It has been suggested that, following surgical trauma and general anaesthesia, an imbalance between pro-inflammatory and anti-inflammatory responses represents a crucial event underlying the transient postoperative immunological deficit . Among the anti-inflammatory mediators interleukin-10 (IL-10) seems to be mainly involved because an alteration in its induction has been demonstrated during the post-surgical period .
Nitric oxide (NO) is an highly reactive molecule whose biological role as well as clinical impact has been a matter of intensive studies in the past decade. NO is involved in several immunological processes including regulation of lymphocyte function . Furthermore, plasma levels of NO have been found to be impaired in the early postoperative period , and Tsuei and colleagues have suggested that IL-10 might be an important contributive factor regulating NO metabolism in post-trauma immune dysfunction .
Recent reports have focused on the crucial role of NO in the anaesthetic state. NO works as humoral mediator in excitatory synaptic transmission and its inhibition results in sedation. The observation that some anaesthetic agents were able to suppress excitatory transmission by blocking NO generation has been substantiated by various experimental trials [6,7].
The objective of this study was to investigate plasma concentrations of NO and IL-10 in the early postoperative period and to evaluate whether different anaesthesia techniques could influence systemic NO and IL-10 responses. Furthermore, we explored the possibility that a correlation between circulating NO and IL-10 concentration could occur during the early postoperative period.
Materials and methods
Thirty-two subjects, ASA I-II scheduled to undergo elective major surgery were enrolled. All participants provide written inform consent and the study protocol was approved by the Hospital's Ethics Committee. Criteria for exclusion were as follows: age <25 or >75 yr, pregnancy, renal and liver insufficiency, oncologic disease, infection including HIV infection, homeopathy, immunological dysfunction and treatment with nitro-derivate compounds or immunosuppressive drugs. Patients were allocated randomly into two groups using the sealed envelope technique. Group I (n = 15) received propofol (2 mg kg−1) and fentanyl (3 μg kg−1) for induction followed by propofol-fentanyl-air-oxygen for maintenance. Group II (n = 17) received thiopental (4 mg kg−1) for induction and sevoflurane-air-oxygen for maintenance of anaesthesia. All anaesthetics were administered by the same anaesthesiologists. All patients were premedicated with oral diazepam 0.2 mg kg−1. Sevoflurane was administrated using a vaporizer (Sevoflurane Vapor 19.2, Drager Medizintechnik, Lubeck, Germany) at a range of 1-3% and propofol using a target-controlled infusion pump (Terufusion, Terumo, Rome, Italy). Target induction concentrations were 4-6 μg mL−1 and anaesthesia was maintained with propofol (4-8 μg mL−1) with air and oxygen. In Group I intraoperative analgesia was secured with boluses of fentanyl adjusted by clinical signs and haemodynamic responses to surgical stimuli. Muscle relaxation was achieved by means of rocuronium (0.6-1.2 mg kg−1) and after tracheal intubation, the lungs were ventilated maintaining normocapnia. Electrocardiogram, pulse oximetry (Nellcor UltraCape N-600, Nellcor Inc, Harvad, CA) and non-invasive blood pressure monitoring (Dinamap, Critikon, Tampa, FL) were applied to all patients. At the end of surgery residual neuromuscular block was antagonized with neostigmine 2.5 mg and atropine 1.5 mg.
Fluid replacement during and after the operation was given according to a local protocol. Haemodynamic variables were maintained within 20% of preoperative values by adjusting the maintenance anaesthetic and fluid replacement accordingly.
In the anaesthetic room a large vein on the patient's forearm was cannulated. Blood samples to determine plasma concentrations of NO and IL-10 were collected before surgery (t0), at the end of surgery (t1) and after 24 h (t2). Ten healthy volunteers (from the medical staff) constituted the control group. Plasma IL-10 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (TEMA Ricerca s.r.l, Bologna, Italy). All samples were assayed in duplicate and the results were averaged at the end of the experiment. The lower assay limit of detection for IL-10 was 3 pg mL−1. Circulating NO concentration was measured by means of a total NO assay kit (Assay Designs, Inc., Ann Arbor, MI). This method involves the enzymatic conversion of nitrate to nitrite, by the enzyme nitrate reductase, followed by the colorimetric detection at 540 nm of nitrite as a coloured azo dye product of the Griess reaction. The kit allows for the total determination of both NO products in the sample by conversion of all the sample nitrate into nitrite, followed by the determination of the total concentration of nitrite in the sample. The detection limit of this technique was 1.35 μmol L−1.
Due to the small sample population, the group data were tested for variation from normality using the Kolmogorov-Smirnov, Shapiro-Wilks and Anderson- Darling tests. Results have been analysed by using both parametric (analysis of variance (ANOVA) for repeated measures with Bonferroni's test) and non-parametric tests (Kruskal-Wallis test and U-test). Correlation between plasma NO and IL-10 changes at the different time points was performed using Bravais-Pearson's correlation coefficient. A value of P < 0.05 was considered to indicate significance. All data are expressed as mean ± standard deviation (SD). Statistical analysis was carried out by means of the Statistica 6.0 software package (StatSoft Inc., Tulsa, OK).
The patient characteristics of the two groups, duration of anaesthesia and surgery, type of interventions as well as intraoperative anaesthetic dosages are summarized in Tables 1 and 2. There were no significant differences between the two groups and no significant perioperative complications. Blood loss and fluid replacement during surgery and in the first 24 h after operation were similar in each group.
