Wang, Zhi-Hua MD; Ni, Xin-Li MD, PhD; Li, Jian-Nan MD; Xiao, Zhao-Yang MD, PhD; Wang, Chen MD, PhD; Zhang, Li-Na MD; Tong, Li MD; Dong, Hai-Long MD, PhD
Anesthesia has been used in the clinical setting for >100 years, and a variety of general anesthetics have been discovered or synthesized. Delayed emergence from general anesthesia frequently occurs in elderly patients,1 but the underling mechansim is not yet fully understood.
In recent years, orexin, a peptide also called hypocretin, has been identified as one of the awakening peptides that is important in regulation of the anesthesia arousal cycle. Orexin has 2 subtypes, orexin-A and orexin-B.2 It is widely accepted that the plasma orexin-A concentration increases during the emergence state and facilitates the arousal process.3,4 Thus, we hypothesized that the cause of delayed emergence in elderly patients might be attributed to an age-related decrease of orexin-A. In this clinical study, we compared the emergence time and examined plasma orexin-A levels in elderly and young patients anesthetized with sevoflurane-remifentanil undergoing elective lumbar surgery. Unexpectedly, in contrast to our prediction, the results showed higher levels of plasma orexin-A in elderly patients than in young patients before, during, and after recovery from anesthesia.
The trial was approved by the institutional ethics committee of Xijing Hospital (number: 20110707-7) and registered at ClinicalTrials.gov (NCT01478906). Written informed consent was obtained from all patients. Forty-one patients with ASA physical status I or II undergoing elective lumbar surgery were enrolled. Inclusion criteria were patients with a body mass index of 20 to 25 kg/m2, ASA physical status of I or II, and those scheduled for elective lumbar surgery under general anesthesia. Exclusion criteria were a history of depression, brain trauma, narcotics allergy, drug addiction, obstructive sleep apnea syndrome, an operation time <2 hours or longer than 4 hours, or blood bleeding >500 mL during the operation. Patients in the 56 to 64 year age range were also excluded. Patients aged 30 to 55 years were assigned to the young group, and those aged 65 to 77 years were assigned to the elderly group.
Anesthesia and Clinical Observations
Anesthesia was induced with 1 to 2 mg/kg propofol and 2 to 3 μg/kg fentanyl, followed by 1 mg/kg rocuronium bromide for muscle relaxation. After endotracheal intubation was performed, anesthesia was maintained with sevoflurane (inhaled concentration: 1.5% and 2.0%), fentanyl (total 6 μg/kg), and remifentanil, (targeted concentration: 2–6 ng/mL) along with an oxygen/air mixture (FIO2 = 0.5). Muscle relaxation was maintained with intermittent administration of rocuronium. The inhaled concentration of sevoflurane and remifentanil target organ concentration (2–6 ng/mL) was titrated to maintain the Bispectral Index between 45 and 65 during the operation.5 Arterial blood pressure was measured from a catheter in the radial artery, and both blood pressure and heart rate were maintained within 20% of their baseline values. The end-tidal carbon dioxide (ETCO2) level was maintained between 30 and 40 mm·Hg by adjusting the tidal volume and/or respiratory rate. After surgery was completed, we adjusted the end-tidal concentration of sevoflurane to 0.8 minimum alveolar concentration (MAC) and targeted concentration of remifentanil to 2 ng/mL and then terminated the administration of all anesthetics while the oxygen flow was increased to 6 L/min. The 0.8 MAC sevoflurane for 30- to 60-year-old adults is 0.015 atm, whereas it is about 0.012 atm for the elderly by using the age calculation for MAC sevoflurane by Mapleson (MAC = a × 10bx, x = difference in age [years from 40]; b = −0.00269; a = MAC at age 40 years.)6 A nonsteroidal anti-inflammatory drug (parecoxib, 40 mg IV) was administrated 30 minutes before terminating all anesthetics to decrease the incidence of emergence agitation.7 The times from terminating all anesthetics to eyes opening (TEO) and to extubation (ET)8,9 were recorded, as well as the values of end-tidal sevoflurane concentration at these 2 time points.
