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The effects of neurosurgical stress on peripheral lymphocyte subpopulations
Original Contributions The Effects of Neurosurgical Stress on Peripheral Lymphocyte Subpopulations Yoshiyuki Hori, MD,* Takae Ibuki, MD, PhD,† Toyoshi Hosokawa, MD, PhD,‡ Yoshifumi Tanaka, MD, PhD§ Department of Anesthesiology, Kyoto Prefectural University of Medicine, and Department of Anesthesia, Kyoto Saiseikai Hospital, Nagaokakyo, Kyoto, Japan
*Chief, Department of Anesthesia, Kyoto Saisekai Hospital †Associate Professor of Anesthesiology ‡Professor of Anesthesiology §Professor and Chairman, Department of Anesthesiology, Kyoto Prefectural University of Medicine Address correspondence to Dr. Ibuki at the Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. Received for publication January 7, 2002; revised manuscript accepted for publication July 2, 2002.
Study Objective: To observe changes in the peripheral lymphocyte subpopulations as an index of cellular immunity during neurosurgical procedures. Design: Clinical study. Setting: Operating room of a university hospital. Patients: 11 patients with early intracranial disease who were scheduled to undergo elective neurosurgery with general anesthesia. Patients in the control group (n ⫽ 10) underwent minor surgeries such as ophthalmologic, otorhinolaryngological, or orthopedic surgeries. Interventions: Blood was sampled before anesthesia induction (t0) for baseline and at 1 hour (t1) and 2 hours (t2) following surgical incision. Measurements: Detection and quantification of lymphocyte subpopulations were performed at each time point using single-label and double-label analyses of monoclonal antibodies against lymphocyte membrane surface markers. Main Results: Significant changes in patients who underwent a neurosurgical procedure included: the percentage of total T cells (CD3⫹) from 57.54 ⫾ 3.50% at t0 to 51.41 ⫾ 4.26% at t1 and 46.29 ⫾ 4.02% at t2; the percentage of inducer T cells (CD4⫹, Leu8⫹) from 27.39 ⫾ 2.26% at t0, to 23.26 ⫾ 2.30% at t1 and 20.82 ⫾ 2.70% at t2; the CD4/CD8 ratio, from 1.78 ⫾ 0.25% at t0 to 1.35 ⫾ 0.12% at t1 and 1.22 ⫾ 0.17% at t2. The percentage of suppressor T cells (CD8⫹, Leu15⫹) increased significantly from 10.8 ⫾ 1.07% at t0 to 13.64 ⫾ 1.62% at t1, and 14.82 ⫾ 1.24% at t2. The percentages of the natural killer cell subsets also increased significantly. Control group patients who underwent minor surgeries showed no significant changes. Conclusions: Neurosurgery-induced significant suppression of cellular immunity was demonstrated in peripheral lymphocyte subpopulations, probably from the surgical stress on the central nervous system. © 2003 by Elsevier Science Inc. Keywords: Flow cytometry; immune response; lymphocytes; subpopulation of; neurosurgery.
Introduction The concept that multiple changes in immune function are induced by anesthesia, surgery, and other kinds of injury has been presented in the works of pioneer investigators, as reviewed by Salo.1 These changes include lymphopenia
Journal of Clinical Anesthesia 15:1– 8, 2003 © 2003 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0952-8180/03/$–see front matter doi 10.1016/S0952-8180(02)00455-5
Original Contributions
and impaired function of T lymphocytes, B lymphocytes, and monocytes,2–5 such as blastogenic responsiveness to mitogens, antibody-dependent lymphocyte cytotoxicity, and delayed hypersensitivity skin response.3,5,6 –10 However, most of the studies were focused on differential effects of anesthetic drugs,11–13 anesthetic methods,14 degree of surgical stress,15 or patients’ ages.13,16 The surgeries studied involved the thoracic, abdominal, orthopedic, or gynecologic areas but few studies on the brain.17 The brain has long been considered to be an immunologically privileged site, and involvement of the brain in systemic immunologic processes has been overlooked. However, the results of recent work suggests focusing on the brain as the major site of immune response.18 In our previous studies, we reported immunosuppressive changes in the peripheral lymphocyte subpopulations following surgical stress to extracranial areas during major surgeries.15,19 Until now, only little has been known about the effect of neurosurgical stress on the peripheral lymphocyte subpopulations. In this study, we tested the hypothesis that surgical stress to the intracranial region induces a suppressive effect on the systemic immune system.
Patients and Methods Patients Eleven patients with benign intracranial diseases, who were scheduled to undergo elective open cranial surgery, were selected for this study. This study was approved by the Institutional Committee on Human Research of the Kyoto Prefectural University of Medicine, and all human subjects signed written informed consent. Diseases included arteriovenous malformation, aneurysms, and benign brain tumors. These patients (5 males and 6 females, mean age 52.1 ⫾ 5.18 yrs) were all in good general condition without any complications from the primary disease. No patient had been taking medication, including steroids, before surgery. The control group included 10 patients (5 males and 5 females, mean age 59.1 ⫾ 4.9 yrs) who were scheduled for minor surgeries for retinal detachment, parotid gland tumor, or habitual dislocation of the shoulder joint.
