in the control group. This finding may be due to higher levels of stress hormones caused by altered light/dark cycles and the short-term effects on wound healing. Unfortunately, this study did not evaluate levels of stress hormones.

  There should be further clinical studies on this subject, and the short-term and long-term effects should be investigated separately. The hormone levels should also be included as a parameter.

  In conclusion, this study found that a CRD created by altered light/dark cycles in rats positively affected the anastomotic bursting pressure. This may be due to the effects of cytokines such as IL-1, IL-6, and TNFα and stress hormones that affect fibroblasts and increase collagen synthesis in the proliferative phase. Nevertheless, wound healing remains a multifactorial and complex process.

Affiliations: Mesut Sipahi, MD; and Ergin Arslan, MD are from the Department of General Surgery, Faculty of Medicine, Bozok University, Yozgat, Turkey. Kürşad Zengin, MD; and Serhat Tanik, MD are from the Department of Urology, Faculty of Medicine, Bozok University, Yozgat, Turkey. Anıl Çubukçu, MD is from the Department of General Surgery, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey.

Address correspondence to:
Kürşad Zengin, MD
Department of Urology
Faculty of Medicine
Bozok University
Yozgat, Turkey
kursadzengin@gmail.com

Disclosure: The authors disclose no financial or other conflicts of interest.