Orexin is a peptide first reported in 1998.1 The peptide consists of 2 subtypes, orexin-A and orexin-B. Neurons expressing orexin are primarily located in the lateral hypothalamus, especially in the perifornical area, and widely project throughout the central nervous system.2 Accumulated evidence suggests that orexinergic neurons play a critical role in the promotion and the maintenance of wakefulness. Genetic and pharmacologic blockade of orexin impairs the arousal process3–7 and leads to narcolepsy.8–10
In our previous studies, we have demonstrated that activation of orexinergic signals in the basal forebrain increases cortical acetylcholine release during isoflurane anesthesia in rats and also facilitates the emergence from sevoflurane anesthesia.11,12 Accumulated evidence makes basal forebrain an attractive nucleus for future investigations in anesthesia–arousal regulation. One report13 demonstrated that wake-active orexinergic neurons were inhibited by isoflurane and sevoflurane and that the emergence time was delayed if the action of orexin was blocked. Intracerebroventricular injection of orexin-A significantly decreased the emergence duration of IV anesthetics such as barbiturates and ketamine.5,14 However, there are few data on the effects of orexinergic signals, especially the signals in basal forebrain, on the anesthesia–arousal circle of propofol anesthesia.
Our hypothesis for this study was that propofol anesthesia may directly alter the activity of orexinergic neurons and the release of orexin. Since orexin-A induces more potent effects on arousal than does orexin-B,15 the current study clarifies the possible role of orexin-A in regulating propofol anesthesia. We first assessed the effects of propofol anesthesia on the orexinergic neurons and blood orexin-A concentrations. Furthermore, we also studied the effects of intrabasalis administration of orexin-A or its antagonist on the induction time, emergence time, and the electroencephalogram (EEG) with propofol anesthesia.
Male Sprague–Dawley (SD) rats, weighing 260 to 280 g, were provided by the Animal Center of the Fourth Military Medical University (Xi'an, China). The animals were housed in a controlled condition with constant temperature (24°C ± 0.5°C) and relative humidity (60% ± 2%) on a light-controlled schedule (lights on between 6:00 AM and 6:00 PM). The rats had free access to food and water. The experimental protocol used in this study was approved by the Ethics Committee for Animal Experimentation and was conducted according to the Guidelines for Animal Experimentation at our institute. To eliminate the influence of the diurnal rhythm of sleep–wakefulness on results, we performed all experiments between 10:00 and 17:00 hours.
To identify the changes of the activity of orexinergic neurons in the hypothalamus and the alteration of orexin-A concentrations in plasma during propofol anesthesia, we killed rats at 0, 30, 60, and 120 minutes of propofol infusion as well as at the time the righting reflex returned after the termination of propofol anesthesia. Blood samples were collected at each time point, and the plasma concentrations of orexin-A was detected by radioimmunoassay. The brains were harvested and processed for double immunofluorescent staining for orexin-A and c-Fos.
Rats were anesthetized with propofol for 30 minutes. To elucidate the effect of orexin-A on regulating the induction and the emergence times of propofol anesthesia, orexin-A (30 or 100 pmol), or the orexin-1 receptor antagonist, SB-334867A (5 or 20 μg), were microinjected into the basal forebrain 15 minutes before propofol infusion or 15 minutes before termination of propofol infusion, respectively. The durations of induction and emergence were judged by the time of loss (LORR) and the time to return of the righting reflex. The influence of orexin-A signal activation in the basal forebrain on the anesthetic depth of propofol was assessed by EEG analysis. The EEG was recorded before and after orexin-A microinjection under propofol anesthesia. The changes of EEG patterns after orexin-A microinjection were analyzed.