Anesthesia and Blood Sampling All patients in both groups underwent surgery with the same anesthetic method. Thirty minutes before anesthesia induction, the patient was premedicated with intramuscular injections of atropine sulfate (0.01 mg/kg) and hydroxyzine (1 mg/kg). General anesthesia was induced with intravenous injections of thiopental sodium (5 mg/ kg) and succinylcholine (1 mg/kg), followed by endotracheal intubation. Anesthesia was maintained with a mixture of 66% nitrous oxide, 33% oxygen, and 0.5% to 1.0% enflurane. Supplementary doses of fentanyl citrate and pancuronium bromide were injected intravenously as needed. Local anesthesia was not used in either the control or the neurosurgical groups before skin incision or head pin insertion. Peripheral venous blood was sam2
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pled from patients before the induction (t0) and at 1 hour (t1) and 2 hours (t2) following surgical incision. In the neurosurgical group, manipulation of the brain tissue had already been begun by 1 hour. Samples were collected in EDTA-filled sampling tubes and used in the analysis of peripheral lymphocytes.
Analysis of Lymphocyte Subpopulations Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ), washed with Hank’s balanced salt solution, and adjusted to 2 ⫻ 107 cells per mL of phosphate-buffered saline to make up a final volume of 1 mL per test tube. Leu-series monoclonal antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA) were used to identify and quantify lymphocyte subpopulations with laser flow cytometry (Ortho-spectrum-3, Ortho Diagnostic Systems, Co. Ltd., Tokyo, Japan) by single-label analysis with CD3 (Leu4), CD4 (Leu3a), CD8 (Leu2a), and CD19 (Leu12). In addition, pairs of monoclonal antibodies such as CD4 and Leu8, and CD8 and CD11 (Leu15), were used in double-label analysis to classify functional subpopulations of lymphocytes (helper, inducer, suppressor, and cytotoxic). We used two monoclonal antibodies, CD16 (Leu11) and CD57 (Leu7), to identify natural killer (NK) subsets. The change in NK cell activity was evaluated by observing the cytotoxicity of NK cells against K-562 target cells. The relationship between Leu antibodies and peripheral lymphocyte subpopulations is shown in Figure 1. The baseline with which all fluctuations were compared was defined as the concentrations of lymphocyte subpopulations before anesthesia induction. The data in Figure 2, Figure 3, and Figure 4 are, therefore, given in terms of fractional change from the baseline value. In this manner, each patient served as his/her own control. The CD4/CD8 ratio was calculated as the proportion of the cells positive to the CD4 antibody divided by the proportion of cells positive to the CD8 antibody. Values are presented as means ⫾ standard error (SE). The obtained data were analyzed by one-way analysis of variance with repeated measures. If the null hypothesis was rejected, multiple comparisons were performed with Bonferroni’s test. p ⬍ 0.05 was set to determine statistically significant differences.
Results As shown in Figure 2, in the neurosurgery group, the percentage of total peripheral T cells, defined by CD3 (Leu4) reactivity, decreased significantly from the baseline value of 57.54 ⫾ 3.50% to 51.41 ⫾ 4.26% at t1, with fractional change of 10.7%, and this change was maintained at t2. In contrast, the proportion of B cells, defined by CD19 (Leu12⫹), decreased slightly during the 2-hour period of surgery with no statistical change. There was no significant change in the minor surgery group. The CD4 (Leu3a⫹) subset, shown in Figure 3 (upper panel), decreased significantly from 38.74 ⫾ 2.94% to 32.17 ⫾ 2.62% at t1, with fractional change of 20.0%, and
Neurosurgical stress on lymphocytes: Hori et al.
Figure 1. The relationship between Leu antibodies and peripheral lymphocyte subpopulations are shown. The superscript ⫹ or – after each marker indicates that the measured lymphocyte subset is marker-positive or marker-negative.
this change was maintained at t2 but the CD8 (Leu2a) subset (middle panel) showed no change. Consequently, the CD4/CD8 (Leu3a/Leu2a) ratio decreased significantly from the baseline value of 1.78 ⫾ 0.25 to 1.22 ⫾ 0.17% at t2 (lower panel). In the minor surgery group, there were no significant changes in the CD4, CD8 subset or the CD4/CD8 ratio. Double-label analysis showed that helper T cells (Leu3a⫹, Leu8–)(Figure 4, a) did not change but inducer T cells (Leu3a⫹, Leu8⫹)(Figure 4, b) decreased significantly. Although the proportions of CD8 positive cells did not change, cytotoxic T cells (Leu2a⫹, Leu15–) (Figure 4, c) decreased slightly but significantly at t2 and suppressor T cells (Leu2a⫹, Leu15⫹)(Figure 4, d) increased significantly during neurosurgery. No significant changes in these lymphocyte subsets occurred in the minor surgery group. Natural killer cell subsets showed marked fluctuation (Figure 5). A statistically significant increase in Leu11⫹, Leu7– cells, which possess the greatest cytotoxic activity, occurred during the neurosurgical procedure from 5.51 ⫾ 0.90% at t0 to 7.97 ⫾ 1.61% at t2. The Leu11– , Leu7⫹ cells showed no significant change (data not shown). The Leu11⫹, Leu7⫹ cells, which possess moderate cytotoxic activity, showed a marked increase from 19.29 ⫾ 2.86% at t0 to 30.26 ⫾ 3.88% at t2. In contrast, there were no significant changes in NK cell subsets in the minor surgery group.