Blood Collection and Assays of Orexin-A
Arterial blood (2.0 mL) was collected from the radial artery at the following time points: before anesthesia (T0), during anesthesia (1 hour after anesthesia induction, T1), during emergence from anesthesia (5 minutes after extubation, T2), and during recovery from anesthesia (30 minutes after tracheal extubation, T3). The blood sample was centrifuged at 3000 rpm for 15 minutes at 4°C to separate plasma and then stored at −80°C until assayed for orexin-A concentration.
Orexin-A was measured by using a radioimmunoassay kit (Peninsula Laboratories Inc., San Carlos, CA), as previously described.10 Briefly, the sample and the standard peptide were incubated at 4°C for 24 hours with rabbit antihuman orexin-A antiserum and then further incubated at 4°C for 24 hours after the addition of 125I-orexin-A. Goat anti-rabbit IgG antiserum was added to the reaction mixture and then incubated at room temperature for 90 minutes. After centrifugation at 1700g for 20 minutes, the radioactivity of precipitates was counted by using a γ-counter.
The blood glucose levels were measured with the glucose oxidase method by a glucose analyzer at the corresponding time points.11
All data distributed normally are expressed as the mean ± SEM. The 95% confidence interval (CI) for the differences are showed as mean difference (lower, upper). All statistical analysis was performed by using SPSS version 16 software (IBM Corp., Armonk, New York). P < 0.05 was considered to indicate a significant difference. Bonferroni measure was used to correct the P values in multiple comparisons via division of comparison times.
The sample size was decided as follows. We did a preliminary observation before the clinical trial. We found that the difference in mean of the ET was about 5 minutes; standard deviation (SD) was 5 minutes. Setting 80% as power, α = 0.05, and then sample size was estimated by PASS 11.0 software (NCSS, Kaysville Utah). The result for estimation of sample size is 16. Considering that approximately 20% of patients might be missed in subsequent processes, we evaluated 20 patients in the young group and 21 in the elderly group at the beginning of the trial. We had also referred some previous reports in which plasma orexin-A was measured. Kushikata et al.12 enrolled about 12 patients. Matsumura et al.13 enrolled 8 to 19 patients in different groups.
Since the data of plasma orexin-A changes were of equal covariances and the residuals were distributed normally (Homogeneity test showed that the P value of Box’s Test of Equality of Covariance Matrices was 0.635 and Kolmogorov-Smirnov test showed the P value was 0.359), the changes of plasma orexin-A were analyzed by 2-way repeated-measures analysis of variance. In addition, since the data for the proportional change of plasma orexin-A in both elderly and young groups were also normally distributed (tested by 1-sample Kolmogorov-Smirnov test and the P value is 0.466), the analysis between the 2 groups was done by t test.
Student t test (data are equal variances) or t′ test (data are unequal variances) was applied to compare differences between the 2 groups, respectively, based on the variances of data reported by SPSS. The results of TEO and ET were shown as median (P25%, P75%), and the differences between the 2 groups were analyzed by Mann-Whitney U test. The data were also transformed to log-TEO and log-ET and tested by Student t test again. The mean differences (95%) were then inverse-log transformed to ratio (95% CI).
Forty-one patients (20 young and 21 elderly) were suitable for study inclusion. Seven patients were excluded: in the young group, 4 patients were excluded for the following reasons: one had been enrolled in another study, one refused to sign the consent form, one lost >500 mL blood during the operation, and one had emergence agitation. In the elderly group, 3 patients were excluded for the following reasons: one withdrew his consent, one had a blood specimen that was hemolyzed, and one had an operation time that was longer than 4 hours. Ultimately, data from 34 patients, 16 young and 18 elderly, were analyzed (Fig 1). Demographic characteristics and clinical features of patients in both groups are shown in Table 1. No significant difference, other than age, was found for these variables between the 2 groups.
TEO and ET data are shown as median (P25%, P75%), and the differences between the 2 groups were analyzed by Mann-Whitney U test. TEO in the elderly group (11.5 [8.25–15.00]) was significantly longer than that in the young group (7.5 [5.25–9.00], P = 0.003). Similarly, ET in the elderly group (15 [10.25–19.20]) was also significantly longer than that in the young group (10 [9–12.3], P = 0.014, Fig. 2). Analyzed by t test after log transformation, the log-TEO (1.0 ± 0.05 in elderly vs 0.9 ± 0.03 in young, P = 0.013) and log-ET (1.2 ± 0.04 in elderly vs 1.0 ± 0.03 in young, P = 0.007) values were also significantly longer in the elderly than in the young group. The ratio of young/elderly and 95% CI were 0.731 (0.401−0.913) for ET and 0.689 (0.516−0.922) for TEO.
