Intracerebroventricular injection of erythropoietin enhances sleep in the rat

Brain Research Bulletin 61 (2003) 541–546

Intracerebroventricular injection of erythropoietin enhances sleep in the rat Fabio Garc´ıa-Garc´ıa, James M. Krueger∗ Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology (VCAPP), College of Veterinary Medicine, Washington State University, P.O. Box 99164-6520, Pullman, WA, USA Received 12 May 2003; received in revised form 17 June 2003; accepted 17 June 2003

Abstract Systemic injection of erythropoietin (EPO) over several days reduces sleep fragmentation in patients with periodic limb movements in sleep (PLMS). However, there are no studies concerning the effects of EPO on spontaneous sleep. In this study, we determined the effects of intracerebroventricular (i.c.v.) administration of EPO on spontaneous rat sleep. Three doses of EPO (25, 75, and 125 ng) were injected i.c.v. at the onset of the dark period. All doses of EPO increased non-rapid eye movement sleep (NREMS). In addition, high and low doses of EPO (125 and 25 ng) increased rapid eye movement sleep (REMS), but the medium dose of EPO (75 ng) inhibited REMS. Electroencephalogram slow-wave activity during NREMS also increased following the two higher doses of EPO. In contrast, EPO injection during the light period failed to affect sleep. Brain temperature (Tbr) was not affected by any dose of EPO. These results suggest that EPO could be part of the cytokine network involved in sleep regulation. © 2003 Elsevier Inc. All rights reserved. Keywords: EEG; Cytokine; Sleep factors; Brain

1. Introduction Erythropoietin (EPO) is produced primarily by the kidneys and promotes the growth, differentiation and survival of erythroid progenitors as well as supporting the viability of erythroid cells during maturation [21]. The EPO receptor is a member of the cytokine receptor family and they are found in the hippocampus, temporal cortex, amygdala and cerebellum of human, monkey and rodent brains [13,16,26,27]. The hypoxia-responsive production of EPO in the adult brain suggests that this hormone acts as a neurotrophic and neuroprotective factor after brain injury. For example, EPO infusion into the lateral cerebral ventricles of gerbils prevents ischemia-induced learning disabilities and rescues hippocampal CA1 neurons from ischemia-induced damage [33]. The observation that EPO is produced by astrocytes, and binds to EPO receptors on adjacent neurons, suggests that EPO has a paracrine action on neurons, independent of its endocrine role in the erythropoietic system [6,20,21]. Systemically administered EPO crosses the

∗

Corresponding author. Tel.: +1-509-335-2090; fax: +1-509-335-4650. E-mail address: [email protected] (J.M. Krueger).

0361-9230/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0361-9230(03)00191-6

blood–brain barrier [2,8] and is present in human cerebrospinal fluid [8]. The administration of human recombinant EPO (rHuEPO) has become an important clinical therapeutic adjunct to the treatment of patients with chronic anemia or renal dysfunction. The majority of patients receiving this drug respond satisfactorily with a significant rise in hemoglobin concentrations, and an improvement in health-related quality of life. For instance, sleep determined by subjective assessment improves after rHuEPO treatment [31,35]. Furthermore, rHuEPO given to patients with periodic limb movements in sleep (PLMS) reduces both sleep fragmentation and the total number of arousals induced by PLMS, thereby improving the quality of sleep and daytime alertness [5]. Because these findings suggest that EPO could play a role in sleep regulation, the objective of the present study was to determine the effects of EPO administration on spontaneous rat sleep.

2. Material and methods Male Sprague–Dawley rats (approximately 3 months old) were used and housed on a 12-h light:12-h dark cycle (light on at 09:00 h) and kept at 23±1 ◦ C. These experiments were

