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Immune Cell Populations Decrease During Craniotomy Under General Anesthesia

Liu, Shujing, MD; Wang, Baoguo, PhD; Li, Shuqin, MD; Zhou, Yali, PhD; An, Lixin, MD; Wang, Yajie, PhD; Lv, Hong, MD; Zhang, Guojun, MD; Fang, Fang, MD; Liu, Zhizhong, PhD; Han, Ruquan, PhD; Jiang, Tao, PhD; Kang, Xixiong, PhD

doi: 10.1213/ANE.0b013e3182278237
Neuroscience in Anesthesiology and Perioperative Medicine: Research Reports
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SDC

BACKGROUND: Postoperative infections are common and potentially fatal complications in neurosurgical intensive care medicine. An impairment of immune function after central nervous system surgery is associated with higher risk of infection and postoperative complications. The aim of our study was to investigate how the immune cell population changes during the anesthesia process in patients undergoing craniotomy surgery.

METHODS: Patients undergoing craniotomy who had an inhaled general anesthetic were studied. Blood samples were collected before anesthesia and 30, 45, 60, 120, and 240 minutes after anesthesia began. Blood counts for neutrophils, monocytes, and lymphocytes were determined along with lymphocyte subpopulations (T cells, inducer and helper T cells, suppressor and cytotoxic T cells, natural killer cells, and B cells). Plasma concentrations of interleukin (IL)–2, IL-4, IL-6, and IL-10 were also measured along with tumor necrosis factor-α and interferon-γ. Data were analyzed by SPSS 13.0 software using repeated-measures analysis of variance followed by a Bonferroni correction.

RESULTS: Eighteen patients were enrolled in this study. In the comparison of the immune cell counts during neuroanesthesia, we found that at 30 minutes after anesthesia induction, neutrophils, monocytes, and lymphocytes decreased 18% (95% confidence interval [CI]: 11.0%–24.6%), 34% (95% CI: 16.2%–51.1%), and 39% (95% CI: 29.0%–48.9%) compared with their levels before anesthesia. At extubation the neutrophils returned to the base level. It also showed that natural killer cells decreased significantly during anesthesia. The concentration of cytokines in peripheral blood did not change significantly.

CONCLUSION: Our results showed that anesthesia and surgery upset the balance of the immune system during craniotomy, and a significant decrease in immune cell populations emerged after induction under general anesthesia.

Published ahead of print August 3, 2011

From Laboratory Diagnosis Center, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.

Funding: This study was funded by National Basic Research Program of China, and the program title was “Basic Research on Clinical Acupuncture Analgesia” (2007CB512500). The title of the subprogram was “Study on the Mechanism of Acupuncture Anesthesia for Craniotomy” (2007CB512503).

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Xixiong Kang, PhD, Beijng Tiantan Hospital, Capital Medical University, Laboratory Diagnosis Center, Beijing Tiantan Hospital, No.6 Tiantan Xili, Chongwen Men District, Beijing, 100050, China. Address e-mail to tt kangxixiong@sina.cn.

Accepted May 17, 2011

Published ahead of print August 3, 2011

Anesthesia and surgery modulate complex immune responses in patients undergoing major surgery.1 Cell-mediated immune balance is affected by anesthetics and surgery, and this could increase postoperative infections.2 The impairment of immunological function could increase the risk of developing postoperative complications, such as sepsis and multiple organ failure.3 In particular, postoperative infections have been reported to be important in neurosurgical management because of the potentially devastating clinical results.4

The natural killer (NK) cell population is a strong indicator of innate immune competence, and many postoperative immune function studies have focused on it.5 Previous studies also investigated the function of adaptive immunity after surgery, including total lymphocyte counts, lymphocyte subset proliferation, and a shift of the cytokine profile.6 For many years, in vitro and in vivo studies have focused on immune function after surgery. However, no studies have investigated the effects of anesthesia and surgery on immune function during surgery, especially the effects of anesthesia before surgery.

In this study, we investigated the immune cell numbers of neutrophils, monocytes, lymphocytes, and lymphocyte subsets including T cells, inducer and helper T cells, suppressor and cytotoxic T cells, NK cells, and B cells during neuroanesthesia. We also examined the concentration of the proinflammatory cytokines (interleukin [IL]–6 and tumor necrosis factor [TNF-α]), T helper 1 cytokines (IL-2 and interferon [IFN]–γ), and T helper 2 cytokines (IL-4 and IL-10) in peripheral blood.