Plasma Orexin-A Levels
Two-way repeated-measures analysis of variance was performed on plasma orexin-A mean values at different time points. Analysis for plasma orexin-A from T0, 1, 2, and 3 (young N = 16; elderly n =18) revealed significant age effect (F = 13.896, P = 0.001), time effect (F = 16.087, P < 0.0001), and a nonsignificant interaction (F = 0.477, ns).
The basal level of plasma orexin-A was significantly higher in elderly than in young patients (P < 0.0001) (Fig 3). After the induction of general anesthesia (T1), the plasma orexin-A values were not significantly changed in either the elderly or young group compared with their corresponding values at T0 (CI, elderly: 0.18 [−4.6 to 4.9]; young:−1.8 [−5.3 to 1.8], P = 0.64 and 0.85, respectively). At the emergence state (T2), plasma orexin-A increased in both the elderly and young groups, compared with their corresponding T0 levels (P < 0.0001, both). During the recovery state (T3), plasma orexin-A declined, and no significant differences were found compared with the T0 values in either the elderly (CI, −1.6 [−6.0 to 2.8], P = 0.3470) or young (CI, −3.4 [−7.5 to 0.8], P = 0.509) groups. The level of plasma orexin-A in the elderly group was significantly higher than in young group (28.7 ± 1.2 vs 22.0 ± 1.3, P = 0.001). The proportion of the orexin-A increase during emergence in the young group (71.0% ± 12.9%) was higher than in the elderly group (37.3% ± 5.9%, P = 0.019). Although glucose was slightly increased and the Bispectral Index was decreased after anesthesia in both the young and elderly groups, there was no significant difference between the 2 groups at each corresponding time point (Table 2). Also, no significant difference was found between the elderly and young groups for fasting times (11.60 ± 0.63 vs 11.80 ± 0.62 hours, P = 0.82), surgical start time point (11:37 ± 149 vs 11:31 ± 160 minutes, P = 0.91), and stop time point (14:21 ± 144 vs 14:19 ± 166 minutes, P = 0.97).
End-Tidal Concentration of Sevoflurane
No difference in the end-tidal concentration of sevoflurane (MAC was corrected for age) was found between the young group and elderly group at TEO (CI, 0.03 MAC [−0.012 to 0.078 MAC], P = 0.130) or ET [CI, 0.05 MAC [−0.004 to 1.004 MAC], P = 0.07).
Our previous studies have shown that orexinergic neuronal system is involved in anesthesia arousal regulation. Activation of the orexin signaling pathway, especially the orexin-A pathway, can shorten the emergence time after the termination of inhaled and IV anesthetics.14,15 To explore the correlation between changes in orexin release and the delayed emergence from general anesthesia in elderly patients, we measured changes in plasma orexin-A, a recognized neuropeptide responsible for arousal from sevoflurane-remifentanyl anesthesia.
The phenomenon that elderly patients require more time to awaken from anesthesia has been well recognized.1 In this clinical trial, we noticed that the times from stopping all anesthetics to TEO and to ET were longer in elderly than in young patients, confirming the effect of age on recovery from anesthesia. To exclude the possible influence of an age-dependent difference in anesthetic metabolism on emergence time, we choose sevoflurane-remifentanil since these drugs have a rapid onset and offset. Moreover, the end-tidal concentration of sevoflurane was monitored, and age-adjusted MAC was maintained. The results showed that the concentrations of sevoflurane were at the same MAC level in both groups before the TEO and ET.