For propofol infusion and blood sample collection, the rats were anesthetized with 2% pentobarbital (50 mg/kg, i.p) 3 days before the experiments to cannulate the jugular or femoral vein. After making an incision in the skin overlying the jugular vein or femoral vein, the vessel was separated by blunt dissection. Ligatures were made at the caudal and rostral ends of the vein.16 A polyethylene tube (I.D. 0.58 mm, O.D. 0.965 mm, Becton Dickinson, Franklin Lakes, NJ) was inserted into the jugular vein or femoral vein. The other end of polyethylene tube was threaded subcutaneously over the shoulder or thigh, bored from subcutaneous tissue, and fixed onto the backs of the rats. Propofol was infused into the jugular vein by an infusion pump at a dose of 60 mg/kg/h.17 The infusion speed was constant from the beginning to the ending. All blood samples were obtained from the femoral vein.
A heparin saline–filled polyethylene catheter (MRE-040; Eicom Japan) was inserted into the femoral artery for measurement of arterial blood pressure. Mean arterial blood pressure, heart rate, and blood gases were monitored during the experiments. During propofol infusion, the animals were allowed to breathe spontaneously, and the rectal temperature was maintained at 37°C with a heating pad.
For microinjection of orexin or its antagonist into the basal forebrain, 5∼7 days before the experiment, the rats were anesthetized with pentobarbital (50 mg/kg, i.p.). A guide cannula (HTX-32T-24-05, Smallparts Inc., Logansport, IN) was stereotaxically implanted into the basal forebrain and fixed with dental cement. The coordinates of the basal forebrain (including the nucleus basalis and substantia innominata) were adopted according to the atlas of Paxinos and Watson18: 1.4 mm posterior to bregma; 2.5 mm lateral to the midline; and 8.5 mm deep from the surface of the brain. The coordinates of the tip of the guide cannula were 2 mm superficial to the tip of the inner needle for microinjection. On the experimental day, the rats were transferred to a cube cage (25 cm internal diameter, 30 cm in height) for intrabasalis microinjection.
Rats were killed by injection of 1.5 mL 10% KCl into the jugular vein cannula at 0, 30, 60, or 120 minutes during propofol anesthesia and at the time the righting reflex reappeared after the end of propofol infusion (n = 5 for each). All rats were transcardially perfused with normal saline (100 mL) followed by 4% paraformaldehyde (500 mL) in 0.1 M phosphate buffer, pH 7.4. The brains were harvested and postfixed in 4% paraformaldehyde for 3 hours and then moved into 20%∼30% sucrose until the tissue sank. The diencephalon, defined by the optic chiasma rostrally and the mammillary bodies caudally,19 was cut into 30-μm-thick coronal sections with a cryostat and collected in serial order.
Correct placement of the tip of the guide cannula for the microinjection was confirmed by histological examination in each animal. Sections were chosen alternatively, and half of the sections were incubated in 4% normal goat serum for 1 hour to eliminate nonspecific staining. The sections were then incubated with a mixture of a polyclonal antibody against c-Fos raised in rabbits (at 1:1000, sc-52, Santa Cruz Biotechnology Inc., CA) and a mouse monoclonal antibody against orexin-A (at 1:1000, MAB763, R&D Systems, Minneapolis, MN) in a diluent solution containing 3% goat serum and 0.2% Triton X-100 in phosphate buffered saline (PBS) for 40 to 48 hours at 4°C. Subsequently, after washing 3 times in PBS, the sections were incubated with fluorescent secondary antibodies comprising DyLight 594 Goat Anti-Rabbit IgG (at 1:200, DI-1594, Vector Laboratories, Inc., Burlingame, CA) and Horse Anti-Mouse IgG (at 1:200, FI-2000, Vector Laboratories) for 1 hour at room temperature. Sections were then washed 3 times in PBS, mounted onto slides, and examined under a fluorescent or confocal microscope.
Cell counting was performed by one researcher who was blinded to the animal grouping and confirmed by a second blinded researcher. About 15 sections were stained with c-Fos and orexin for each rat. We chose 2 representative sections at the mid-tuberal level of the hypothalamus with the largest number of orexin neurons in the perifornical/lateral hypothalamic area for cell counts within a horizontally oriented 0.7 mm × 0.5 mm box20 (Fig. 1). The c-Fos immunoreactive (IR) nuclei, orexin-IR neurons, and double-labeled orexin/c-Fos-IR neurons on one side of the brain were examined at 100× or 200× magnification.