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METHODS

Patient Management

In this study, 18 adult patients scheduled for elective neurosurgery were enrolled. The study protocol was approved by the IRB of our hospital, and written informed consent was obtained from all patients. Surgical procedures began at 8:30 AM and all cases were resection of mass lesions. Patients with immune, renal, or central nervous system dysfunction, as well as patients with congestive heart failure, exogenous hormone therapy (including steroids), malnutrition, diabetes, malignancy, infection, or inflammation were not included in the study.

General anesthesia was initiated with a combined infusion of 2 mg/kg propofol, 0.1 μg/kg sufentanil, and 0.1 mg/kg vecuronium. After tracheal intubation, patients' lungs were ventilated with oxygen-enriched air and the minimum alveolar concentration of sevoflurane to achieve end-tidal concentrations of 1.8% to 2%. During surgery, anesthesia was maintained by adjusting the end-tidal concentration of sevoflurane to keep arterial blood pressure and heart rate within 15% of preoperative values. Intermittent administration of 0.05 mg/kg vecuronium was performed to maintain muscle relaxation. In cases of hypotension, mean arterial blood pressure (<20% of baseline), bradycardia (heart rate <50 beats per minute [bpm]), or hypertension (mean arterial blood pressure >10% of baseline values), 6 mg ephedrine, 0.5 mg atropine, or 0.2 to 0.5 mg nicardipine was given for these problems, respectively.

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Sampling

Blood samples were taken immediately after entering the operating room (before anesthesia, T0), as well as 30 minutes (30 minutes after anesthesia induction, T1), 45 minutes (45 minutes after anesthesia induction, T2), 60 minutes (craniotomy start, T3), 120 minutes (tumor resection, T4), and 240 minutes (before extubation, T5) after anesthesia began. The blood samples were collected in ethylene diamine tetraacetic acid (EDTA) tubes (Becton Dickinson, Frankline Lakes, NJ) for blood cell counting and lymphocyte analysis and in SSTTMII advance tubes (Becton Dickinson, Oxford, UK) for cytokine concentration testing.

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Blood Cell Counts

A complete blood count was performed on all blood samples using an automated hemoanalyzer. The following variables were determined: the total white blood cell count and differential white blood cell counts, including total lymphocyte count, percentage lymphocytes, and hemoglobin concentration.

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Flow Cytometry

Lymphocyte subsets were analyzed by flow cytometry (Becton-Dickinson Immunocytometry Systems, San Jose, CA) with fluorescent-labeled antibodies specific to the cell markers. The following antibodies to lymphocyte antigens were used, and cell types were determined: CD3CD19+ (B cells), CD3+CD19 (T cells), CD3+CD4+ (inducer and helper T cells, Th), CD3+CD8+ (suppressor and cytotoxic T cells, Ts), and CD3CD16+CD56+ (NK cells). The proportions of B, T, Th, Ts, and NK cells were multiplied by the total number of lymphocytes, and cell counts were reported as number of cells per microliter (cells/μL).

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Multiplex Cytometric Bead Assay

A cytometric bead assay kit (Becton Dickinson) was used to measure levels of IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α in plasma according to described protocols.7

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Statistical Analysis

Results are expressed as means ± SD or median. Data of immune cells and cytokines were analyzed using repeated-measures analysis of variance with time as within-subject variables (4 comparisons) and tumor type and sex as between-subjects variables (5 comparisons, T1 to 5 with T0). In the analysis, Mauchly's test was used to judge whether there were relations among the repeatedly measured data. When P < 0.05, we did Greenhouse–Geisser to correct the results. All reported P values have been Bonferroni corrected. Data were analyzed by SPSS 13.0 software.

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RESULTS

Clinical Results

Patient characteristics and operative data are given in Table 1. There was no operative morbidity or mortality. Of the 18 patients, only 1 blood sample was not collected. Moreover, lymphocytes subsets tests were not performed in 2 patients.

Table 1

Table 1

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Tumor Type and Sex Influence

There were no significant differences between groups with regard to tumor type and sex. Repeated-measures analysis of variance was conducted to investigate the effect of tumor types and sex on the distribution of immune cells during surgery. The results showed that there were no statistically significant effects of tumor type or sex on immune cell populations and cytokine concentrations during surgery.