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approved by the Animal Care and Use Committee of Washington State University in accordance with Washington state and USA law. Precautions were taken to minimize pain or discomfort. Animals were provided electroencephalogram (EEG) and electromyogram (EMG) electrodes, a brain thermistor and a lateral intracerebroventricular (i.c.v.) cannula [22]. Briefly, stereotaxic surgery was performed using ketamine–xylazine (87 and 13 mg/kg) anesthesia. The guide cannula was placed in the left lateral ventricle (A: −0.8, L: 1.5, V: 3.5–4.0 mm from the bregma). The EEG electrodes were placed over the frontal and parietal cortices and an EMG electrode was implanted in the dorsal neck muscle. The thermistor (30 k
; model 44008 Omega Engineering, Stanford, CT) was placed on the dura mater over the parietal cortex to measure brain temperature (Tbr). The leads from the EEG, EMG electrodes and the thermistor were routed to a Teflon pedestal. The pedestal, guide cannula, and leads were attached to the skull with dental acrylic. After 10 days of recovery, the animals were placed in sleep recording chambers for 3-days of acclimation before starting experiments. Water and food were accessible ad libitum throughout the experiment. EEG signals (128 Hz sampling rate) were amplified, passed through filters, digitized and collected by a computer and stored on compact discs. Power density values were calculated using fast-Fourier transformation for consecutive 10-s epochs in 1 Hz bins for the frequency range 0.25–25.0 Hz. The average power of EEG slow-wave activity (SWA) (0.5–4 Hz) during non-rapid eye movement sleep (NREMS) throughout the entire 23 h control-recording period was normalized to 100% for each animal. Then, EEG SWA data were converted to a percentage of control values. The states of vigilance were determined for 10 s epochs by the usual criteria: NREMS (high-amplitude EEG slow waves, low muscle tone), rapid-eye movement sleep (REMS) (highly regular theta EEG activity, loss of muscle tone with occasional twitches), and wakefulness (EEG activities similar to REMS and high EMG activity). Three different doses of rHuEPO (R&D Systems, Minneapolis, MN) dissolved in 5 ␮l pyrogen free saline (PFS) were tested. The rats received 5 ␮l of PFS i.c.v. as con-

trol and the next day the rats received one of three doses of rHuEPO at the onset of the dark period: 25 ng (n = 7), 75 ng (n = 7) and 125 ng (n = 7). The injections took place between 20:30 and 21:00 h and rats were recorded for the next 23 h. Another group of rats received one of two doses of rHuEPO at light onset: 75 ng (n = 6) and 125 ng (n = 6). The injections took place between 10:30 and 11:00 h; these rats were recorded for the next 21 h. The amount of time spent in each vigilance state was calculated for 2-h intervals for graphical display. All analyses were performed with two-way ANOVA for repeated measures across the entire recording period and for 3 h time blocks followed by the Student–Newman–Keuls test. For the power spectrum analysis data the EEG power density values were summed in four frequency bands: delta (0.25–4.0 Hz), theta (4.0–8.0 Hz), alpha (8.0–12.0 Hz), beta (12.0–25.0 Hz) wave activities, and then one-way ANOVA for repeated measures was performed for these four frequency bands. A significant level of P < 0.05 was accepted.

3. Results Administration of EPO at dark onset dose-dependently augmented NREMS [ANOVA for the entire 23-h period, time–treatment interaction: 25 ng, F(1, 6) = 19.46, P < 0.01; 75 ng, F(1, 6) = 12.16, P < 0.01; 125 ng, F(1, 6) = 12.35, P < 0.01] (Table 1, Fig. 1). After the two higher doses of EPO (75 and 125 ng) NREMS occurred during the first 12 h after injection (dark period) while NREMS during the subsequent light period (13–23 h postinjection) was near that of control animals. In contrast, after the lowest dose of EPO (25 ng) NREMS was not increased during the dark period (0–12 h postinjection) although there was a slight increase during the subsequent light period (13–23 h) [ANOVA for the 11-h light period, time–treatment interaction, F(1, 6) = 15.78, P < 0.01]. These changes in NREMS were mainly due to an increase in the average duration of NREMS episodes after 25 and 125 ng EPO (control 2.95 ± 0.10 min versus EPO 25 ng 3.28 ± 0.16, control 2.93 ± 0.12 min versus EPO 125 ng 3.18 ± 0.05)

Table 1 Erythropoietin injection at dark onset enhances spontaneous rat sleep Doses EPO (ng) 0 25 0 75 0 125

NREMS (min) 23-h period 519.67 567.53 539.83 591.10 503.89 593.08

± ± ± ± ± ±

25.69 19.53∗ 14.56 11.35∗ 23.99 17.68∗

Tbr (◦ C)

REMS (min) Dark period (12 h) 211.74 222.20 223.48 278.67 187.69 280.31

± ± ± ± ± ±

29.08 25.71 15.21 23.29∗ 19.36 26.24∗

Light period (11 h) 307.93 345.33 316.35 312.43 316.19 312.78

± ± ± ± ± ±

9.57 11.6∗ 6.85 21.4 5.70 10.27

23-h period 123.62 155.50 134.08 105.90 124.0 141.53

± ± ± ± ± ±

11.72 4.16∗ 8.53 20.0∗ 13.86 6.51∗

Dark period (12 h) 38.69 52.67 52.21 36.77 35.61 64.89

± ± ± ± ± ±

5.66 6.50∗ 5.65 10.5∗ 5.62 7.64∗

Light period (11 h) 84.93 102.83 81.88 69.13 88.39 76.64

± ± ± ± ± ±

10.97 9.7∗ 7.89 13.5∗ 12.32 12.34

23-h period 35.19 35.39 35.28 35.33 35.08 35.65

± ± ± ± ± ±

0.12 0.27 0.21 0.14 0.07 0.21

Sleep data (means ± S.E.) are expressed as minutes spent in non-rapid eye movement sleep (NREMS) or rapid eye movement sleep (REMS) for each time period. Brain temperatures (Tbr) are expressed as mean values collected in 10 s intervals for the 23 h recording period. n = 7 rats per group; EPO, erythropoietin. ∗ P < 0.05 vs. corresponding vehicle treatment.

