dcta anaesth. scand. 1979, 23, 344-348
Effect of Halothane Anaesthesia on Primary Antibody
Response in the Chicken M. SALO,M. VILJANEN, L. KANCAS and 0.-P. LEHTONEN Departments of Anaesthesiology, Medical Microbiology and Pharmacology, UniverTity of Turku, Turku, Finland
‘Ihe effect o f a single anaesthesia of 2 hours’ duration with I%, vjv Iialothane and of 1, 2 and 3 hours’ duration with 2%, v/v halothane was studied on the primary antibody response in line-bred chickens. I n contradistinction to earlier studies, tlie immunisation was performed during anaesthesia to resemble antigenic exposure in surgical practice. The IgG and IgM antibodies against the antigens, bovine serum albumin (BSA) and formalin-killed Brucella abortris organisms (Brncelh), were quantified by the enzyme-linked immunosorbent assay (ELISA). The halothane concentration of the blood was measured by gas chromatography at the end of anaesthesia. Anaesthesia with 1% and 2%, v/v halothane given in the antigen-processing phase of humoral immune response had no effect on the concentrations of IgGand IgM-anti-USA and IgG- and IgM-anti-Brucella antibodies in the primary antibody response. Received 1.7 December 1978, nccepted,fir publication 26 Februniy 197.9
et al. 1967, VILJANEN Halothane is a potent anaesthetic which responses (WINCARD affects every phase of the cell cycle (GRANT et al. 1973). Immunisation in the above-mentioned et al. 1974). A decrease of mitogen-induced transformation of lymphocytes in oitro has studies was carried out some days before the been reported when halothane is added anaesthesia, which was thus carried out in the directly to the culture medium (BRUCE proliferative phase of the humoral immune et al. 1967, HUMPHREY 1972, CULLEN et al. 1972), and in uiuo in the response (WINGARD et al. 1973, WINct al. 1973, SALOet al. et al. 1969a,b, VILJANEN chicken (VILJANEN 1978) and possibly in man (ESPANOL et al. GARL, 1973, BANDOH & FUJITA 1976). 1974). Halothane also inhibits cell-mediated However, in surgical practice bacterial et al. 1976) and anti- contamination and other antigenic exposures cytotoxicity (CULLEN body-dependent cellular cytotoxicity (VOSE are most likely to occur during anaesthesia & KIMBER1977). In the humoral immune and surgery. Therefore, in the present study, response, halothane decreases the number of the effect of halothane anaesthesia was studied splenic plaque-forming cells (WINGARII et al. on the primary antibody response in a model 1967, HUMPHREY et al. 1969a,b, WINGARD where immunisation was performed during 1973). I t also decreases the DNA synthesis the anaesthesia. This effect was studied on of splenic cells after immunisation with both T-dependent and T-independent antisheep red blood cells (SRBC) (BANDOH& body responses. T h e concentrations of classFUJITA1976)) but it has no or only a faint specific antibodies were measured by ELISA decreasing effect on the agglutination titres to allow separate quantitation of IgG and in the primary and secondary antibody I g M class antibodies. 0001-51 72/79/040344-0S$02.50/0
0 1 9 7 9 Thc Scandinavian Socirty of Anaesthesiologists
HALOTHANE AND PRIMARY ANTIBODY RESPONSE
MATERIAL AND METHODS
Iinmuiiisatioir The chickens were immunised 15 min after the onsrt of the anaesthesia intraperitoneally with 5 mg bovinr serum albumin (BSA, fraction V, Armour Pharmaceutical Co. Ltd., Eastbourne, England) and 10' formalin-killed Brucella abortus organisms (Brucella). Blood samples were drawn for antibody titrations 6 and 12 days after the anaesthesia. These sampling days were chosen from a preliminary experiment with 12 chickens anaesthetised with 2% v/v halothane for 2 hours (blood halothane concentration 0.2 16 k 0.031 g/l) and with 12 non-anaesthetised rontrol chickens, which were immunised as mentioned before.
