Effect of intravenous general anaesthesia with epidural block on the expression of pre-endogenous opioid peptide genes

Abstract

Objective

To measure the plasma concentrations of three endogenous opioid peptides and the levels of preproenkephalin (PPE) and preprodynorphin (PPD) mRNA in peripheral blood lymphocytes of patients during scheduled surgery performed under intravenous general anaesthesia combined with an epidural block.

Methods

Patients were anaesthetized and arterial blood was collected at 0 (baseline), 20, 40, 60, and 80 min during surgery. The plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A were measured using radioimmunoassay. Reverse transcription–polymerase chain reaction was used to measure the levels of PPD and PPE mRNA in peripheral blood lymphocytes collected during surgery.

Results

Fifteen patients participated in this prospective study. The plasma concentrations of β-endorphin were significantly lower at all time-points compared with the baseline value. The plasma concentrations of leucine-enkephalin and dynorphin A were significantly lower at 40, 60, and 80 min compared with baseline. The PPD/β-actin ratio was significantly lower at 80 min compared with baseline, while the PPE/β-actin ratio showed no significant change.

Conclusion

The level of mRNA from two pre-endogenous opioid peptide genes either decreased or remained unchanged during surgery under intravenous general anaesthesia with epidural block, suggesting that patients remained pain free during surgery.

Keywords

Pre-endogenous opioid peptides β-endorphin leucine-enkephalin dynorphin A intravenous general anaesthesia epidural block

Introduction

It is widely recognized that endogenous opioid peptides, expressed by pre-endogenous opioid peptide genes, are associated with pain and stress.¹ These genes are expressed in response to pain, producing pre-endogenous opioid peptides (pre-EOPs) that undergo further cleavage and post-translational modification into the endogenous opioid peptides (EOPs).² For example, dynorphin is derived from preprodynorphin (PPD), β-endorphin is derived from proopiomelanocortin, and enkephalin is derived from preproenkephalin (PPE).^3–9 As powerful analgesics, EOPs are produced in the central neurons, suprarenal glands, spleen, thymus gland, lungs and lymphatic cells.^10–13 In mammals, EOPs are natural endogenous substances that perform the same function as opioids.² For example, dynorphin improves the pain threshold.^14–16 It has also been reported that blood EOP concentrations increased significantly when the human body was exposed to acute pain;^17,18 and decreased with chronic pain.¹⁹ EOPs, which were first discovered in the pig brain by Hughes,²⁰ are efficient analgesics. For example, 200 ng of enkephalin injected into the rat cerebral ventricle showed obvious analgesic effects.²¹ EOPs are also associated with learning and memory, cardiovascular activity, reproduction, the endocrine system and immunity.^22,23

It is well recognized that the intense pain associated with surgical operations can activate the pre-EOP genes that encode for the pre-EOPs.²⁴ In our opinion, the expression of the pre-EOP genes may reflect the particular type of anaesthesia in use during surgery.²⁴ Our previous research demonstrated that some pre-EOP genes were highly expressed in patients undergoing surgery with the use of intravenous and inhalational general anaesthesia, suggesting that these surgical patients were still exposed to pain and stress whilst being anaesthetized.²⁴ This finding could be explained by the fact that somatosensory pain is still being successfully conducted to the central nervous system. It is well known that epidural anaesthesia can block the transmission of all somatosensory pain impulses towards the central nerve system, thus ensuring pain relief during surgery. In order to investigate whether patients are truly pain free under general anaesthesia with epidural block, this current study investigated the changes in the plasma concentrations of three EOPs and the mRNA levels of two pre-EOP genes in peripheral blood lymphocytes during surgery.

Patients and methods

Patient population

This prospective study enrolled consecutive patients scheduled to undergo pectoral or abdominal surgery in the Department of Anaesthesiology, Cancer Hospital, Shanghai Medical College, Fudan University, Shanghai, China between January 2007 and December 2009. All patients were American Society of Anesthesiologists Physical Status Classification System 1–2.²⁵ All patients were interviewed before enrolment to ensure that they were not experiencing any cancer pain prior to their operation.

Ethics approval was provided by the Ethics Committee of Fudan University (clinical trial registration number: 0331-2006; Cancer Hospital, Shanghai Medical College, Fudan University) and written informed consent was obtained from all study participants.