Radioimmunoassay for Plasma Orexin-A Concentration
Blood samples (0.5 mL) were obtained from the femoral vein in each rat at 0, 30, 60, or 120 minutes of propofol infusion and at the time when the righting reflex was restored after the termination of propofol infusion (n = 5 for each time point). The blood was transferred to a tube containing EDTA (1 mg/mL blood) and Trasylol (500 kallikrein inhibitor units/mL blood; Bayer, Leverkusen, Germany) and centrifuged by 3000g for 15 minutes at 4°C. The plasma was then separated and immediately frozen and stored at –80°C.
The rat orexin-A concentrations were detected by using a radioimmunoassay as described previously.21 In our study, the general protocol for RK-003-30 orexin-A/hypocretin-1 radioimmunoassay kit (Phoenix Pharmaceuticals Inc., Burlingame, CA) was used. According to the specifications of the kit, the antiserum was raised in the rabbit and exhibits 100% cross-reactivity with human orexin-A, but does not cross-react with human neuropeptide Y, α-melanocyte-stimulating hormone, human leptin, or human orexin-B. The measured range of the radioimmunoassay kit is 10 to 1280 pg/mL, and the concentration of the peptide in unknown samples can be determined by a “standard curve.”
Influence of Orexin-A or SB-334867A on Induction and Emergence Time
To investigate the effect of orexin-A on the induction of propofol anesthesia, we divided the animals into 3 groups: 30 or 100 pmol orexin-A (American Peptide, Sunnyvale, CA, both dissolved in 0.3 μL of Ringer's solution) groups or the Ringer's control group (n = 6). The solutions were injected into the basal forebrain with a Hamilton syringe connected to polyethylene tubing (I.D. 0.1 mm, O.D. 0.4 mm, 50 cm in length, Smallparts Inc., Logansport, IN) using a micropump (Pump 11 Plus, Harvard Apparatus, Farmingdale, NY). The injection speed of the solution with the micropump was 0.06 μL/min and lasted about 5 minutes. Injections were made 15 minutes before propofol infusion. The LORR was checked every 15 seconds and recorded as the induction time.
To determine the effect of orexin-A on the emergence from propofol anesthesia, we evaluated the time for recovery of the righting reflex in the same rats 3 days later. Rats were anesthetized with propofol for 30 minutes. At 15 minutes before the termination of propofol infusion, orexin-A (30 or 100 pmol), or Ringer's solution was injected into the basal forebrain respectively (n = 6 for each). The return of the righting reflex was checked every 15 seconds after termination of propofol infusion and recorded as the emergence time.
To study the effect of the orexin-1 receptor antagonist, SB-334867A (Tocris Bioscience, Ellisville, MO), on the induction or the emergence from propofol anesthesia, we used 0.3 μL SB-334867A (5 μg or 20 μg dissolved in vehicle, n = 6 in each) or 0.3 μL vehicle solution (10% encapsin and 2% dimethylsulfoxide in sterile water), respectively. The injection procedure and the method for observation of the righting reflex were the same as described above.
Implantation of Electrodes and EEG Recording
To record epidural EEG signals, 4 stainless steel screws were fixed over the frontal (3.9 mm anterior, 2.0 mm lateral to the bregma) and the occipital cortices (7.4 mm posterior, 5.0 mm lateral to the bregma) bilaterally 5 days before the experiment. The reference electrode was placed on the frontal bone (5.5 mm anterior and 0.8 mm lateral to bregma on the right). The leads were connected to a socket that was fixed with dental cement to the skull with the electrodes and the EEG screws.