Although the mechanisms associated with the action of general anesthetics are not well understood, increasing evidence indicates that anesthetics may affect specific neurotransmitters and their receptors in the central nervous system.16 Several investigators reported that a new neurotransmitter named orexin (especially orexin-A) plays an important role in emergence from anesthesia. Hirota et al.4 reported that the exogenous administration of orexin reduced anesthesia time, which was reversed by an orexin receptor antagonist. Kushikata et al.12,17 showed that plasma orexin-A increased at emergence from both sevoflurane-fentanyl and propofol-fentanyl anesthesia in patients undergoing eye surgery. Kelz et al.3 reported that inhibition of orexinergic signaling delayed emergence from anesthesia. Our previous animal studies also indicated that orexinergic neurons affected emergence from sevoflurane or propofol anesthesia, and the effect of orexin-A was more potent than orexin-B.14,15 Orexin-B levels are much less stable and more difficult to measure. Therefore, we chose to measure the plasma orexin-A concentration instead of both subtypes of orexins in the current trial and found it increased significantly in both young and elderly patients during emergence from sevoflurane-remifentanyl anesthesia. These results are consistent with the results of Kushikata et al.,12,17 who found the same trend for plasma orexin-A levels in patients undergoing ophthalmologic surgery.
Because orexin-A is responsible for the emergence of patients from general anesthesia, we originally hypothesized before the experiment that the delayed emergence of elderly patients might be attributed to lower orexin-A levels. However, we found in the present study that plasma orexin-A levels were higher at T0, T1, T2, and T3 in the elderly. Our results confirm the findings of Matsumura et al.,13 who found that healthy elderly patients had higher basal levels of orexin-A. For ethical reasons, we did not invasively measure the cerebrospinal fluid level of orexin-A in the current trial. This is a limitation of our protocol design. Although we measured the orexin-A concentration in peripheral blood, previous studies showed that plasma orexin-A may reflect central nervous system release.12,13,18
Although orexin-A can facilitate emergence from anesthesia, it is not clear why older patients with higher levels of plasma orexin-A should experience delayed recovery from anesthesia. One possible explanation is that the relative increase during emergence in older patients is lower (37.3% ± 5.9%) than in young patients (71.0% ± 12.9 %). Another reason for this phenomenon might be attributed to the change of density of orexin receptors in the orexinergic neural projection area in elderly patients. Previous studies have suggested that the expression of orexin receptors may be responsible for the relation between the age-related decline in orexin function and food intake.19 Takano et al.20 reported that the decrease in OX1R protein expression may be responsible for orexin-A’s lack of stimulation of food intake in older rats. Stanley and Fadel21 demonstrated that an intrinsic reduction in orexin innervation of cholinergic cells is responsible for the orexin regulation of septohippocampal cholinergic activity in aged animals.
It is possible that there are some other mechanisms. Orexin directly excites histaminergic neurons in the tuberomammillary nuclei.22 Terao et al.23 found histamine receptor mRNA levels showed an age-related decrease in the mouse brain. In our unpublished data in animal experiments, we found that, compared with the young group, the basal level of orexin-A was higher whereas the orexin receptor-1 was lower in aged rats, indicating that delayed emergence from anesthesia might result from the lower density of orexin receptors. A reduction of orexin receptor-1 density in the elderly might have been responsible for delayed recovery from anesthesia in the present study, despite higher plasma orexin-A levels.
In conclusion, we found that although the emergence time in elderly patients was indeed longer than that of young patients, the plasma orexin levels in the former were higher than in the latter at each corresponding time point during the anesthesia arousal cycle. Therefore, the expression of orexin is not responsible for delayed emergence from general anesthesia of aged patients, and the mechanisms underlying this phenomenon need further exploration.
Name: Zhi-Hua Wang, MD.
Contribution: This author helped conduct the study, analyze the data, and write the manuscript.
Attestation: Zhi-Hua Wang has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Xin-Li Ni, MD, PhD.
Contribution: Xin-Li Ni and Zhi-Hua Wang contributed equally to this study.
Attestation: Xin-Li Ni has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files
Name: Jian-Nan Li, MD.
Contribution: This author helped conduct the study.
Attestation: Jian-Nan Li has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Zhao-Yang Xiao, MD, PhD.
Contribution: This author helped conduct the study.
Attestation: Zhao-Yang Xiao has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Chen Wang, MD, PhD.
Contribution: This author helped conduct the study.
Attestation: Chen Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Li-Na Zhang, MD.
Contribution: This author helped conduct the study.
Attestation: Li-Na Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Li Tong, MD.
Contribution: This author helped conduct the study.
Attestation: Li Tong has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Hai-Long Dong, MD, PhD.
Contribution: This author helped design and conduct the study and write the manuscript.
Attestation: Hai-Long Dong has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
This manuscript was handled by: Gregory J. Crosby, MD.
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