Anaesthesiology methods

Following epidural acupuncture, 10 ml of 0.5% bupivicaine was injected into the cavity. With the epidural anaesthesia at full effect, a Graseby™ 3500 syringe anaesthesia pump (Smiths Medical, Ashford, UK) for target controlled infusion (TCI) and a HXD-I series block-type multifunctional monitor (Huaxiang, Heilongjiang, China) to record the bispectral index (BIS) and mean arterial pressure (MAP) were installed. The feedback stopping indices of propofol TCI were set at BIS <50 and MAP <70 mmHg. The following drugs were injected intravenously for tracheal intubation: 0.04–0.06 mg/kg midazolum, 3–4 µg/ml propofol TCI, 2–3 µg/kg fentanyl and 0.1 mg/kg vecuronium. Anaesthesia was maintained with 1.5–2.5 µg/ml propofol TCI. Arterial blood (4 ml) was collected from each patient at the surgical time points of 0 (baseline), 20, 40, 60, and 80 min, and 0.3 M disodium ethylenediaminetetra-acetic acid (80 µl) was added to each sample to provide anticoagulation. Aprotinin (2000 U; Livzon Pharmaceutical Group, Zhuhai, China) was added to the blood samples to prevent protein degradation and the plasma was processed immediately by centrifugation at 2000 g for 15 min at 25℃ using an L-500 centrifuge (Shanghai Zhaodi Biotechnology, Shanghai, China). Peripheral blood lymphocytes were separated from 2.5 ml arterial blood drawn at the surgical time points of 0 and 80 min. The sample of arterial blood was mixed with disodium ethylenediaminetetra-acetic acid (80 µl), 5 ml of lymphocyte separating medium (Lymphocyte Separation Medium; Biomars, Beijing, China) and 0.9% w/v sodium chloride (2.5 ml), then centrifuged at 2000 g for 20 min at room temperature using an L-500 centrifuge (Shanghai Zhaodi Biotechnology). The purified lymphocytes were mixed with 1 ml of TRIzol® reagent (Sangon Biotech, Shanghai, China) to extract total RNA following the manufacturers’ instructions, and then the samples were stored at −70℃ until further analysis.

All the patients were asked if they had experienced any pain or sensorium during surgery or if they did so shortly after palinaesthesia (i.e. waking up from anaesthesia).

Radioimmunoassay and RT–PCR analysis

The plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A were measured using commercially available radioimmunoassay kits following the manufacturers’ instructions (The Second Military Medical University, Shanghai, China).

Reverse transcription–polymerase chain reaction (RT–PCR) was used to measure the levels of PPD and PPE mRNA in peripheral blood lymphocytes. Total RNA was extracted from peripheral blood lymphocytes that were separated from 2.5 ml of arterial blood as described above and reverse transcribed to cDNA using an RT–PCR kit (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. The PCR primers were prepared by one of the authors (Z.-Y.C.) in the Department of Anaesthesiology, Cancer Hospital, Shanghai Medical College, Fudan University, Shanghai, China using Primer Premier 5 (PREMIER Biosoft, Palo Alto, CA, USA). The PCR primer sequences for PPD were: 5′-GGTCCCAAACCTATCAATCC-3′ (sense) and 5′-TGGCACCATCGTTCAGC-3′ (antisense); producing a PCR amplification product of 627 base pairs (bp). The PCR primer sequences for PPE were: 5′-TGGCTTGCGTAATGGAAT-3′ (sense) and 5′-CTCTGGGCGACCTACTCTT-3′ (antisense); producing a PCR amplification product of 529 bp. The PCR primer sequences for the internal control β-actin mRNA were: 5′-TGTGGCATCCACGAAACTA-3′ (sense) and 5′-TCATACTCCTGCTTGCTGATC-3′ (antisense); producing a PCR amplification product of 275 bp. The PCR was performed in a thermal cycler (Shanghai Naze Instrument, Shanghai, China). The cycling programme involved preliminary denaturation at 94℃ for 2 min, followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 60℃ for 60 s, and elongation at 68℃ for 120 s, followed by a final elongation step at 68℃ for 7 min. The PCR products were separated by electrophoresis in 1% agarose gel, stained with ethidium bromide and the density of the bands was calculated using an ImageMaster™ VDS-CL system (Amersham Pharmacia Biotech, Little Chalfont, UK).

Statistical analyses

All statistical analyses were performed using the SPSS® statistical package, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows®. All data are presented as mean ± SD. The data collected at all of the surgical time points after 0 min were compared with the baseline data at 0 min using analysis of variance or Student’s t-test. A P-value <0.05 was considered to be statistically significant.

Results

Fifteen patients (13 male and 2 female) with a mean ± SD age of 45.31 ± 7.23 years (range 38–56 years) participated in this study. The BIS was controlled at near 50 during surgery in all patients. None of the patients complained of pain or sensorium during surgery or shortly after palinaesthesia. The changes in the plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A during surgery are presented in Table 1. The plasma concentrations of β-endorphin were significantly lower at all time-points compared with the baseline value at 0 min (P < 0.05 for all comparisons). The plasma concentrations of leucine-enkephalin and dynorphin A were significantly lower at 40, 60, and 80 min compared with the baseline value at 0 min (P < 0.05 for all comparisons).

Table 1.

Plasma concentrations of three endogenous opioid peptides in patients during scheduled pectoral or abdominal surgery performed under general anaesthesia with an epidural block (n = 15).

