Effect of two types of anesthesia on postoperative recovery of patients with gastric cancer and changes in the levels of their T lymphocyte subsets

Purpose: To investigate the effects of two types of anesthesia on postoperative recovery of patients with gastric cancer (GC), and to assess changes in the levels of their T lymphocyte subsets. Methods: Patients with GC (200 cases) were randomly assigned to two groups of 100 patients each: general anesthesia (GA) group and GA + epidural anesthesia (GAEP) group. Fasting venous blood samples (2 mL) were collected from the patients before anesthesia, and at different time-points: zero time (T0), 1 h (T1), day 1 (T2), and day 3 (T3) after surgery. CD3+, CD4+, CD8+T, and regulatory T cells (Tregs) were determined using a flow cytometer. Results: Percent CD3+, CD4+, CD8+T, and ratio of CD4+ to CD8+ in both groups at 1 h and day 1 after operation were significantly lower than their values before anesthesia (p < 0.05). There were significant differences in CD3+ and CD4+ levels between the two groups on day 3 (p < 0.05). Percent peripheral CD4+ CD25+T, and ratio of CD4+ CD25+ to CD4+ in both groups also significantly increased at 1 h and day 1 after operation, while percent peripheral CD4+, CD25+T and CD4+ CD25+ to CD4+ ratio in GAEP group on day 1 after operation were significantly lower than those of GA group (p < 0.05). Conclusion: These results suggest that combination of general and epidural anesthesia partly controls the expressions of Tregs, thereby enhancing postoperative recovery of GC patients.


INTRODUCTION
In China, gastric cancer (GC) is ranked first in the incidence of all malignant tumors, and it is the most frequently occurring cancer in the elderly aged 50 years and above [1,2].Operation-induced trauma, use of different anesthesia and postoperative pain have been shown to have negative impact on postoperative recovery and immunity of patients.Since GC patients have reduced immunity, the inhibition of autoimmune function usually induces complications such as postoperative infection and pyemia which can endanger their lives [3,4].
T-Lymphocyte subsets, especially regulatory Tlymphocytes (Tregs), partly reflect the state of a patient's immunity [5].Tregs are grouped into two: naturally-occurring Tregs and inducible or adaptive Tregs, and their immunosuppressive functions are closely related to autoimmune disorders [6,7].The aim of the present study was to investigate the effects of two types of anesthesia on postoperative recovery of patients with GC and to assess changes in the levels of their T lymphocyte subsets.

EXPERIMENTAL Materials and equipment
Flow cytometer (CytoFLEX) was purchased from Beckman Coulter, Inc, USA.High speed freezing centrifuge (DL-5M) was obtained from Changsha Xiangyi Centrifuge Co. Ltd., China.Mouse antihuman CD3-FITC, CD4-FITC, CD8-FITC and CD25-PE monoclonal antibodies were products of Abcam Biotechnology Co., UK.This research was approved by the Ethical Committee of Changzhou First People's Hospital (approval no.20180312), and performed according to the guidelines of Declaration of Helsinki promulgated in 1964 as amended in 1996 [8].

Patients and general information
A total of 200 patients with GC were recruited over a 2-year period for this study.They were randomly assigned to two groups of 100 patients each: general anesthesia (GA) group and GA + epidural anesthesia (GAEP) group.Patients in GA group consisted of 68 males and 32 females weighing between 46 and 81 kg (mean weight = 62.74 ± 5.71 kg), and aged 41 to 67 years (mean age = 59.84 ± 5.67 years.Group GAEP consisted of 70 males and 30 females weighing between 43 and 78 kg (mean weight = 61.27± 4.57 kg), and aged 42 to 66 years (mean age = 54.07 ± 6.27 years).Based on the American Society of Anesthesiologists (ASA) grading system, the patients were between grade I and II.There were no significant differences in the clinical data of patients in the two groups.The patients signed written informed consent with their family members.

Inclusion and exclusion criteria
The inclusion criteria were: (1)

Group GA
The patients were injected intramuscularly with 0.1 g of phenobarbital sodium and 0.5 mg atropine before surgery.Puncture was operated and a catheter inserted into the epidural space between T7 and T8.Thereafter, they received vecuronium bromide (0.12 mg/kg), fentanyl (2 -3 μg/kg), and propofol (2 mg/kg) as intravenous induction agents, while mechanical ventilation was carried out via an endotracheal tube.This was immediately followed with the administration of pancuronium bromide to maintain their muscular relaxation, and propofol by continuous intravenous drip (2 -4 mg/kg/h).In addition, enroflunine (1 -2 %) was given by inhalation to maintain anesthesia, and the end-expiratory concentration was maintained at 0.4 -0.9 MAC.In the radical operation for GC, patients also received additional 3 μg/kg fentanyl and 0.05 -0.08 mg/kg vecuronium, with 1 % lidocaine administered at 2 mL/h post-operation via a selfcontrolled analgesia pump.

Group GAEP
The patients were injected intramuscularly with 0.1 g phenobarbital sodium and 0.5 mg atropine before surgery.A puncture was made and a catheter inserted into the epidural space between T7 and T8; 3 mL of lidocaine (2 %) was injected.After 5 min, an anesthesia plane test was performed on the patients.After determination of the anesthetic effect and endotracheal intubation, an additional 4 -6 mL of 2 % lidocaine was administered through the epidural space, and thereafter maintained at 3 -5 ml/h until the completion of surgery.The block planes were located at T4 to T10.