.liiiiiials
The experimental animals wrre 4-week-old line P L2.hite Leghorn chickens, homozygous for thr major histocompatibility locus, genotype BZBZ. .4tiaesthesia
The chickens were divided into experimental groups, as shown in Table I . They were anaesthetised with halothane in a mixture of 30% oxygen and 70%, nitrogen at a gas flow rate of 3 I/min. using a Drager\.apor vaporizer (Dragerwerk Liibeck, Liibeck, Germany). The concentration of halothane in the blood was nirasured at the end of anaesthesia by gas chromatography (KANCAS et al. 1976) (Table I ) . The oxygen percentage in the box where the chickens were anaesthetised was 22-25%, measuied with the Berkman Oxygen Analyser Model D2 (Beckman Instruments Ltd., Glenrothes, Fife, Scotland). No C 0 2 could be measured in this box with a rapnograph (Godart NV, Bilthoven, England). The chickens were warmed during the anaesthesia with an infra-red lamp. The rectal temperatures were measured with an electric thermometer (Thermorapid, Medeor G.m.b.H., Hamburg. Germany) at the end of the anaesthesia (Table I ) . M'ith this experimental arrangement there was no impairment of acid-base balance (SALOet al. 1978). The control chickens were managed in the same way as the anaesthetised chickens, except for the anaesthesia. Two chickens died during anaesthesia: one during the anaesthesia with 2% v/v halothane for 2 hours and the other during the anaesthesia with 2% v/v lialothane for 3 hours. The anaesthesia with 1% v / v halothane in the inspired gas mixture caused only a light anaesthesia in the chickens (they startled when the box was knocked). By contrast, with 2% v/v halothane a proper thirdstage surgical anaesthesia was achieved.
Jliiantitatiou o j antibodieJ
A modification of the enzyme-linked immunosorbent assay (ELISA) of ENCVALL & PERLMANN (1972) was applied for quantitation of chicken IgG and IgM antibodies against BSA and Brucella abortus organisms. Anti-heavy-chain antibodies against chicken IgG and IgM (anti-y and anti-p) were produced in sheep and purified as described earlier (VIL,JANEN et al. 197.5). The method for conjugation of these antibodies with alkaline phosphatase (Sigma Chemicals, St. Louis, Mo., USA) was essentially the same as described & PERLMANN (1972). by ENCVALL The antibody quantitations were carried out in disposable polystyrene cuvettes, which were combined to form 9-cuvette blocks to help handling and measurement (Finnpipette-Labsystems, Helsinki, Finland) (LEINIKKI & PASSILA1976). The antigens were diluted with 0.05 M phosphate buffered saline (PBS p H 7.5) for coupling into the cuvettes. Two hundred microliters of BSA (10 mg/ml) or formalin-killed Brucella abortus organisms (107/ml) were added into the cuvettes. The cuvette blocks were incubated a t 37°C for 3 h and washed three times with 0.9% NaCl with 0.05% Tween 20 (NaC1-Tw). In order to decrease t h e non-specific absorption of proteins onto the solid phase, it was saturated by incubating the cuvettes at 37°C for 16 h with 200 p I of I:< normal sheep
'Table 1 The experimental groups. Blood halothane concentration and rectal temperature of the chickens at the end of anaesthesia. Arithmetic meansf s.d.
Group Control 1 "A v/v halothane 2% v/v halothane 2% v/v halothane 2% v / v halothane
***
2 I1 1h 2 11 3h
345
Number of chickens
Blood halothane concentration (g/l)
Rectal temperature ('C)
8 8 8 7 7
-
0.133 fO.019 0.217 f0.017 0.224k 0.020 0.222 k 0.023
40.3 f0.8 40.2 f0.5 39.6 k 0.6 37.9f 1.4*** 35.7 k 2.5***
= P
Effect of Halothane Anaesthesia on Primary Antibody
Response in the Chicken M. SALO,M. VILJANEN, L. KANCAS and 0.-P. LEHTONEN Departments of Anaesthesiology, Medical Microbiology and Pharmacology, UniverTity of Turku, Turku, Finland
‘Ihe effect o f a single anaesthesia of 2 hours’ duration with I%, vjv Iialothane and of 1, 2 and 3 hours’ duration with 2%, v/v halothane was studied on the primary antibody response in line-bred chickens. I n contradistinction to earlier studies, tlie immunisation was performed during anaesthesia to resemble antigenic exposure in surgical practice. The IgG and IgM antibodies against the antigens, bovine serum albumin (BSA) and formalin-killed Brucella abortris organisms (Brncelh), were quantified by the enzyme-linked immunosorbent assay (ELISA). The halothane concentration of the blood was measured by gas chromatography at the end of anaesthesia. Anaesthesia with 1% and 2%, v/v halothane given in the antigen-processing phase of humoral immune response had no effect on the concentrations of IgGand IgM-anti-USA and IgG- and IgM-anti-Brucella antibodies in the primary antibody response. Received 1.7 December 1978, nccepted,fir publication 26 Februniy 197.9