The EEG signal was continuously monitored before and after the microinjection of Ringer's solution, or 30 or 100 pmol orexin-A during propofol anesthesia (n = 5 for each group). In each group, if the EEG changed from the burst and suppression pattern to an arousal pattern, the rat was defined as an aroused rat. The arousal pattern of the EEG was characterized by low-amplitude, high-frequency signals.
All data were expressed as the mean ± SEM. Radioimmunoassay statistical analysis was performed by 1-way analysis of variance (ANOVA) followed by the Student Neuman–Keuls test or the paired t test, as appropriate. Other data were analyzed with 1-way ANOVA with post hoc Bonferroni multiple-comparison testing. All statistical analyses were performed using the SPSS 12.0 for Windows (SPSS, Chicago, IL), and a 5% probability of type I errors was used to determine statistical significance. In all cases, P < 0.05 was taken as the level for significant difference.
Propofol Anesthesia Inhibits c-Fos Expression in Orexinergic Neurons
Since expression of the immediate-early gene c-Fos is a reliable indicator of transcriptional activation at the single cell level,22 in the current study, c-Fos expression was used to determine whether orexinergic neurons were functionally inhibited during propofol anesthesia. Orexin-A-immunoreactive neurons are located in the hypothalamus, especially in the perifornical area (Fig. 1). Table 1 and Figure 2 show the effect of propofol on orexinergic neurons. The total number of orexin-A-positive neurons was approximately 152 orexin-A neurons per section, which did not change during the whole course of propofol anesthesia. Within the total orexin-positive neurons, the percentage of orexin and c-Fos double-labeled neurons at 30 minutes of propofol anesthesia was reduced to 7.3% ± 0.4%, which is significantly less than that in the preanesthesia state (36.8% ± 1.0%, P < 0.05). As the anesthesia was prolonged, the percentage of double-labeled neurons further decreased to 2.3% ± 0.3% at 60 minutes, and there were no double-labeled neurons observed at 120 minutes of propofol anesthesia. However, the percentage of orexin-A and c-Fos dual-labeled neurons reappeared at a level of 25.0% ± 0.6% when the rats emerged from anesthesia and the righting reflex was recovered.
Plasma Concentrations of Orexin-A Are Decreased by Propofol Anesthesia
Plasma orexin-A concentrations were measured in all of the blood samples. In the preanesthetic state (before the propofol infusion), the concentration of orexin-A in the plasma obtained from the femoral vein was 24.96 ± 0.641 pg/mL (mean ± SEM). At 30, 60, and 120 minutes after propofol infusion, the concentrations of orexin-A were 19.25 ± 0.562 pg/mL, 12.31 ± 0.291 pg/mL, and 12.33 ± 0.451 pg/mL, respectively (n = 5 for each time point). These data indicate a significant decrease of orexin-A levels under propofol anesthesia. When the righting reflex was recovered, the concentration of orexin-A returned to 39.67 ± 0.886 pg/mL (n = 5), which was higher than the preanesthesia state (Fig. 3).
Intrabasalis Microinjection of Orexin-A Aroused Rats in Cortical EEGs Under Propofol Anesthesia
A normal awake state of EEG tracings is shown in Figure 4A. The microinjection of 100 pmol orexin-A resulted in a change in EEG activity from the burst and suppression patterns (Fig. 4B) to an arousal pattern (Fig. 4C) during propofol anesthesia 3 to 5 minutes after injection. No significant difference was found after microinjection of Ringer's solutions or 30 pmol orexin-A. Two of the 5 animals after 30 pmol orexin-A and all 5 animals after 100 pmol orexin-A microinjection showed arousal pattern changes in the EEG. There were no aroused EEG patterns in rats microinjected with Ringer's solution (Table 2).
Microinjection of Orexin-A Had No Effect on Induction Time But Facilitated Emergence from Propofol Anesthesia
The microinjection of orexin-A did not affect the induction time of propofol anesthesia at the 30 or 100 pmol doses (Fig. 5A). However, microinjection of 100 pmol of orexin-A shortened the emergence time from propofol anesthesia (40% less time in comparison with the Ringer's control) (P < 0.01) (Fig. 5B). No significant changes in the emergence time were observed when 30 pmol orexin-A was administered (Fig. 5B).