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Fig. 1. EPO i.c.v. injections at dark onset enhance non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS), and electroencephalogram (EEG) slow-wave activity (SWA) during NREMS in rats. Open circles and closed circles represent pyrogen free saline (PFS) and EPO treatment, respectively. Horizontal shade bars denote dark phase of the day. Data are means ± S.E.

and an increase in the average number of NREMS episodes after the 75 ng EPO dose (control 116.62 ± 3.44 min versus EPO 75 ng 183.0 ± 8.76). The low (25 ng) and high (125 ng) doses of EPO given at the onset of the dark period also markedly enhanced REMS with an increase in the number and duration of REMS episodes [ANOVA for the entire 23-h period, time–treatment interaction, 25 ng, F(1, 6) = 5.79, P < 0.001; 125 ng, F(1, 6) = 3.94, P < 0.003] (Table 1, Fig. 1). In contrast, the 75 ng EPO dose reduced total time spent in REMS [F(1, 6) = 2.31, P < 0.05] (Table 1, Fig. 1). EEG SWA during NREMS was significantly enhanced after the two high EPO doses given at dark onset [ANOVA

for the entire 23-h period, time–treatment interaction, 75 ng, F(1, 6) = 95.79, P < 0.001; 125 ng, F(1, 6) = 63.93, P < 0.001] (Fig. 1). The low EPO dose (25 ng) increased EEG SWA slightly but this effect was not significant. In addition, EEG power densities were performed for the period 2–6 h after EPO injection (Fig. 2). During NREMS EEG delta activity (0.25–4.0 Hz) increased after the higher doses of EPO [75 ng, F(1, 6) = 38.09, P < 0.001; 125 ng, F(1, 6) = 67.72, P < 0.001]. EEG power densities during REMS also were enhanced in the delta and theta (4.0–8.0 Hz) frequency bands but only after the high EPO dose (125 ng) [ANOVA: delta, F(1, 6) = 69.65, P < 0.01; theta, F(1, 6) = 67.72, P < 0.01] (Fig. 2).

Table 2 Effects of erythropoietin injection at light onset on spontaneous rat sleep Doses EPO (ng)

NREMS (min)

0 75 0 125

537.69 548.08 568.75 544.17

21-h period ± ± ± ±

35.79 25.12 18.24 8.98

Tbr (◦ C)

REMS (min) Light period (9 h) 312.94 313.36 337.39 325.75

± ± ± ±

18.68 13.62 10.39 10.25

Dark period (12 h) 224.75 234.72 231.36 218.42

± ± ± ±

21.58 14.74 10.80 9.75

21-h period 89.0 84.86 95.44 100.86

± ± ± ±

4.67 5.02 9.64 7.16

Light period (9 h) 59.25 50.89 57.42 63.36

± ± ± ±

3.46 5.95 6.93 4.10

Dark period (12 h) 29.75 33.97 38.03 37.50

± ± ± ±

4.82 5.17 4.96 3.45

21-h period 35.08 35.04 35.09 35.01

± ± ± ±

0.09 0.14 0.11 0.09

Sleep data (means ± S.E.) are expressed as minutes spent in non-rapid eye movement sleep (NREMS) or rapid eye movement sleep (REMS) for each time period. Brain temperatures (Tbr) are expressed as mean values collected in 10 s intervals for the 21 h recording period. n = 6 rats per group; EPO, erythropoietin.

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Fig. 2. Power spectrum analysis of the EEG in non-rapid eye movement sleep (NREMS) (left) and rapid eye movement sleep (REMS) (right) after EPO injection. Closed circles represent EPO treatment. (A) EPO (25 ng); (B) EPO (75 ng); (C) EPO (125 ng).

Unlike the results obtained with dark onset EPO administration, administration of EPO at light onset failed to affect sleep. The 75 ng dose of EPO tended to increase NREMS during the first 6 h after injection (Table 2, Fig. 3) while the high dose of EPO (125 ng) slightly decreased NREMS (Table 2, Fig. 3). However, neither effect was shown to be statistically significant. Brain temperature was not affected by any dose of EPO given at light or dark onset (Tables 1 and 2). Although behavior was not systemically analyzed, no gross abnormalities were observed after EPO administration.

