Stress-induced immune responses, known for more than a century as leukocytosis after physical work , have been studied extensively in physical stresses since immune-phenotyped flow cytometry was established [2,3]. More recently, laboratory stress studies [4,5] found that acute psychological stresses may elicit changes in leukocyte numbers similar to those occurring in physical stresses. Both types of stresses evoke – mainly by release of catecholamines – leukocytosis resulting from a release of natural killer cells (NK-cells), of CD8+ T-cells, of monocytes and of neutrophils [3,6].
However, there is little proof that laboratory stress models can be applied to daily clinical routines . The extent of immune responses induced by painful events occurring in clinical day-to-day life has not yet been sufficiently investigated. As a likely inductor of an immunological stress response the setting of retrobulbar anaesthesia prior to intraocular surgery allows the study of a representative for a short-term painful anaesthetic procedure under highly standardized conditions.
Most patients requiring ophthalmological surgery can be considered healthy with regard to their immune system. Thus, the present study aimed to evaluate the impact of the painful process of retrobulbar anaesthesia on cell numbers of leukocyte and lymphocyte subsets in apparently healthy elderly individuals. The setting of retrobulbar anaesthesia was previously shown to double plasma epinephrine concentration within 2 min, thus inducing a more significant release of catecholamines than the following surgical intervention . We, therefore, expected the retrobulbar block to provoke a distinct catecholamine-induced leukocytosis as an indicator of pain-induced stress.
A group of 16 female patients [age, 72.9 years (range, 61–82 years); body weight, 68.6 kg (range 50–96 kg); ASA (American Society of Anesthesiologists) physical status grade I (n =4) or II (n =12)] volunteered to participate in the study. All patients were scheduled to undergo intraocular surgery with retrobulbar anaesthesia [cataract surgery (n =12) or retinal surgery (n =4)]. Patients suffering from insulin-dependent diabetes mellitus, acute infections, malignant tumours, severe ischaemic heart disease [CCS (Canadian Cardiovascular Society) grade III–IV] or any neurological, immunological, haematopoetical or other severe organic disease were excluded from the study.
Concomitant medication consisted of digitalis (6 patients), diuretics (5), ACE inhibitors (4), calcium channel blockers (3), L-thyroxin (2), allopurinol (2), nitrates (2), fibrates (2), theophylline (1), anti-depressive agents (1) and sulphonylurea (1). No patients had received anxiolytic or sedative premedication.
The study was conducted between August and October 1995. Written consent was obtained from all patients. The protocol and consent form for the study were reviewed and approved by the regional ethics committee in Saarbrücken, Germany, August 16, 1995.
All patients were taken to the surgery unit at 07:30 h (range 07:20–08:30 h) in the supine position. Through a venous cannula inserted into a peripheral vessel, blood samples were taken five times: directly after the placing of the venous cannula, after an interval of 30 min (one minute before the setting of the retrobulbar block) and 2, 15 and 45 min after the setting of the retrobulbar block. Surgery commenced thereafter.
Heart rate, systolic and diastolic arterial pressures were registered simultaneously (Invivo 4500plus1 Pulse Oximeter, Omega 1445, Invivo Research Inc, Birmingham, USA).
For the block of the eyelid according to O'Brien , 1.5 mL of a bupivacaine solution [bupivacaine 0.75% (Astra, Wedel, Germany)] with the addition of 0.05 mL naphazoline nitrate 1:30000 (Novartis, Basel, Switzerland) and 7.5 IE hyaluronidase (Pharma Dessau, Dessau, Germany) per mL bupivacaine mixed with 0.5 mL of the short-acting local anaesthetic articaine 2% (Hoechst Marion Roussel, Frankfurt, Germany) was used. For the method of retrobulbar injection according to Atkinson , 3.5 mL of this bupivacaine solution and 1.5 mL of articaine 2% were used. Thereafter, pressure on the eye of about 40 mmHg was applied for 10 min.
Serum cortisol was measured by radioimmunoassay (Travenol Baxter, Unterschleißheim, Germany).
Leukocyte subpopulations were determined using 3-colour flow cytometry (direct immunofluorescence technique). Samples of 20 μL EDTA blood were incubated (15 min at room temperature, in darkness) with an adequate amount of fluorescein isothiocyanate (FITC), phycoerythrin (PE) and/or R-phycoerythrin-cyanine 5 (PE-Cy5/TRI) – conjugated monoclonal antibody (anti-UCHT1-PE-Cy5 (CD3), anti-13B8.2-FITC (CD4), anti-B9.11-PE (CD8), anti-RMO52-PE (CD14), anti3G8-FITC (CD16), anti-L130-FITC (CD18), anti-Immu19.2/RMO52-FITC/PE (CD45/CD14), anti-UCHL-1-PE (CD45RO), anti-B159-PE (CD56) (all from Immunotech, Hamburg, Germany); anti-MEM-18-TRI (CD14), anti-CR3-PE (CD11b), anti-HL38-TRI (HLA-DR) (all from Medac, Hamburg, Germany)).