Microinjection of the Orexin-1 Receptor Antagonist SB-334867A Delayed the Emergence from Propofol Anesthesia
Intrabasalis microinjection of 2 different doses (5 μg and 20 μg) of the selective orexin-1 receptor antagonist SB-334867A or the same volume of vehicle failed to alter the induction time of propofol anesthesia (Fig. 6A). However, microinjection of 20 μg SB-334867A markedly delayed the emergence process, in comparison with that in the vehicle group (P < 0.01) (Fig. 6B).
The present study demonstrated that propofol (2,6-diisopropylphenol) anesthesia induced a significantly reduced activation of orexinergic neurons and a decreased plasma level of orexin-A. However, the number of activated orexinergic neurons and the concentration of immunoreactive orexin-A was restored when the rats recovered from anesthesia. Moreover, microinjection of 100 pmol orexin-A into the basal forebrain facilitated the emergence from propofol anesthesia and evoked an arousal pattern on the EEGs. Inversely, administration of the orexin-1 receptor antagonist SB-334867A delayed the emergence from propofol anesthesia but did not change the induction time. These findings indicate that the orexinergic signal pathway is involved in anesthesia–arousal regulation of propofol anesthesia. The orexin-A signals in the basal forebrain play an important role in this regulation.
Accumulated evidence suggests that expression of c-Fos in orexinergic cells in the hypothalamus is significantly increased by modafinil, an antinarcoleptic drug,9 and by sleep deprivation.23 This phenomenon indicates that orexinergic neurons play an important role in the maintenance of wakefulness. On the other hand, the expression of c-Fos in orexinergic neurons was dramatically reduced with inhalation anesthetics, while the activation of adjacent melanin-concentrating neurons was not affected.13 In the present study, we investigated the changes in activation of orexin-A-positive neurons during propofol anesthesia. The activation of orexin-A-positive neurons was observed by orexin and c-Fos double immunofluorescence staining. We found that propofol anesthesia markedly reduced the number of c-Fos-positive orexinergic neurons after propofol infusion. Moreover, there were no double-labeled neurons when the anesthesia time was prolonged to 120 minutes. Interestingly, when the righting reflex returned after termination of anesthesia, 68% of the activities of orexinergic neurons were restored. These findings indicate that activation of orexinergic neurons was reversibly inhibited by propofol anesthesia.
Propofol acts mainly at γ-aminobutyric acid A (GABAA) receptors to exert its anesthetic effects. A previous study reported that injection of the GABAA receptor agonist muscimol into the tuberomammillary nucleus caused the LORR, but injection of propofol itself caused sedation without LORR.24 These data imply that other neuronal pathways may be involved in the mechanisms of propofol anesthesia. Therefore, propofol may act, at least in part, by enhancing the inhibitory actions of GABA on other neurons in specific nuclei. Because the orexinergic neurons in the hypothalamus are important for sleep–arousal regulation, they are likely candidates. Zecharia et al. found that orexinergic neurons are selectively inhibited by GABAergic drugs in vivo during anesthesia.16 A recent study found that intracerebroventricular injection of orexin-A decreased the time to emerge from propofol anesthesia mediated by the OX-1 receptor, a selective orexin-A receptor.25 However, Gompf et al. found that orexinergic neurons remain active during halothane anesthesia and mice lacking orexinergic neurons emerge from halothane anesthesia in a manner indistinguishable from their sibling controls.26 These findings indicate that the anesthesia induced by different general anesthetics may be mediated by a different awake-promoting pathway. Since the orexinergic signals are projected to almost all functional areas of the brain, the orexinergic signals in which nucleus are crucial for the regulation of anesthesia–awake are still unclear. The results of our current study further support the hypothesis that the orexinergic signals activation in basal forebrain is one of the targets of recovery from propofol anesthesia.