Endogenous opioid peptides	Time after start of surgery
Endogenous opioid peptides	0 min	20 min	40 min	60 min	80 min
β-Endorphin, pg/ml	46.3 ± 11.1	31.3 ± 13.3*	26.9 ± 15.4*	18.9 ± 15.7**	19.8 ± 14.7*
Leucine-enkephalin, pg/ml	321.7 ± 40.1	280.5 ± 29.3	259.6 ± 18.8*	192.3 ± 25.3**	135.6 ± 30.2**
Dynorphin A, pg/ml	130.5 ± 25.0	108.4 ± 36.2	55.8 ± 22.2*	49.3 ± 17.8*	55.3 ± 14.3*

Data presented as mean ± SD.

P < 0.05, **P < 0.01 compared with the value at 0 min; analysis of variance.

From the results of RT–PCR, the PPD/β-actin ratio decreased significantly at 80 min compared with the ratio at 0 min (P < 0.05), while the PPE/β-actin ratio showed no significant change (Table 2).

Table 2.

The semiquantitative levels of preproenkephalin (PPE), preprodynorphin (PPD) and β-actin mRNA in the peripheral blood lymphocytes of patients during scheduled pectoral or abdominal surgery performed under general anaesthesia with an epidural block (n = 15).

Surgical time-point	PPE	β-Actin	PPE/β-actin ratio	PPD	β-Actin	PPD/β-actin ratio
0 min	26576.11 ± 9840.90	25955.62 ± 9785.55	1.07 ± 0.26	33883.55 ± 1347.09	22702.74 ± 1403.78	1.29 ± 0.35
80 min	33086.55 ± 1767.79	28517.60 ± 1054.90	1.19 ± 0.44	25683.79 ± 1479.44	33650.03 ± 977.95	0.71 ± 0.28*

Data presented as mean ± SD.

P < 0.05 compared with the value at 0 min; Student’s t-test.

Discussion

In recent years, controlling and monitoring the depth of general anaesthesia has become an important area of research.²⁶ A number of indices, including BIS, spectral edge frequency, heart rate variability and auditory evoked potentials, have been considered as reflecting the depth of general anaesthesia.^27–29 To control the depth of anaesthesia in the current study, BIS and MAP were set as the feedback indices for stopping the propofol TCI. No pain or sensorium were reported to have been experienced during the operation and following palinaesthesia in all patients in this current study. The plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A were either significantly lower or showed no significant change from the baseline values during surgery, which explains why the patients reported experiencing no pain or sensorium during surgery.

In the current study, the PPD/β-actin mRNA ratio decreased significantly at 80 min compared with the baseline ratio at 0 min, while the PPE/β-actin mRNA ratio showed no significant change. These current findings suggest that the expression of these two pre-EOP genes was not increased during surgery performed under intravenous general anaesthesia and an epidural block. When the current results are compared with those of our previous research,²⁴ which showed a higher level of expression of pre-EOP genes in peripheral blood lymphocytes and a significant increase in the plasma concentrations of β-endorphin, leucine-enkephalin and dynorphin A during surgery performed under intravenous and inhalational general anaesthesia, they suggest that the patients in the current study were truly pain free when operated upon under general anaesthesia administered with an epidural block.

The active area of general anaesthetics runs from the peripheral nerve terminal to the cerebral cortex, where neurons are densely packed. The centre of the nociceptive reflex arc can be the neurons in the spinal cord or other areas of the central nerve system containing different types of neurons. It is well known that various types of neurons respond differently to anaesthetics. When a patient undergoes intravenous and inhalational general anaesthesia, the somatosensory impulses of pain, temperature and touch can still be successfully conducted towards the cortex. Surgical procedures can produce somatosensory impulses that stimulate the reticular formation of the central nervous system. Therefore, the electroencephalographic indices and depth of general anaesthesia can be unstable, as indicated by the fact that the BIS changes markedly when the skin is cut, which might explain why there is no one index that perfectly reflects the depth of general anaesthesia.²⁴

For the patient who is operated upon under intravenous general anaesthesia combined with an epidural block, no somatosensory pain impulses are conducted towards the brain during surgery because the active area of local anaesthetics is the spinal nerve root. As a consequence, the electroencephalographic indices would be stable so that the depth of anaesthesia could be readily controlled, as demonstrated in the current study.

This current study had a number of limitations. First, the half-life of EOPs in plasma is not very long because they are short peptides, so it might be more physiologically correct to measure the change in their concentrations in the cerebrospinal fluid. Secondly, the expression of the pre-EOP genes in the brain might better reflect the changes in response to surgery and type of anaesthesia. This current study was limited to the use of peripheral blood lymphocytes from arterial blood. Future research will focus on the expression of pre-EOP genes in the brain during surgery performed using different types of anaesthesia in animal models.

In conclusion, this current study demonstrated that the plasma concentrations of three endogenous opioid peptides, β-endorphin, leucine-enkephalin and dynorphin A, in patients during scheduled pectoral or abdominal surgery were either significantly lower or remained unchanged compared with baseline. In addition, the relative level of PPD mRNA was significantly lower at 80 min compared with baseline, while the relative level of PPE mRNA showed no significant change during surgery. Taken together, these findings suggest that the patients in this current study were truly pain free when operated upon under general anaesthesia administered with an epidural block.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.

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