The cells were incubated for 7 min at room temperature in darkness with 1 mL of lysing solution [No. 92–0002, diluted in a 1:9 ratio with distilled water (Becton-Dickinson, Heidelberg, Germany)]. Stained and fixed cells were washed [2000 r.p.m., at room temperature, phosphate-buffered saline (Cell Wash, Becton-Dickinson)], stored at 4°C in the dark and measured in a flow cytometer (FACScan, Becton-Dickinson) within 4 h.
Data acquisition and analysis were made with FACScan research software (PC-Lysis 1.0, Becton-Dickinson) using live gates in the forward scatter vs. side scatter dot plot for leukocyte separation. Absolute cell numbers of leukocyte subpopulations were calculated on the basis of total leukocytes (Sysmex microcellcounter, model F-800, TOA Medical Electronics Company, Ltd, Kobe, Japan). Concentrations of haemoglobin and packed cell volume were used to calculate a dilution factor respecting plasma volume changes .
All data are expressed as means and standard deviation (SD). Variables were tested for normal distribution with the Kolmogorov–Smirnov test. Normally distributed variables were analysed by one-way analysis of variance (1+5 repeated measurement design). The Scheffé post hoc comparison was then applied to determine the significance of differences in means. Calculations were done with STATISTICA software (Stat Soft, Tulsa, USA). The level of significance was taken as P < 0.05.
The retrobulbar block induced an increase in systolic arterial pressure within 2 min (+15.2 mmHg, P < 0.01; Table 1). Diastolic arterial pressure [+4.3 mmHg, NS (not significant)] and heart rate [+ 3.8 beats minute−1 NS] had a tendency to increase. Cardiovascular variables returned to baseline values within 15 min.
There was a decrease in serum cortisol during the preoperative phase (Table 2). A minor rise in cortisol was observed with a latency of 15 min after the retrobulbar block.
Leukocytes and major subpopulations
The setting of the retrobulbar block induced a small but significant increase in leukocytes (P < 0.01; Table 3), mainly caused by a rise in the neutrophil count (+5.5%, P < 0.01). Total lymphocytes (+4.4%) did not change significantly.
Regarding the entire observation period, numbers of leukocytes rose continuously as a result of a constant increase in neutrophils, reaching statistical significance 1 min before and 15 min after the retrobulbar block. Numbers of lymphocytes fell continuously over the preoperative period, only to be interrupted by the short-term release of lymphocytes induced by the retrobulbar block.
During the first 30 min after the patients' arrival at the surgery unit, numbers of lymphocytes declined (−10.1%, P < 0.05). This effect was more pronounced for NK-cells (−23.1%, P < 0.01, Figure 1) than for total numbers of T-cells (−8.6%, P < 0.05). Among all T-cell subsets studied, CD8+ cells declined the most (−14.1%, P < 0.05). Decreases in non-MHC-restricted cytotoxic T-cells (CD3+ CD16/CD56+ −14.6%) and in CD4+CD8+ T-cells (−14.3%) did not reach statistical significance due to the small cell numbers of these subsets. B-cells (−1.6%) and CD4+-cells (−8.4%) did not change significantly.
The setting of retrobulbar anaesthesia evoked a release of certain lymphocyte subsets: NK-cells increased significantly (+22.1%, P < 0.01). Among T-cell-subsets, CD8+ cells (+8.1%, NS), non-MHC-restricted cytotoxic T-cells (CD3+ CD16/CD56+ +16.4%, NS) and CD4+CD8+ T-cells (+10.0%, NS) had a tendency to increase. Within 15 min, these lymphocyte subsets returned to counts similar to those measured immediately before the block. Neither CD4+-T-cells nor B-cells rose considerably after the retrobulbar block.
We examined to what degree short-term, stressful anaesthetic procedures induce acute immunological stress reactions. Even though such immunological stress reactions are known to occur in laboratory models of acute psychological stress [4,5], there is surprisingly little evidence that laboratory stress models – mostly studied among young and healthy volunteers – are useful clinically . As a model for a stressful anaesthetic procedure, the painful setting of the retrobulbar anaesthesia was studied, as it allowed highly standardized study conditions.