Discussion A novel finding in this study was that a localized surgical injury to the brain, an area that heretofore was regarded as immunologically segregated, triggered immunosuppression, as shown by changes in peripheral lymphocyte subpopulations, including NK cells. In humoral immunity, B
cells have a major role in producing antibodies following the detection of an exogenous antigen by macrophages. Both the helper T cells, through the enhancement of antibody synthesis, and the suppressor T cells, through the inhibition of the antibody synthesis, have a regulatory influence on the function of B cells. In cellular immunity, helper T cells are involved directly or via the action of killer helper factors in the induction of cytotoxic T cells and lymphokines following an invasion by exogenous antigens. Furthermore, suppressor T cells control the induction of cytotoxic T cells and directly affect their differentiation and proliferation. Thus, interactions between helper T cells and suppressor T cells are involved in humoral immunity as well as in cellular immunity. Therefore, we regarded the functional analysis of peripheral T lymphocyte subpopulations as an indicator of immune function, and we observed time-dependent changes in the subpopulations with a two-color analytical method. The change in NK cell activity was evaluated by observing the cytotoxicity of NK cells against K-562 target cells. The Leu11 antibody has been used as a marker for NK cells. However, the Leu11 positive cells are heterogeneous and can be used to divide NK cells into subsets.20,21 Lanier et al.21 used two monoclonal antibodies, Leu7 and Leu11, to divide NK cells into three subsets with different levels of cytotoxicity. With this method, the change in the number of NK cells of each subset can be determined rapidly and accurately without the use of radioisotopes. In our study, the percentage changes in NK cell subsets increased significantly during the neurosurgical procedure, although NK cell activity was not examined. Tønnensen et al.22 studied changes in NK cell activity during anesthetic maneuvers, upper abdominal surgery,23 and coronary artery bypass grafting (CABG)24 relative to an endocrine stress response. Previous studies have indicated J. Clin. Anesth., vol. 15, February 2003
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Figure 2. The percentage changes from preinduction levels of total peripheral T cells (upper panel) and total peripheral B cells (lower panel) are shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
Figure 3. The percentage changes from preinduction levels of CD4⫹ cells (upper panel), CD8⫹ cells (middle panel), and the CD4/CD8 ratio (lower panel) are shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
enhanced NK cell activity following premedication and during anesthesia and surgery22,23,25,26 and only a slight increase during minor surgery.27 Some studies have also demonstrated that selective increases in epinephrine enhance NK cell activity,28 –30 whereas increases in cortisol suppress the activity, both in vivo and in vitro.31 Tønnensen et al.24 even suggested selectively using epinephrine to stimulate the NK cell system during CABG under cardiopulmonary bypass, an operation that evokes a marked endocrine stress response. Furthermore, in our study, the increase in NK cell subpopulations during a neurosurgical procedure was, to our knowledge, quantified for the first time. Therefore, we suspect that NK cell activity is also increased during neurosurgical procedures and that the increase in NK cell subpopulations, possibly induced by
mobilization from extravasal spaces or lymphoid tissues into the systemic circulation, may be one of the factors contributing to the increased activity. Mechanisms underlying the fluctuations in peripheral lymphocyte subpopulations are complex and require further study. One of the most important problems in this kind of study was the preoperative use of glucocorticoids for the alleviation of brain edema, because it is well known that glucocorticoids are immunosuppressive. In most cases of malignant brain tumors, preoperative use of glucocorticoids is standard care. Furthermore, it is believed that malignant tumors in the brain affect the preoperative immune system just as such malignant tumors are known to affect other systemic functions. In fact, in other studies of the effect of neurosurgical stress on the immune system,17 patients were studied who had a variety of
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Neurosurgical stress on lymphocytes: Hori et al.