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White Blood Cell Counts

Leukocytes decreased to their minimum levels early after induction (all 4 P < 0.023), and increased to preoperative levels at the end of the surgery (Table 2). The lowest levels were reached after induction for neutrophils (decreased 18% [95% CI: 11.0%–24.6%] in comparison with T0), monocytes (decreased 34% [95% CI: 16.2% – 51.1%] in comparison with T0) and lymphocytes (decreased 39% [95% CI: 29.0%–48.9%] in comparison with T0) (Fig. 1). Neutrophils increased to preoperative levels by T5 (Table 2).

Table 2

Table 2

Figure 1

Figure 1

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Lymphocyte Subset Counts

T lymphocytes in peripheral blood decreased (significant effect of time, all 4 P < 0.028) and remained decreased at T5 (Table 2). This effect was mainly a result of a significant decrease of Ts cells. Ts cells decreased 43% (T1), 30% (T2), 33% (T3), 38% (T4), and 32% (T5) during surgery (Fig. 2). In contrast, Th cells decreased significantly at T1. At T2 to 5, there was no significant difference in Th cells in comparison with T0.

Figure 2

Figure 2

NK cell counts decreased, and the minimum level observed at T1 decreased 66% in comparison with T0. At the end of the surgery, the NK cells were still 48% below the preoperative level (Fig. 2). B cells decreased slightly and recovered at T5 (Table 2).

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Cytokine Concentrations

Circulating IL-6 was unaltered in the surgery and anesthesia. The release of Th1 cytokines (IL-2 and IFN-γ) and Th2 cytokines (IL-4 and IL-10) was not significantly changed during the intraoperative period. There was also no significant change in the production TNF-α (Table 3).

Table 3

Table 3

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DISCUSSION

In this study, a transient but significant alteration in the distribution of white blood cell populations and lymphocyte subsets was observed in brain tumor patients after anesthesia induction. In addition, Ts and NK cells decreased more significantly during anesthesia. Although anesthesiologists are aware of the risk for infection from surgical exposure, our findings indicate for the first time that there may be immune suppression that happens before surgery from the effects of anesthesia.

Previous studies have demonstrated that the distribution of white blood cell population and lymphocyte subsets was associated with surgery types8 and sex differences.9 Therefore, we selected patients undergoing craniotomy as the participants in our study. A cross-over study was conducted to investigate the effect of tumor types and sex on the distribution of immune cells during surgery. Our results showed that there were no effects of tumor type or sex on immune cell populations and cytokine concentrations during surgery. Therefore, the changes in the distribution of immune cells in the current study were time dependent. The same result was observed in Kempuraj et al.'s research. In their study, the trend of CD4+ T cell and CD8+ T changes was similar in benign and malignant brain tumor patients.10

The immune system can be conceptualized as 2 integrated systems, the innate and adaptive systems. There is generally a balance between the innate and adaptive immune systems, such that suppression of one is likely associated with enhancement of the other. The innate responses to an antigen include presenting the antigen for antibody production and the production of cytokines to stimulate adaptive responses. However, our study showed that all of the immune cells were decreased after induction and that this decrease was longer lasting for lymphocytes. This result indicated that the balance between the innate and adaptive immune systems was disturbed during craniotomy.

In the present study, the inhibitory effect on immune cells was most significant after finishing anesthesia induction, which was confirmed in our study by the reduction of neutrophil, monocyte, and lymphocyte counts. One possible cause of the immune cell decrease was the effect of blood loss and hemodilution. But after correcting these effects by monitoring the hemoglobin level, the result was the same. Another possible cause was the influence of anesthesia and surgery. Our results indicated that the most obvious changes of immune cell distribution were related to anesthesia induction. The period of anesthesia induction was the interval between induction of general anesthesia and maintenance before the onset of surgical incision.5 In addition, previous studies had also confirmed that anesthetics could induce dose-dependent and time-dependent immunosuppressive effects on neutrophil, monocyte, and lymphocyte cells.11,12 Furthermore, other studies showed that cortisol and adrenocortico tropic hormone plasma concentrations decreased after induction of anesthesia.13 Because the immune system was affected by the hypothalamic–pituitary–adrenal axis and the sympathetic nervous system,14 the decrease of immune cell counts may be the result of a suppressed stress system. These findings suggested that anesthesia and surgery had differential effects on patients that could have substantial clinical consequences. Previous studies also showed that general anesthesia significantly reduced the stress response after loss of consciousness, whereas surgery significantly increased inflammatory responses.15

The NK cell has been a focus of many postoperative immune function studies.16,17 Without prior sensitization, NK cells recognize and kill an array of virally infected and tumor targets, and initiate protection from some types of bacterial pathogens.18 Our investigation of circulating NK cells revealed that the number of NK cells was decreased during craniotomy. Additionally, other studies show that NK cell activity is suppressed within hours of surgery and lasts for days.19,20 A comparison with previous research indicated that a similar suppression of NK cells was also observed during craniotomy.