In the present study, setting the retrobulbar block induced a significant increase in peripheral blood leukocytes, mainly due to rising numbers of neutrophils and of NK-cells. Furthermore, cytotoxic, non-MHC-restricted T-cells, CD8+ T-cells and CD4+ CD8+ T-cells had a tendency to increase. In contrast, cell numbers of CD4+ T-cells and B-cells remained unchanged. Within 15 min, lymphocyte subset counts fell to baseline values, whereas neutrophils continued to rise during the whole study period.
Similarly, in laboratory stress models, acute psychological stresses [4,5] provoke increases in NK-cells and in CD8+ cells. Likewise, NK-cells, CD8+ cells and cytotoxic, non-MHC-restricted T-cells rather than B-cells or CD4+ cells increase during acute physical stress [2,3].
Acute psychological and physical stresses induce interactions between the neuroendocrine and the immune system, which are mainly mediated by a stress-induced release of catecholamines. In accordance, the setting of the retrobulbar block is known to provoke a release of catecholamines within 2 min, resulting in a short-term doubling of plasma epinephrine .
By activating β2-adrenoceptors expressed on certain leukocyte subsets, catecholamines cause lymphocytes and NK-cells adhering to the vessel wall (‘marginal pool’ ) to detach from it and to enter the circulating blood . This demargination of cells is probably due to changes in the avidity state of adhesion molecules [3,14,15].
Infusions of epinephrine or β-adrenergic agonists mimic stress-induced effects on lymphocyte subsets , whereas the application of β-adrenergic antagonists blunts the lymphocytosis after acute stress . Interestingly, lymphocyte subtypes, which show the most pronounced stress-induced increases in cell numbers (NK-cells and CD8+ cells), have a larger density of β2-receptors than CD4+ cells, B-cells and monocytes .
To summarize, the acute sympatho-adrenal stress reaction after the painful retrobulbar block induces changes in immunological variables, as it leads to changes in cardiovascular variables (increase in arterial pressure and in heart rate) . The return of arterial blood pressure and cell counts of lymphocyte subsets to baseline values within 15 min reflects the short-term half-life period of plasma catecholamines.
Beyond this sympatho-adrenal stress reaction, we found a release of cortisol evoked by the block with a latency of 15 min . Former studies measured serum cortisol levels 2 min after retrobulbar anaesthesia and therefore failed to observe this increase in glucocorticoids . This rise is partly concealed by the physiological circadian decline of serum cortisol after the early morning high, coinciding with our study period. Corticosteroids can cause neutrophilia  and lymphopenia  with a latency of about 1 h. The demargination of leukocytes observed within 2 min after retrobulbar anaesthesia can thus not be explained by the stress-induced release of glucocorticoids.
A potential drawback of our study is that the baseline values 30 and 1 min before retrobulbar anaesthesia already reflect a patient's state of mind influenced by preoperative anxiety. Thus, even before setting the retrobulbar anaesthesia, catecholamines may have reached a higher level and may have induced some demargination of leukocytes. Whereas the first time point of blood sampling reflects the anxious moments upon arrival at the surgery unit, the second blood sample is drawn after 30 min of rest, which may explain the significant decrease in arterial blood pressure and in numbers of T-lymphocytes and of NK-cells.
Circadian rhythms interfere with the stress-induced changes in leukocyte counts: the continuous increase in neutrophils and the decline of lymphocyte subsets observed during the preoperative period corresponds to the physiological increase in numbers of neutrophils and the physiological fall in lymphocyte cell counts between 08:00 h and 12:00 h, the examination period chosen in this study [22,23].
This pain-induced mobilization of leukocytes into the circulation is considered to be a non-specific sign of activation of the immune system, allowing for rapid transport of leukocytes to any locus of potential harm. Whether this catecholamine-induced immediate leukocytosis with acute stress goes along with any important changes in cell function on single cell level , is discussed controversially. Although the acute changes in cell counts of leukocyte subsets we observed in this study might seem too minor to have an impact on the patient's overall health, there could be a cumulative adverse effect on immune function in patients who have to submit to painful and stressful procedures repetitively. This might contribute to the increased incidence of infectious complications in these patients.
Further studies are needed to find out whether anxiolytic premedication might modify the immunological stress reaction.
The authors would like to thank Hans-Josef Müller for excellent technical assistance. The participation of patients and the nursing staff at the Department of Ophthalmology, Homburg, is also gratefully acknowledged.