Figure 4. The percentage changes from preinduction levels of (a) helper, (b) inducer, (c) cytotoxic, and (d) suppressor T cells are shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
diseases such as benign and malignant tumors and aneurysm, among others, and the patients were taking steroid therapy. However, in our study, so as to observe the effect of neurosurgery on the immune system exclusively, we limited the study population to only those patients with benign intracranial diseases who had not received any preoperative or intraoperative administration of glucocorticoids. Immune function in the brain is considered to be totally different from that in other organs because of the existence of the blood-brain barrier and the lack of a lymphoid system. Animal studies have shown disorders in immune specificity to brain tumors. It has been reported that brain tumors in humans induce such immunologic changes as a decrease in the number of lymphocytes,32,33 decreased activity of lymphocytes,34,35 delayed reaction to a hypersensitivity skin test,32 increased activity of suppressor cells,35 and a decrease in the CD4/CD8 ratio.36 These changes can be caused by tumors in other regions as well as by brain tumors; however, in cases of tumors in other regions, such changes occur at a more advanced, not early stage. Conversely, brain tumors can affect the immune state before they induce systemic changes because in most cases, tumors are restricted to the intracranial space
throughout their invasion, a phenomenon specific to brain tumors. Only a few reports address the direct effect of neurosurgical procedures on the immune system.17 Many studies have been performed on changes in the immune state during anesthesia and surgery; an extensive review of the effects of anesthesia and surgery on the immune response was made by Salo.1 According to his study, the numbers of T and B lymphocytes tend to decrease depending on the extent of surgery, although no changes were observed in the numbers or proportions of these cells during minor surgery. His review also indicated that CD4/CD8 proportions remain either unchanged or change in favor of CD8-positive cells after operations of increasing trauma severity. In this study, we found fluctuations in the immune system following neurosurgical manipulation. A decrease in the ratio of the number of T lymphocytes to the total number of lymphocytes was observed, which suggests a decrease in the number of T lymphocytes because reportedly there is no significant change in the total number of lymphocytes during surgery.19 From these observations, neurosurgical stimulation may be considered a major surgical stress. The CD4/CD8 ratio decreased following neurosurgical manipulation in this J. Clin. Anesth., vol. 15, February 2003
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Figure 5. The percentage change from the preinduction level of natural killer (NK) cell subsets is shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
study. Although this ratio has been regarded as an important index of cellular immunity and most of the previous studies have focused on the CD4/CD8 ratio, it is now controversial in basic immunology. However, in this study, by using single-label and double-label analyses of lymphocyte membrane surface markers, we were able further to differentiate each subpopulation as helper, inducer, suppressor, or cytotoxic T cells. Therefore, it can be demonstrated that the decrease in the ratio was induced by both a decrease in the inducer T cells and an increase in the suppressor T cells. Changes in the immune system as shown in this study are similar to those reported in other major surgeries.1,19 Although the precise mechanism of the immune response is not clear, the message of surgical stress may be transmitted to immune cells to induce the response. Many 6
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bioactive substances may be involved in this process. Glucocorticoids and catecholamines, which influence the redistribution of leukocytes, and glucocorticoids, which trigger antiinflammatory as well as immunosuppressive effects, may be key substances in perioperative immunologic changes. In clinical studies, increased levels of both cortisol and catecholamine have been proven to induce changes in the immune system similar to those observed during surgery,37,38 although no obvious correlation between serum levels of cortisol and these changes was demonstrated.39 Only a few studies have been undertaken to study the changes of immune function during the perioperative period of neurosurgery. Asadullah et al.17 demonstrated immunodepression following neurosurgical procedures by showing a decrease in monocytic human leukocyte antigen-DR expression and the association with a preceding intracranial inflammatory response. The preceding inflammatory response shown by the increase in interleukin (IL)-6 and IL-8 in the cerebrospinal fluid occurred a couple of hours after the end of surgery. In their prospective study, they related the very low expression of monocytic human leukocyte antigen-DR expression and the possibility of infection. Compared with their study, our study included only patients who were not taking perioperative steroid medication, and it was focused on the immunologic changes during the neurosurgical maneuver, namely the very acute phase. For the first time we have, to our knowledge, shown that such immunosupressive changes can occur immediately after the beginning of manipulation of brain tissue, even without the use of steroids. It is not evident whether such immunosuppressive changes are induced by the intracranial inflammatory response or from other mechanisms, but it is likely that operative manipulation of brain tissue is the major stimulus to the patients. Generally, nociceptive stimulation can be regarded as the painful stimulation to the tissue during surgery. In fact, in cases of abdominal surgery, immunosuppression was inhibited by an epidural block.40 However, in cases involving the brain, pain sensation is not the major nociceptive stimulation to the body, because brain tissue itself lacks pain sensation. Brain may be specific in the point that immune function can be suppressed with less painful stimulation. Although the exact mechanism of the immunosupression is not clear, it is suspected that immunosuppressive changes in the lymphocyte subpopulations were induced by the activation of the hypothalamic-pituitary-adrenal (neuroendocrine) axis response induced by the intracranial inflammatory response. The significance of the phenotypic changes observed in this study is unclear. However, immunosuppression during and after surgery may contribute to subsequent infectious complications or metastasis of a malignancy. As was mentioned previously, in abdominal surgery, immunosuppression could be partly inhibited by an epidural block.40 Gogos et al.41 suggested that a cyclo-oxygenase inhibitor may improve immunocompetence in patients undergoing major surgical procedures during the perioperative period. Further investigation is necessary to clarify the mechanism underlying the immunologic fluctuation so as to
Neurosurgical stress on lymphocytes: Hori et al.
determine its clinical implications, and to ascertain which anesthesia method and course of perioperative management would prevent immune dysfunction after neurosurgical procedures. Especially for the brain, some unclarified stimulus may be more important than nociceptive stimulus. In conclusion, we have measured the lymphocyte subpopulations during neurosurgery and proved that immunosuppressive changes such as the decrease in total peripheral T cells, CD4 positive cells, CD4/CD8 ratio, cytotoxic T cells, inducer T cells, and the increase in suppressor T cells, Leu11⫹Leu7– cells and Leu11⫹Leu7⫹ cells were induced possibly by neurosurgical stress to the brain.