Previous studies have also revealed that surgery resulted in alterations within the specific, adaptive immune system, which primarily affects T helper cells.21 Our results showed that during neurosurgery, although the Th cell counts decreased, they recovered early before the end of surgery. A previous study was consistent with our results, which showed that Th cells were not inhibited by propofol.22 Moreover, it has been indicated that Th cell responses are most beneficial in terms of an appropriate and effective response to trauma and infection.23,24 As a result, Th cells may play an immunomodulatory role at the end of a craniotomy operation.

Because immune cells have receptors that stimulate cytokine synthesis,25 the concentrations of circulating cytokines were also observed in this study. This analysis showed that IL-6 was increased at the end of the surgery, but there were no significant differences. Previous studies also show an increased IL-6 response after surgery, and suggest that immediate cytokine responses might be relevant for the later onset of severe infections and sepsis.26 A previous study shows that proinflammatory mediator protein levels are significantly increased in situ after acute brain injury, whereas anti-inflammatory cytokine protein levels remain unchanged in humans.13 Therefore, our study suggested that the inflammatory response was activated at the end of surgery. At the same time, we also tested IL-2 and IFN-γ (Th1 production) as well as IL-4 and IL-10 (Th2 production). IL-2/IL-4 is an important marker reflecting the Th1/Th2 balance. Th1/Th2 balance has been used to explain most of the immunological phenomena observed in autoimmune diseases, infections, and tumors. Our study showed that the ratio of IL2/IL4 was not changed significantly during anesthesia. More research is needed to explain the association between cytokines and anesthesia as well as surgery.

Taken together, our investigation showed that anesthesia and surgery upset the balance of the immune system during craniotomy and that the most significant changes of immune cells are due to anesthesia induction. More studies investigating new anesthesia induction drugs with less immune suppression are expected.

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ACKNOWLEDGMENTS

This manuscript was handled by Gregory J. Crosby, MD.

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REFERENCES

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2. von Dossow V, Sander M, MacGill M, Spies C. Perioperative cell-mediated immune response. Front Biosci 2008;13:3676–84
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4. Herzer G, Trimmel H. Neuroanaesthesia, principles of optimized perioperative management. Anesthetist 2010;59:371–82
5. Page GG. Surgery-induced immunosuppression and postoperative pain management. AACN Clin Issues 2005;16:302–9
6. Ni Choileain N, Redmond HP. Cell response to surgery. Arch Surg 2006;141:1132–40
7. Ng PC, Li K, Wong RPO, Chui K, Wong E, Li G, Fok TF. Proinflammatory and anti-inflammatory cytokine responses in preterm infants with systemic infections. Arch Dis Child Fetal Neonatal Ed 2003;88:209–13
8. Sablotzki A, Ebel H, Mühling J, Dehne MG, Nopens H, Giesselmann H, Hempelmann G. Dysregulation of immune response following neurosurgical operations. Acta Anaesthesiol Scand 2000;44:82–7
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DISCLOSURES

Name: Shujing Liu, MD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Shujing Liu 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: Baoguo Wang, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Baoguo Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Shuqin Li, MD.

Contribution: This author helped conduct the study.

Attestation: Shuqin Li has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Yali Zhou, PhD.

Contribution: This author helped analyze the data.

Attestation: Yali Zhou has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Lixin An, MD.

Contribution: This author helped conduct the study.

Attestation: Lixin An has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Yajie Wang, PhD.

Contribution: This author helped design the study and analyze the data.

Attestation: Yajie Wang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Hong Lv, MD.

Contribution: This author helped analyze the data.

Attestation: Hong Lv has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Guojun Zhang, MD.

Contribution: This author helped analyze the data.

Attestation: Guojun Zhang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Fang Fang, MD.

Contribution: This author helped analyze the data.

Attestation: Fang Fang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Zhizhong Liu, PhD.

Contribution: This author helped analyze the data.

Attestation: Zhizhong Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Ruquan Han, PhD.

Contribution: This author helped design the study.

Attestation: Ruquan Han has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Tao Jiang, PhD.

Contribution: This author helped design the study.

Attestation: Tao Jiang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Xixiong Kang, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Xixiong Kang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

© 2011 International Anesthesia Research Society