Although this study has been performed as a pilot study on a small cohort of patients, the results we obtained were at once very clear. NO circulating concentrations were significantly reduced at times t1 and t2 compared with preoperative time in both groups (Group I: 13.76 ± 1.51 and 14.33 ± 1.52 mmol L−1 at t1 and t2, respectively, vs. 30.35 ± 2.70 mmol L−1 at t0, P < 0.0001 and P < 0.0001, respectively; Group II: 11.38 ± 0.95 and 12.52 ± 1.11 mmol L−1 at t1 and t2, respectively, vs. 28.23 ± 2.50 mmol L−1 at t0, P < 0.0001 and P < 0.0001, respectively). Preoperatively plasma NO values were similar to those of healthy volunteers. Furthermore, there was no significant difference between Groups I and II with respect to mean values of NO recorded at each time point (Fig. 1).
Plasma IL-10 concentrations showed significant elevation at t1 (26.35 ± 3.42 and 26.18 ± 3.22 pg mL−1 in Groups I and II, respectively) and t2 (75.39 ± 8.33 and 69.91 ± 7.33 pg mL−1 in Groups I and II, respectively) compared with baseline values (4.93 ± 0.31 and 5.50 ± 0.33 pg mL−1 in Groups I and II, respectively) peaking at t2 (t1 vs. t0: P = 0.03; t2 vs. t0: P < 0.0001; t1 vs. t2: P < 0.001 in Group I; t1 vs. t0: P = 0.02; t2 vs. t0: P < 0.0001; t1 vs. t2: P < 0.001 in Group II). A similar profile of plasma IL-10 levels was recorded in all patients. No significant difference with respect to mean values of IL-10 was observed between the two groups throughout the study time (Fig. 2). Plasma IL-10 levels were 4.50 ± 1.22 pg mL−1 in the control group of healthy subjects, not different from those measured in patients at the preoperative time.
Finally, we found that there was no correlation between the decrease of circulating NO at t1 and t2 with the elevation of plasma IL-10 concentrations (r = −0.11 and −0.15 at t1 and t2 times, respectively in Group I; r = 0.58 and 0.16 at t1 and t2 times, respectively in Group II).
We have demonstrated that in patients undergoing surgery under general anaesthesia circulating NO decreased and remained low at 24 h following operation. Such a decrease has been paralleled by an overregulation of the anti-inflammatory IL-10 cytokine, plasma concentrations of which were increased at the same time points at which a fall of plasma NO concentration occurred. No correlation was observed between the two phenomena. We also found that these changes were not influenced by the different anaesthesia techniques, as a similar value in the concentration of both NO and IL-10 was detected in patients treated with either total intravenous anaesthesia or an inhalational method.
The increased production of IL-10 in the early postoperative period is in line with findings of other researchers [8,9]. Such an increase could simply be related to expanding the T-2 lymphocyte population of which IL-10 is a product, to the detriment of the T-1 lymphocyte cells in patients undergoing surgical trauma . However, other factors have been involved as determinants of IL-10 surgery-induced increases including blood loss, stress hormones and anaesthetic drugs even though the ultimate cause of such an increase has not so far been elucidated [8,9]. In a previous study we also noticed an elevated plasma IL-10 concentration during the postoperative period closely associated with an aberrant rate of lymphocyte apoptosis .
The significantly reduced level of NO plasma concentration following surgical trauma is also in accordance with the results of other investigators and different injury-mediated events could account for the circulating NO decrease in the postoperative phase. Fujioka and colleagues suggested that hypoperfusion could be a main cause of the defect in perioperative NO production as they observed a significant negative correlation between serum nitrite/nitrate concentration and plasma lactate . Unfortunately, we cannot support this hypothesis as our patients had normal plasma lactate during the study period.
Some researchers underscore the role of an unknown inhibitor of NO synthesis as well as of high amount of blood glucocorticoids and glucagon. In their opinion, these could be alternative factors involved into the trauma-induced reduction of NO. In fact, those substances are able to inhibit in vitro NO generation in some different cellular lines [12,13]. Recently a close relationship has been found between the post-injury NO decrease and immune cell arginase. Arginase expression was increased in splenic immune cells of trauma model mice as well as in patients undergoing elective general surgery [5,14,15]. The arginase substrate arginine participates in the production of essential proteins in repairing wounds following surgical trauma. Since arginine plays a crucial role in NO metabolism, its availability can be insufficient for NO generation in the postoperative period . In short, post-surgical arginine depletion might be a main cause of the decreased plasma level of NO .
Several studies underline the link between IL-10 and the NO pathway. Ochoa and colleagues observed that the increase of IL-10 was directly dependent on the elevated arginase activity after trauma suggesting a crucial role of IL-10 in mediating NO production . This assumption was supported by Chang and Zdon, who hypothesized that IL-10 could down regulate NO synthetase activity in septic animal models undergoing surgical stress . In the present investigation we failed to demonstrate any evidence of a relationship between circulating IL-10 and NO.
Anaesthetic agents have been also involved as contributive factors to the postoperative fall in circulating NO. A previous study showed that intravenous and inhalational anaesthetics including thiopental and halothane were able to cause a significant decrease of NO synthetase activity from human polymorphonuclear leucocytes . Recent work has provided a new insight into the ability of anaesthetic substances to influence NO metabolism in the brain [6,7]. In the current study we found that the plasma level of NO was unaffected by the anaesthesia regimen as patients who received two different types of compounds to induce and maintain general anaesthesia exhibited similar changes in postoperative NO serum concentrations. Thus, these results could suggest that in patients undergoing an operative major procedure the decrease of NO plasma concentration is due by a great extent to the surgical stress.
In conclusion, although we only studied on small sample population, which has some limitations, our data strongly suggests that in the early postoperative period, no relationship could link the increase in circulating IL-10 with the altered production of NO. Moreover, the increase of IL-10 as well as the reduction in NO serum level seem to be two events independent by the type of agents used for general anaesthesia.
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