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38. Davis JM, Albert JD, Tracy KJ, et al: Increased neutrophil mobilization and decreased chemotaxis during cortisol and epinephrine infusions. J Trauma 1991;31:725–32. 39. Ide H, Kakiuchi T, Furuta N, et al: The effect of cardiopulmonary bypass on T cells and their subpopulations. Ann Thorac Surg 1987;44:277–82. 40. Hashimoto T, Hashimoto S, Hori Y, Nakagawa H, Hosokawa T: Epidural anaesthesia blocks changes in peripheral lymphocytes subpopulation during gastrectomy for stomach cancer. Acta Anaesthesiol Scand 1995;39:294 –8. 41. Gogos CA, Maroulis J, Zoumbos NC, Salsa B, Kalfarentzos F: The effect of parenteral indomethacin on T-lymphocyte subpopulations and cytokine production in patients under major surgical operations. Res Exp Med 1995;195:85–92.
*Chief, Department of Anesthesia, Kyoto Saisekai Hospital †Associate Professor of Anesthesiology ‡Professor of Anesthesiology §Professor and Chairman, Department of Anesthesiology, Kyoto Prefectural University of Medicine Address correspondence to Dr. Ibuki at the Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan. Received for publication January 7, 2002; revised manuscript accepted for publication July 2, 2002.
Study Objective: To observe changes in the peripheral lymphocyte subpopulations as an index of cellular immunity during neurosurgical procedures. Design: Clinical study. Setting: Operating room of a university hospital. Patients: 11 patients with early intracranial disease who were scheduled to undergo elective neurosurgery with general anesthesia. Patients in the control group (n ⫽ 10) underwent minor surgeries such as ophthalmologic, otorhinolaryngological, or orthopedic surgeries. Interventions: Blood was sampled before anesthesia induction (t0) for baseline and at 1 hour (t1) and 2 hours (t2) following surgical incision. Measurements: Detection and quantification of lymphocyte subpopulations were performed at each time point using single-label and double-label analyses of monoclonal antibodies against lymphocyte membrane surface markers. Main Results: Significant changes in patients who underwent a neurosurgical procedure included: the percentage of total T cells (CD3⫹) from 57.54 ⫾ 3.50% at t0 to 51.41 ⫾ 4.26% at t1 and 46.29 ⫾ 4.02% at t2; the percentage of inducer T cells (CD4⫹, Leu8⫹) from 27.39 ⫾ 2.26% at t0, to 23.26 ⫾ 2.30% at t1 and 20.82 ⫾ 2.70% at t2; the CD4/CD8 ratio, from 1.78 ⫾ 0.25% at t0 to 1.35 ⫾ 0.12% at t1 and 1.22 ⫾ 0.17% at t2. The percentage of suppressor T cells (CD8⫹, Leu15⫹) increased significantly from 10.8 ⫾ 1.07% at t0 to 13.64 ⫾ 1.62% at t1, and 14.82 ⫾ 1.24% at t2. The percentages of the natural killer cell subsets also increased significantly. Control group patients who underwent minor surgeries showed no significant changes. Conclusions: Neurosurgery-induced significant suppression of cellular immunity was demonstrated in peripheral lymphocyte subpopulations, probably from the surgical stress on the central nervous system. © 2003 by Elsevier Science Inc. Keywords: Flow cytometry; immune response; lymphocytes; subpopulation of; neurosurgery.
Introduction The concept that multiple changes in immune function are induced by anesthesia, surgery, and other kinds of injury has been presented in the works of pioneer investigators, as reviewed by Salo.1 These changes include lymphopenia
Journal of Clinical Anesthesia 15:1– 8, 2003 © 2003 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0952-8180/03/$–see front matter doi 10.1016/S0952-8180(02)00455-5
Original Contributions
and impaired function of T lymphocytes, B lymphocytes, and monocytes,2–5 such as blastogenic responsiveness to mitogens, antibody-dependent lymphocyte cytotoxicity, and delayed hypersensitivity skin response.3,5,6 –10 However, most of the studies were focused on differential effects of anesthetic drugs,11–13 anesthetic methods,14 degree of surgical stress,15 or patients’ ages.13,16 The surgeries studied involved the thoracic, abdominal, orthopedic, or gynecologic areas but few studies on the brain.17 The brain has long been considered to be an immunologically privileged site, and involvement of the brain in systemic immunologic processes has been overlooked. However, the results of recent work suggests focusing on the brain as the major site of immune response.18 In our previous studies, we reported immunosuppressive changes in the peripheral lymphocyte subpopulations following surgical stress to extracranial areas during major surgeries.15,19 Until now, only little has been known about the effect of neurosurgical stress on the peripheral lymphocyte subpopulations. In this study, we tested the hypothesis that surgical stress to the intracranial region induces a suppressive effect on the systemic immune system.
Patients and Methods Patients Eleven patients with benign intracranial diseases, who were scheduled to undergo elective open cranial surgery, were selected for this study. This study was approved by the Institutional Committee on Human Research of the Kyoto Prefectural University of Medicine, and all human subjects signed written informed consent. Diseases included arteriovenous malformation, aneurysms, and benign brain tumors. These patients (5 males and 6 females, mean age 52.1 ⫾ 5.18 yrs) were all in good general condition without any complications from the primary disease. No patient had been taking medication, including steroids, before surgery. The control group included 10 patients (5 males and 5 females, mean age 59.1 ⫾ 4.9 yrs) who were scheduled for minor surgeries for retinal detachment, parotid gland tumor, or habitual dislocation of the shoulder joint.
