The intensity of c-fos immune reactivity significantly increased in the dorsal horns of rats that had total sleep deprivation for 48 h and 72 h compared to the rats in adequate sleep environment (Figure 2). This increase was observed in the Superficial (lamina I-II), Nucleus Proprius (lamina III-IV) and Neck (lamina V) of the dorsal horn at the L4-5 spinal segment ipsilateral to the hind paw injection of formalin.
(Table 1 & Figure 3)
33
Figure 2. Appearance of spinal cord slice illustrating fos-like immunoreactivity at the L4-5 spinal segment ipsilateral to the hind paw injection of formalin. Four experimental situations are represented: control (A), total sleep deprivation for 24 hours (B), total sleep deprivation for 48 hours (C), and total sleep deprivation for 72 hours (D). The number of c-fos-like immunoreactive neurons increased significantly in the dorsal horns of rats that had total sleep deprivation for 48 h and 72 h.
A B
C D
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Figure 3. Effects of 24 h, 48 h, and 72 h of sleep deprivation on the expression of c-fos neurons in each laminar region (mean ± SEM percentage control). Ratio of c-fos neurons in sleep deprivation group to control group plotted at each lamina:
Laminar I-II = Superficial Lamina, Laminar III-IV = Nucleus Proprius, Laminar V = Neck of the Dorsal Horn. * represented p<0.01 compared with control.
* *
Laminar I-II Laminar III-IV Laminar V Total Laminar Region
% of c -f os n eu ro ns /c ont ro l
24 h study time
48 h study time
72 h study time
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Table 1. Number of c-fos-like immunoreactive neurons was significantly increased in the dorsal horns of rats that had total sleep deprivation for 48 h and 72 h compared with adequate sleep environment. This increase was observed in the Superficial (I-II), Nucleus Proprius (III-IV) and Neck (V) lamina of the dorsal horn.* represents p< 0.05 compared with control.
Sleep Deprivation
(number of positive c-fos neuron per section)
Adequate Sleep
(number of positive c-fos neuron per section) Time
Lamina I-II Lamina III-IV Lamina V Total Lamina I-II Lamina III-IV Lamina V Total
24 h 13.4±3.6 7.8±2.2 9.6±2.3 10.3±1.6 13.5±3.6 8.8±2.5 10.7±3.0 11.0±1.7 48 h 52.8±7.3* 33.0±4.8* 42.4±4.5* 43.0±3.6* 28.3±2.8 16.0±1.8 21.5±2.0 22.0±1.7 72 h 49.5±2.8* 29.5±2.2* 37.1±2.5* 38.7±2.4* 20.8±2.2 11.7±1.4 15.6±2.0 16.0±1.4
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Discussion
Total sleep deprivation of 48 and 72 hours induces a significant increase in c-fos immuno-reactive neurons in the dorsal horns of rats. However, the formalin test induced pain related behaviors do not show a clinical difference between the sleep deprived groups and adequate sleep groups.
Sleep consists of two major parts: REM sleep and NREM sleep, and every stage in sleep has different physiological functions. Sleep deprivation may induce enormous physiological changes such as disturbed liver functions, serum lipid levels and hyperphosphatemia,23 alteration in endocrine and hormonal functions,24,25 leukocytosis and increases in natural killer activity.26 Sleep deprivation could even lead to death as a result of deterioration of body tissue and increase in the ratio of catabolism to anabolism.27 Frequently, there are patients who need to receive anesthesia or in pain conditions suffering previous night/s sleeplessness. Patients from the ICU with no sedation during their previous ICU stay and patients who are too anxious or too painful to be able to sleep are among the many examples.28 As sleep deprivation generates enormous physiological changes,29 it will be valuable to know how it may influence anesthesia and the management of pain.
Previous studies focus mostly on the interaction of REM sleep deprivation and pain sensitivity; the studies on total sleep deprivation were rare. With a blind randomized controlled design we performed this experiment.
Our result presented a biphasic pattern of pain related behaviors that are usually found in formalin test.14 The early phase seems to be caused predominantly by C-fibre activation due to the peripheral stimulus, while the late phase appears to be dependent on the combination of an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord.31 The rating score does
37
not change significantly after sleep deprivation groups compared to the adequate sleep groups. However, the number of c-fos-like immuno-reactive neurons was significantly increased in the dorsal horns of rats that had total sleep deprivation.
This augmentation was observed in the superficial (I-II), nucleus proprius (III-IV) and deep (V) lamina of the dorsal horn. That finding suggested total sleep deprivation affects nociceptive perception in a more complex manner than REM sleep deprivation. The proto-oncogene c-fos when activated makes the immunologically detectable nuclear protein Fos that are in the coresponding locations for nociceptive somatic sensory integration.32 Thus, expression of Fos or c-fos indicates populations of neurons activated or excited by nociceptive input.33 Recent studies showed that activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing.34 Therefore, the increasing expression of c-fos immuno-reactive neurons in the dorsal horn after total sleep deprivation suggests that microglia might play a role in the upstream pathway of sleep deprivation induced hyperalgesia.
Our finding is important as the effects of total sleep deprivation on pain threshold in rats were poorly investigated before. However, we do not have enough information in the literature to explain why formalin test showed no significant difference but on the other hand increased the number of c-fos positive neurons.
Further investigation to study the pathway which regulates c-fos expression in the future may be necessary.
38
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