Because detecting orexin-A release in the brain is technically difficult, we used a radioimmunoassay to measure the orexin-A concentration in the blood at different times after propofol anesthesia. Although orexin-A is expressed in the hypothalamus and also in peripheral tissues, the orexin-A content that is produced by peripheral tissues is negligible when compared with the orexin-A levels produced in the hypothalamus.27 Previous reports found that plasma orexin-A is significantly correlated with the arousal index in patients,28 and orexin-A can rapidly cross the blood–brain barrier by simple diffusion.29 Therefore, the plasma orexin-A quantified in our current study mainly reflects its release from the hypothalamus. Our results showed that the plasma level of orexin-A significantly decreased after the induction of propofol anesthesia. Although the number of orexin-A immunoreactive neurons did not seem to change during propofol anesthesia, the numbers of activated orexin-A neurons significantly decreased. Taken together, we deduce that propofol likely inhibits orexinergic signals and the release of orexin-A during anesthesia. Recent clinical studies also found that the plasma orexin-A concentration increases during emergence from propofol–fentanyl anesthesia30 and sevoflurane–fentanyl anesthesia.31 However, these studies did not find a decrease in the plasma orexin-A level during the induction time. The discrepancy between these clinical studies and our findings in the current study may be due to the fact that diazepam premedication in previous clinical studies may have already reduced plasma orexin-A levels before the induction of anesthesia.
The results of current study showed the reappearance of activated orexin-A neurons in the hypothalamus and that the plasma orexin-A level was restored during emergence from propofol anesthesia. To clarify whether this increase was an accompanying phenomenon caused by deinhibition when propofol infusion was terminated, we further microinjected orexin-A or an orexin-1 receptor antagonist into the basal forebrain to analyze their impact on induction and emergence times. Our results showed that administration of orexin-A shortened the emergence time, while administration of the orexin-1 receptor antagonist delayed the process. Our previous reports showed that the basal forebrain is a crucial brain area in the functional regulation of the orexinergic neuronal pathway during anesthesia–arousal regulation.11,12 Also, the basal forebrain plays a pivotal role in the ventral ascending activating system.32 Furthermore, the EEG patterns showed that microinjection of 100 pmol of orexin-A into the basal forebrain changed the EEGs from a burst and suppression pattern to an arousal pattern under propofol anesthesia. When orexin-A was microinjected into the basal forebrain before anesthesia, we observed an increase in rat locomotor activity and grooming. However, the administration of orexin-A or SB-334867A did not influence the induction time of propofol anesthesia. We speculate that orexin-A may directly activate the orexin-1 receptor in the basal forebrain area to exert an arousal effect, or may activate local cholinergic neurons to increase acetylcholine release as we have shown previously.12 Our data demonstrate that orexinergic signals in the basal forebrain area play a pivotal role in promoting arousal after anesthesia.
In summary, the results of the present study indicate that propofol anesthesia inhibits the activities of hypothalamic orexinergic neurons and decreases the plasma concentration of orexin-A. Since administration of orexin-A into the basal forebrain facilitates the emergence after propofol anesthesia, orexinergic signals in this region may play an important role in the emergence process.
Name: Li-Na Zhang, MM.
Contribution: This author helped conduct the study and prepare the manuscript.
Name: Zhao-Ju Li, MM.
Contribution: This author helped conduct the study and collect the data.
Name: Li Tong, MM.
Contribution: This author helped collect the data and analyze the data.
Name: Chao Guo, MM.
Contribution: This author helped collect the data and analyze the data.
Name: Ji-Yuan Niu, MD.
Contribution: This author helped analyze the data.
Name: Wu-Gang Hou, MD.
Contribution: This author helped process the images.
Name: Hai-Long Dong, MD, PhD.
Contribution: This author helped design the study and prepare the manuscript.
This manuscript was handled by: Marcel E. Durieux, MD, PhD.
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© 2012 International Anesthesia Research Society
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