Anesthesia and Blood Sampling All patients in both groups underwent surgery with the same anesthetic method. Thirty minutes before anesthesia induction, the patient was premedicated with intramuscular injections of atropine sulfate (0.01 mg/kg) and hydroxyzine (1 mg/kg). General anesthesia was induced with intravenous injections of thiopental sodium (5 mg/ kg) and succinylcholine (1 mg/kg), followed by endotracheal intubation. Anesthesia was maintained with a mixture of 66% nitrous oxide, 33% oxygen, and 0.5% to 1.0% enflurane. Supplementary doses of fentanyl citrate and pancuronium bromide were injected intravenously as needed. Local anesthesia was not used in either the control or the neurosurgical groups before skin incision or head pin insertion. Peripheral venous blood was sam2
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pled from patients before the induction (t0) and at 1 hour (t1) and 2 hours (t2) following surgical incision. In the neurosurgical group, manipulation of the brain tissue had already been begun by 1 hour. Samples were collected in EDTA-filled sampling tubes and used in the analysis of peripheral lymphocytes.
Analysis of Lymphocyte Subpopulations Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (Pharmacia Fine Chemicals, Piscataway, NJ), washed with Hank’s balanced salt solution, and adjusted to 2 ⫻ 107 cells per mL of phosphate-buffered saline to make up a final volume of 1 mL per test tube. Leu-series monoclonal antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA) were used to identify and quantify lymphocyte subpopulations with laser flow cytometry (Ortho-spectrum-3, Ortho Diagnostic Systems, Co. Ltd., Tokyo, Japan) by single-label analysis with CD3 (Leu4), CD4 (Leu3a), CD8 (Leu2a), and CD19 (Leu12). In addition, pairs of monoclonal antibodies such as CD4 and Leu8, and CD8 and CD11 (Leu15), were used in double-label analysis to classify functional subpopulations of lymphocytes (helper, inducer, suppressor, and cytotoxic). We used two monoclonal antibodies, CD16 (Leu11) and CD57 (Leu7), to identify natural killer (NK) subsets. The change in NK cell activity was evaluated by observing the cytotoxicity of NK cells against K-562 target cells. The relationship between Leu antibodies and peripheral lymphocyte subpopulations is shown in Figure 1. The baseline with which all fluctuations were compared was defined as the concentrations of lymphocyte subpopulations before anesthesia induction. The data in Figure 2, Figure 3, and Figure 4 are, therefore, given in terms of fractional change from the baseline value. In this manner, each patient served as his/her own control. The CD4/CD8 ratio was calculated as the proportion of the cells positive to the CD4 antibody divided by the proportion of cells positive to the CD8 antibody. Values are presented as means ⫾ standard error (SE). The obtained data were analyzed by one-way analysis of variance with repeated measures. If the null hypothesis was rejected, multiple comparisons were performed with Bonferroni’s test. p ⬍ 0.05 was set to determine statistically significant differences.
Results As shown in Figure 2, in the neurosurgery group, the percentage of total peripheral T cells, defined by CD3 (Leu4) reactivity, decreased significantly from the baseline value of 57.54 ⫾ 3.50% to 51.41 ⫾ 4.26% at t1, with fractional change of 10.7%, and this change was maintained at t2. In contrast, the proportion of B cells, defined by CD19 (Leu12⫹), decreased slightly during the 2-hour period of surgery with no statistical change. There was no significant change in the minor surgery group. The CD4 (Leu3a⫹) subset, shown in Figure 3 (upper panel), decreased significantly from 38.74 ⫾ 2.94% to 32.17 ⫾ 2.62% at t1, with fractional change of 20.0%, and
Neurosurgical stress on lymphocytes: Hori et al.
Figure 1. The relationship between Leu antibodies and peripheral lymphocyte subpopulations are shown. The superscript ⫹ or – after each marker indicates that the measured lymphocyte subset is marker-positive or marker-negative.
this change was maintained at t2 but the CD8 (Leu2a) subset (middle panel) showed no change. Consequently, the CD4/CD8 (Leu3a/Leu2a) ratio decreased significantly from the baseline value of 1.78 ⫾ 0.25 to 1.22 ⫾ 0.17% at t2 (lower panel). In the minor surgery group, there were no significant changes in the CD4, CD8 subset or the CD4/CD8 ratio. Double-label analysis showed that helper T cells (Leu3a⫹, Leu8–)(Figure 4, a) did not change but inducer T cells (Leu3a⫹, Leu8⫹)(Figure 4, b) decreased significantly. Although the proportions of CD8 positive cells did not change, cytotoxic T cells (Leu2a⫹, Leu15–) (Figure 4, c) decreased slightly but significantly at t2 and suppressor T cells (Leu2a⫹, Leu15⫹)(Figure 4, d) increased significantly during neurosurgery. No significant changes in these lymphocyte subsets occurred in the minor surgery group. Natural killer cell subsets showed marked fluctuation (Figure 5). A statistically significant increase in Leu11⫹, Leu7– cells, which possess the greatest cytotoxic activity, occurred during the neurosurgical procedure from 5.51 ⫾ 0.90% at t0 to 7.97 ⫾ 1.61% at t2. The Leu11– , Leu7⫹ cells showed no significant change (data not shown). The Leu11⫹, Leu7⫹ cells, which possess moderate cytotoxic activity, showed a marked increase from 19.29 ⫾ 2.86% at t0 to 30.26 ⫾ 3.88% at t2. In contrast, there were no significant changes in NK cell subsets in the minor surgery group.