1 Schultz G. Experimentelle Untersuchungen über das Vorkommen und die diagnostische Bedeutung der Leukocytose. Deutsch Arch F Klin Med
1893; 51: 234–281.
2 Gabriel H, Kindermann W. Flow cytometry. Principles and applications in exercise immunology. Sports Med
1995; 20: 302–320.
3 Gabriel H, Kindermann W. The acute immune response to exercise: what does it mean? Int J Sports Med
1997; 18 (Suppl. 1): S28–S45.
4 Naliboff BD, Benton D, Solomon GF, et al.
Immunological changes in young and old adults during brief laboratory stress. Psychosom Med
1991; 53: 121–132.
5 Cacioppo JT, Malarkey WB, Kiecolt-Glaser JK, et al.
Heterogeneity in neuroendocrine and immune responses to brief psychological stressors as a function of autonomic cardiac activation. Psychosom Med
1995; 57: 154–164.
6 Pedersen BK, Bruunsgaard H, Klokker M et al.
Exercise-induced immunomodulation – possible roles of neuroendocrine and metabolic factors. Int J Sports Med
1997; 18 (Suppl. 1): S2–S7.
7 Linden W, Rutledge T, Con A. A case for the usefulness of laboratory social stressors. Ann Behav Med
1998; 20: 310–316.
8 Adams HA, Hessemer V, Hempelmann G, Jacobi KW. [Endocrine stress response in cataract operations with local anesthesia]. Klin Monatsbl Augenheilkd
1992; 200: 273–277.
9 O'Brien HD. Anesthesia for cataract surgery. Am J Ophthalmol
1964; 57: 751–760.
10 Atkinson W. Anesthesia in Ophthalmology
. Springfield, IL: Thomas, 1955: 60.
11 Dill DB, Costill DL. Calculation of percentage changes in Volumes of blood, plasma, and red cells in dehydration. J Appl Physiol
1974; 37: 247–248.
12 Athens JW, Haab OP, Raab SO. Leukokinetic studies – IV. The total blood, circulating and marginal granulocyte pools and the granulocyte turnover rate in normal subjects. J Clin Invest
1961; 40: 989–1001.
13 Benschop RJ, Oostveen FG, Heijnen CJ, Ballieux RE. Beta 2-adrenergic stimulation causes detachment of natural killer cells from cultured endothelium. Eur J Immunol
1993; 23: 3242–3247.
14 Rehman J, Mills PJ, Carter SM, et al.
Dynamic exercise leads to an increase in circulating ICAM-1: further evidence for adrenergic modulation of cell adhesion. Brain Behav Immun
1997; 11: 343–351.
15 Gabriel HH, Kindermann W. Adhesion molecules during immune response to exercise. Can J Physiol Pharmacol
1998; 76: 512–523.
16 van Tits LJ, Michel MC, Grosse-Wilde H, et al.
Catecholamines increase lymphocyte beta 2-adrenergic receptors via a beta 2-adrenergic, spleen-dependent process. Am J Physiol
1990; 258: E191–E202.
17 Maisel AS, Fowler P, Rearden A, Motulsky HJ, Michel MC. A new method for isolation of human lymphocyte subsets reveals differential regulation of beta-adrenergic receptors by terbutaline treatment. Clin Pharmacol Ther
1989; 46: 429–439.
18 Benschop RJ, Godaert GL, Geenen R, et al.
Relationships between cardiovascular and immunological changes in an experimental stress model. Psychol Med
1995; 25: 323–327.
19 Testa R, Basso A, Piantanelli L, et al.
Blood catecholamine levels and lymphocyte beta-adrenoceptors following acute noise stress. Boll Soc Ital Biol Sper
1994; 70: 193–198.
20 Bishop CR, Athens JW, Boggs DR, Warner HR, Cartwright GE, Wintrobe MM. Leukokinetic studies. 13. A non-steady-state kinetic evaluation of the mechanism of cortisone-induced granulocytosis. J Clin Invest
1968; 47: 249–260.
21 Rabin BS, Moyana NM, Kusnecov A, Zhou D, Shurin MR, In: Hofmann-Goetz L, ed. Exercise and Immune Function
. Boca Raton: CRC Press, 1997: 21–37.
22 Haus E, Lakatua DJ, Swoyer J, Sackett-Lundeen L. Chronobiology in hematology and immunology. Am J Anat
1983; 168: 467–517.
23 Levi FA, Canon C, Touitou Y, et al.
Circadian rhythms in circulating T lymphocyte subtypes and plasma testosterone, total and free cortisol in five healthy men. Clin Exp Immunol
1988; 71: 329–335.