4. Discussion To our knowledge, this is the first report demonstrating that i.c.v. injection of EPO induces changes in sleep and EEG power. These results are consistent with the previous report of the sleep-consolidation activity of EPO in humans [5]. EPO enhanced rat sleep when given at dark onset. In con-

trast, EPO failed to affect sleep following light onset administration. Dependency on the time of day of administration has been observed for other pro-somnogenic cytokines. For example, administration of interleukin-1␤ (IL-1␤) at dark onset enhances NREMS in rats, whereas the same doses of IL-1␤ suppress NREMS if given during the light period [29]. It seems likely that EPO and other cytokines interact with homeostatic and circadian sleep regulatory processes to affect sleep. There are several possible mechanisms for EPO-induced changes in sleep. The NREMS enhancing action of EPO could result from interactions with other sleep regulatory substances (SRS) such as tumor necrosis factor ␣ (TNF-␣) and IL-1␤ [1,2]. Treatment of dialysis patients with rHuEPO is associated with an increase in circulating TNF-␣ and IL-1␤ [2,25]. Both TNF-␣ and IL-1␤ induce NREMS, whereas their inhibition results in a loss of spontaneous sleep [23,29]. In sleep apnea patients, a condition associated with daytime sleepiness, TNF-␣ and EPO plasma levels are elevated [9,10,28].

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Fig. 3. Effects of EPO i.c.v. injections at light onset on non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS), and electroencephalogram (EEG) slow-wave activity (SWA) during NREMS in rats. Open circles and closed circles represent pyrogen free saline (PFS) and EPO treatment, respectively. Horizontal shade bars denote dark phase of the day. Data are means ± S.E.

Another possible mechanism of EPO-enhanced sleep is that the EPO receptor is coupled to the Janus kinase-2 (Jak2) pathway, which induces the phosphorylation of inhibitory kappa B (I␬B) thereby activating nuclear factor ␬B (NF-␬B) [17]. NF-␬B activation promotes the production of several substances involved in sleep regulation, for example, nitric oxide synthase (NOS), IL-1␤, nerve growth factor, cyclooxygenase-2, the adenosine A-1 receptor and TNF-␣ [19,30,36]. Substances that inhibit sleep, for example, IL-4 inhibit NF-␬B activation [11]. Intracerebral injection of a NF-␬B cell permeable inhibitor inhibits NREMS in rats and rabbits [22]. These findings suggest that EPO-induced Jak2-NF␬B pathway is involved in its effects on sleep. EPO administration also increases EEG SWA during NREMS. An increase in EEG SWA is often used as a measure of sleep intensity, since there is a correlation of enhanced arousal threshold with enhanced EEG SWA [7]. The reported improvement in the quality of sleep in humans after EPO treatment [5] is likely associated with this change in EEG SWA, although this parameter was not determined in those studies. Many other sleep regulatory substances also enhance EEG SWA, for example, IL-1␤, TNF-␣, growth hormone releasing hormone and adenosine [28]. The mechanisms for EPO-induced enhancement of EEG SWA are not clear, however, other pro-somnogenic cytokines such as TNF-␣ increase the expression and/or function of AMPA receptors on neurons [4,15]. An increase in AMPA receptors on cortical interneurons is associated

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with enhanced cortical slow-wave oscillations [3]. EPO can protect cultured cortical neurons from AMPA toxicity [34], but whether this mechanism is involved in EPO-enhanced EEG SWA is unknown. EPO also increases REMS following dark onset administration. This finding is consistent with the previous report demonstrating that EPO treatment increases REMS time in humans [5]. EPO-enhanced REMS may be due to EPO stimulation nitric oxide (NO) production in the brain [18]. Neural NOS colocalizes with cholinergic neurons in the laterodorsal tegmental nucleus (LDT) and pedunculopontine nucleus (PPT), these neurons project to the medial pontine reticular formation (mPRF) and are crucial in REMS generation [32]. Microinjections of NO donor into the cholinergic cell compartments of the PPT increase NREMS [14]. In addition, inhibition of NOS within the mPRF decreases acetylcholine release and inhibits REMS [24]. Finally, mice lacking neuronal NOS have less REMS [12]. In conclusion, data presented here are consistent with previous literature supporting a role for the brain cytokine network in physiological sleep regulation and extends this hypothesis to include EPO as a possible sleep regulatory substance.

Acknowledgements This work was support in part by National Institutes of Health grants NS25378 and NS31453. We thank Dr. Tim Traynor for his comments and suggestion and Richard Brown and Victoria Caussen for their assistance.

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