Discussion A novel finding in this study was that a localized surgical injury to the brain, an area that heretofore was regarded as immunologically segregated, triggered immunosuppression, as shown by changes in peripheral lymphocyte subpopulations, including NK cells. In humoral immunity, B
cells have a major role in producing antibodies following the detection of an exogenous antigen by macrophages. Both the helper T cells, through the enhancement of antibody synthesis, and the suppressor T cells, through the inhibition of the antibody synthesis, have a regulatory influence on the function of B cells. In cellular immunity, helper T cells are involved directly or via the action of killer helper factors in the induction of cytotoxic T cells and lymphokines following an invasion by exogenous antigens. Furthermore, suppressor T cells control the induction of cytotoxic T cells and directly affect their differentiation and proliferation. Thus, interactions between helper T cells and suppressor T cells are involved in humoral immunity as well as in cellular immunity. Therefore, we regarded the functional analysis of peripheral T lymphocyte subpopulations as an indicator of immune function, and we observed time-dependent changes in the subpopulations with a two-color analytical method. The change in NK cell activity was evaluated by observing the cytotoxicity of NK cells against K-562 target cells. The Leu11 antibody has been used as a marker for NK cells. However, the Leu11 positive cells are heterogeneous and can be used to divide NK cells into subsets.20,21 Lanier et al.21 used two monoclonal antibodies, Leu7 and Leu11, to divide NK cells into three subsets with different levels of cytotoxicity. With this method, the change in the number of NK cells of each subset can be determined rapidly and accurately without the use of radioisotopes. In our study, the percentage changes in NK cell subsets increased significantly during the neurosurgical procedure, although NK cell activity was not examined. Tønnensen et al.22 studied changes in NK cell activity during anesthetic maneuvers, upper abdominal surgery,23 and coronary artery bypass grafting (CABG)24 relative to an endocrine stress response. Previous studies have indicated J. Clin. Anesth., vol. 15, February 2003
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Original Contributions
Figure 2. The percentage changes from preinduction levels of total peripheral T cells (upper panel) and total peripheral B cells (lower panel) are shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
Figure 3. The percentage changes from preinduction levels of CD4⫹ cells (upper panel), CD8⫹ cells (middle panel), and the CD4/CD8 ratio (lower panel) are shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
enhanced NK cell activity following premedication and during anesthesia and surgery22,23,25,26 and only a slight increase during minor surgery.27 Some studies have also demonstrated that selective increases in epinephrine enhance NK cell activity,28 –30 whereas increases in cortisol suppress the activity, both in vivo and in vitro.31 Tønnensen et al.24 even suggested selectively using epinephrine to stimulate the NK cell system during CABG under cardiopulmonary bypass, an operation that evokes a marked endocrine stress response. Furthermore, in our study, the increase in NK cell subpopulations during a neurosurgical procedure was, to our knowledge, quantified for the first time. Therefore, we suspect that NK cell activity is also increased during neurosurgical procedures and that the increase in NK cell subpopulations, possibly induced by
mobilization from extravasal spaces or lymphoid tissues into the systemic circulation, may be one of the factors contributing to the increased activity. Mechanisms underlying the fluctuations in peripheral lymphocyte subpopulations are complex and require further study. One of the most important problems in this kind of study was the preoperative use of glucocorticoids for the alleviation of brain edema, because it is well known that glucocorticoids are immunosuppressive. In most cases of malignant brain tumors, preoperative use of glucocorticoids is standard care. Furthermore, it is believed that malignant tumors in the brain affect the preoperative immune system just as such malignant tumors are known to affect other systemic functions. In fact, in other studies of the effect of neurosurgical stress on the immune system,17 patients were studied who had a variety of
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Neurosurgical stress on lymphocytes: Hori et al.