Study indices and determination of levels of T lymphocyte subsets
General data for patients such as operation methods, length of incision and operation time were recorded.The time lag before the first postoperative flatus or defecation, length of stay in the hospital, diarrhea, throat discomfort, incision infection and pulmonary infection were also recorded.Fasting venous blood samples (2 mL) were collected from the patients before anesthesia and at different time points: zero time (T0), 1 h (T1), day 1 (T2), day 3 (T3) after surgery.The peripheral mononuclear cells were prepared using Ficoll density gradient method.Percent CD3+, CD4+, CD8+T, and Tregs were determined by flow cytometry.

Statistical analysis
Numeric data are expressed as mean ± SEM.Statistical analysis was performed using SPSS (version 17.0).Groups were compared using Chi-square ( 2 ) test.Values of p < 0.05 were considered statistically significant.

RESULTS Basic profile of patients
There were no significant differences in sex, age, operation method, length of incision, and operation time of the patients between the two groups (Table 1).

Outcomes of postoperative recovery condition
The length of hospital stay in group GAEP was significantly shorter than that of group GA (p < 0.05).The time of the first postoperative flatus or defecation, incidence of diarrhea, throat discomfort and incision infection in group GAEP were not significantly different from those of group GA (p > 0.05).There was no incidents of pulmonary infection in the two groups (Table 2).

Changes in levels of lymphocyte subsets of the patients
Percent CD3+, CD4+, CD8+T, and ratio of CD4+ to CD8+ in both groups at 1 h and day 1 after operation were significantly lower than their values before anesthesia (p < 0.05).However, at day 3 after operation, percent CD3+, CD4+, CD8+T, and CD4+/CD8+ in group C returned to their levels before anesthesia.There were no significant differences in percent CD3+, CD4+, CD8+T, and CD4+/CD8+ for the two groups at 1 h and day 1 (p < 0.05).However, there were significant differences in percent CD3+ and CD4+ for the two groups on day 3 (p < 0.05).The results are shown in Table 3.

Levels of Tregs in peripheral blood of the patients
Percent peripheral CD4+ CD25+T and ratio of CD4+CD25+ to CD4+ in both groups were significantly increased at 1 h and day 1 after operation.Percent peripheral CD4+ CD25+T and CD4+ CD25+/CD4+ in group GAEP at day 1 after operation were significantly lower than those in group GA (p < 0.05).However, at day 3 after operation, percent peripheral CD4+ CD25+T and CD4+ CD25+/CD4+ of both groups nearly returned to the same level prior to anesthesia (Table 4).

DISCUSSION
In recent years, the effect of different anesthesia on the treatment of cancers has received huge attention.However, a combination of general and epidural anesthesia not only reduces the dose of anesthesia significantly, but also effectively controls the afferent system of the autonomic nerves from nerve root level, and suppresses changes in the concentration of circulatory adrenocorticotropic hormone (ACTH), and other regulatory molecules.Thus, it may decrease the inhibitory effect of general anesthesia on the immune system [9].
In the present study, there were no significant differences in sex, age, operation method, length of incision, and operation time of the patients between the two groups.The length of stay in the hospital of group GAEP was significantly shorter than that of group GA.The time of the first postoperative flatus or defecation, incidence of diarrhea, throat discomfort and incision infection in group GAEP were not significantly different from those of group GA.There was no incident of pulmonary infection in the two groups.The percentages of CD3+, CD4+, CD8+T, and the ratio of CD4+ to CD8+ in both groups at 1 h and day 1 after operation were significantly lower than their values before anesthesia.However, at day 3 after operation, the percentages of CD3+, CD4+, CD8+T, and CD4+/CD8+ of group GAEP returned to their levels before anesthesia.There were no significant differences in the percentages of CD3+, CD4+, CD8+T, and CD4+/CD8+ between the two groups at 1 h and day 1.However, there were significant differences in the percentages of CD3+ and CD4+ between the two groups at day 3. Regulatory T cells (Tregs) refer to CD4+ CD25+, which plays a crucial role in the maintenance of immunologic function, immune response and immune tolerance [13].Some reports have suggested that Tregs are types of T cell which are negatively regulated.In addition, some authors have speculated that the roles of Tregs in peripheral blood of cancer patients are usually augmented, and that when compared with those with low levels, patients with higher Treg levels have poor prognosis [14,15].Tregs can restrict the activities of several kinds of T lymphocyte subsets and natural killer (NK) cells, and thus are able to trigger tumor immune tolerance [16].
In this study, percent peripheral CD4+CD25+T and ratio of CD4+ CD25+ to CD4+ in both groups were significantly increased at 1 h and day 1 after operation.Percent peripheral CD4+ CD25+T and CD4+ CD25+/ D4+ in GAEP group at day 1 after operation were significantly lower than those in GA group.However, on day 3 after operation, percent peripheral CD4+ CD25+T and CD4+ CD25+ to CD4+ ratio in both groups nearly returned to the same level before anesthesia.

Limitations of the study
The limitation of this study is the use of a small sample size and a single population.

CONCLUSION
The results of this study suggest that a combination of general and epidural anesthesia can partly control the expressions of Tregs, thus enhancing postoperative recovery of GA patients.

Table 2 :
Postoperative recovery conditions of patients (mean ± SD) Key: TFPFD: time of first postoperative flatus or defecation; HS: hospital stay; TD: throat discomfort; II: incision infection; PI: pulmonary infection

Table 3 :
Changes in the levels of lymphocyte subsets in patients (n, %)

Table 4 :
Levels of Tregs in peripheral blood of the patients (n, %) b p < 0.05 compared to group G