Figure 4. The percentage changes from preinduction levels of (a) helper, (b) inducer, (c) cytotoxic, and (d) suppressor T cells are shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
diseases such as benign and malignant tumors and aneurysm, among others, and the patients were taking steroid therapy. However, in our study, so as to observe the effect of neurosurgery on the immune system exclusively, we limited the study population to only those patients with benign intracranial diseases who had not received any preoperative or intraoperative administration of glucocorticoids. Immune function in the brain is considered to be totally different from that in other organs because of the existence of the blood-brain barrier and the lack of a lymphoid system. Animal studies have shown disorders in immune specificity to brain tumors. It has been reported that brain tumors in humans induce such immunologic changes as a decrease in the number of lymphocytes,32,33 decreased activity of lymphocytes,34,35 delayed reaction to a hypersensitivity skin test,32 increased activity of suppressor cells,35 and a decrease in the CD4/CD8 ratio.36 These changes can be caused by tumors in other regions as well as by brain tumors; however, in cases of tumors in other regions, such changes occur at a more advanced, not early stage. Conversely, brain tumors can affect the immune state before they induce systemic changes because in most cases, tumors are restricted to the intracranial space
throughout their invasion, a phenomenon specific to brain tumors. Only a few reports address the direct effect of neurosurgical procedures on the immune system.17 Many studies have been performed on changes in the immune state during anesthesia and surgery; an extensive review of the effects of anesthesia and surgery on the immune response was made by Salo.1 According to his study, the numbers of T and B lymphocytes tend to decrease depending on the extent of surgery, although no changes were observed in the numbers or proportions of these cells during minor surgery. His review also indicated that CD4/CD8 proportions remain either unchanged or change in favor of CD8-positive cells after operations of increasing trauma severity. In this study, we found fluctuations in the immune system following neurosurgical manipulation. A decrease in the ratio of the number of T lymphocytes to the total number of lymphocytes was observed, which suggests a decrease in the number of T lymphocytes because reportedly there is no significant change in the total number of lymphocytes during surgery.19 From these observations, neurosurgical stimulation may be considered a major surgical stress. The CD4/CD8 ratio decreased following neurosurgical manipulation in this J. Clin. Anesth., vol. 15, February 2003
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Figure 5. The percentage change from the preinduction level of natural killer (NK) cell subsets is shown. Closed circles correspond to the data from the patients in the neurosurgery group, and open circles correspond to that from the patients in the minor surgery group. *p ⬍ 0.05, **p ⬍ 0.01 significantly different from the preoperative value. Data are given as means ⫾ SEM.
study. Although this ratio has been regarded as an important index of cellular immunity and most of the previous studies have focused on the CD4/CD8 ratio, it is now controversial in basic immunology. However, in this study, by using single-label and double-label analyses of lymphocyte membrane surface markers, we were able further to differentiate each subpopulation as helper, inducer, suppressor, or cytotoxic T cells. Therefore, it can be demonstrated that the decrease in the ratio was induced by both a decrease in the inducer T cells and an increase in the suppressor T cells. Changes in the immune system as shown in this study are similar to those reported in other major surgeries.1,19 Although the precise mechanism of the immune response is not clear, the message of surgical stress may be transmitted to immune cells to induce the response. Many 6
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bioactive substances may be involved in this process. Glucocorticoids and catecholamines, which influence the redistribution of leukocytes, and glucocorticoids, which trigger antiinflammatory as well as immunosuppressive effects, may be key substances in perioperative immunologic changes. In clinical studies, increased levels of both cortisol and catecholamine have been proven to induce changes in the immune system similar to those observed during surgery,37,38 although no obvious correlation between serum levels of cortisol and these changes was demonstrated.39 Only a few studies have been undertaken to study the changes of immune function during the perioperative period of neurosurgery. Asadullah et al.17 demonstrated immunodepression following neurosurgical procedures by showing a decrease in monocytic human leukocyte antigen-DR expression and the association with a preceding intracranial inflammatory response. The preceding inflammatory response shown by the increase in interleukin (IL)-6 and IL-8 in the cerebrospinal fluid occurred a couple of hours after the end of surgery. In their prospective study, they related the very low expression of monocytic human leukocyte antigen-DR expression and the possibility of infection. Compared with their study, our study included only patients who were not taking perioperative steroid medication, and it was focused on the immunologic changes during the neurosurgical maneuver, namely the very acute phase. For the first time we have, to our knowledge, shown that such immunosupressive changes can occur immediately after the beginning of manipulation of brain tissue, even without the use of steroids. It is not evident whether such immunosuppressive changes are induced by the intracranial inflammatory response or from other mechanisms, but it is likely that operative manipulation of brain tissue is the major stimulus to the patients. Generally, nociceptive stimulation can be regarded as the painful stimulation to the tissue during surgery. In fact, in cases of abdominal surgery, immunosuppression was inhibited by an epidural block.40 However, in cases involving the brain, pain sensation is not the major nociceptive stimulation to the body, because brain tissue itself lacks pain sensation. Brain may be specific in the point that immune function can be suppressed with less painful stimulation. Although the exact mechanism of the immunosupression is not clear, it is suspected that immunosuppressive changes in the lymphocyte subpopulations were induced by the activation of the hypothalamic-pituitary-adrenal (neuroendocrine) axis response induced by the intracranial inflammatory response. The significance of the phenotypic changes observed in this study is unclear. However, immunosuppression during and after surgery may contribute to subsequent infectious complications or metastasis of a malignancy. As was mentioned previously, in abdominal surgery, immunosuppression could be partly inhibited by an epidural block.40 Gogos et al.41 suggested that a cyclo-oxygenase inhibitor may improve immunocompetence in patients undergoing major surgical procedures during the perioperative period. Further investigation is necessary to clarify the mechanism underlying the immunologic fluctuation so as to
Neurosurgical stress on lymphocytes: Hori et al.
determine its clinical implications, and to ascertain which anesthesia method and course of perioperative management would prevent immune dysfunction after neurosurgical procedures. Especially for the brain, some unclarified stimulus may be more important than nociceptive stimulus. In conclusion, we have measured the lymphocyte subpopulations during neurosurgery and proved that immunosuppressive changes such as the decrease in total peripheral T cells, CD4 positive cells, CD4/CD8 ratio, cytotoxic T cells, inducer T cells, and the increase in suppressor T cells, Leu11⫹Leu7– cells and Leu11⫹Leu7⫹ cells were induced possibly by neurosurgical stress to the brain.
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