et al. 1967, VILJANEN Halothane is a potent anaesthetic which responses (WINCARD affects every phase of the cell cycle (GRANT et al. 1973). Immunisation in the above-mentioned et al. 1974). A decrease of mitogen-induced transformation of lymphocytes in oitro has studies was carried out some days before the been reported when halothane is added anaesthesia, which was thus carried out in the directly to the culture medium (BRUCE proliferative phase of the humoral immune et al. 1967, HUMPHREY 1972, CULLEN et al. 1972), and in uiuo in the response (WINGARD et al. 1973, WINct al. 1973, SALOet al. et al. 1969a,b, VILJANEN chicken (VILJANEN 1978) and possibly in man (ESPANOL et al. GARL, 1973, BANDOH & FUJITA 1976). 1974). Halothane also inhibits cell-mediated However, in surgical practice bacterial et al. 1976) and anti- contamination and other antigenic exposures cytotoxicity (CULLEN body-dependent cellular cytotoxicity (VOSE are most likely to occur during anaesthesia & KIMBER1977). In the humoral immune and surgery. Therefore, in the present study, response, halothane decreases the number of the effect of halothane anaesthesia was studied splenic plaque-forming cells (WINGARII et al. on the primary antibody response in a model 1967, HUMPHREY et al. 1969a,b, WINGARD where immunisation was performed during 1973). I t also decreases the DNA synthesis the anaesthesia. This effect was studied on of splenic cells after immunisation with both T-dependent and T-independent antisheep red blood cells (SRBC) (BANDOH& body responses. T h e concentrations of classFUJITA1976)) but it has no or only a faint specific antibodies were measured by ELISA decreasing effect on the agglutination titres to allow separate quantitation of IgG and in the primary and secondary antibody I g M class antibodies. 0001-51 72/79/040344-0S$02.50/0
0 1 9 7 9 Thc Scandinavian Socirty of Anaesthesiologists
HALOTHANE AND PRIMARY ANTIBODY RESPONSE
MATERIAL AND METHODS
Iinmuiiisatioir The chickens were immunised 15 min after the onsrt of the anaesthesia intraperitoneally with 5 mg bovinr serum albumin (BSA, fraction V, Armour Pharmaceutical Co. Ltd., Eastbourne, England) and 10' formalin-killed Brucella abortus organisms (Brucella). Blood samples were drawn for antibody titrations 6 and 12 days after the anaesthesia. These sampling days were chosen from a preliminary experiment with 12 chickens anaesthetised with 2% v/v halothane for 2 hours (blood halothane concentration 0.2 16 k 0.031 g/l) and with 12 non-anaesthetised rontrol chickens, which were immunised as mentioned before.
.liiiiiials
The experimental animals wrre 4-week-old line P L2.hite Leghorn chickens, homozygous for thr major histocompatibility locus, genotype BZBZ. .4tiaesthesia
The chickens were divided into experimental groups, as shown in Table I . They were anaesthetised with halothane in a mixture of 30% oxygen and 70%, nitrogen at a gas flow rate of 3 I/min. using a Drager\.apor vaporizer (Dragerwerk Liibeck, Liibeck, Germany). The concentration of halothane in the blood was nirasured at the end of anaesthesia by gas chromatography (KANCAS et al. 1976) (Table I ) . The oxygen percentage in the box where the chickens were anaesthetised was 22-25%, measuied with the Berkman Oxygen Analyser Model D2 (Beckman Instruments Ltd., Glenrothes, Fife, Scotland). No C 0 2 could be measured in this box with a rapnograph (Godart NV, Bilthoven, England). The chickens were warmed during the anaesthesia with an infra-red lamp. The rectal temperatures were measured with an electric thermometer (Thermorapid, Medeor G.m.b.H., Hamburg. Germany) at the end of the anaesthesia (Table I ) . M'ith this experimental arrangement there was no impairment of acid-base balance (SALOet al. 1978). The control chickens were managed in the same way as the anaesthetised chickens, except for the anaesthesia. Two chickens died during anaesthesia: one during the anaesthesia with 2% v/v halothane for 2 hours and the other during the anaesthesia with 2% v/v lialothane for 3 hours. The anaesthesia with 1% v / v halothane in the inspired gas mixture caused only a light anaesthesia in the chickens (they startled when the box was knocked). By contrast, with 2% v/v halothane a proper thirdstage surgical anaesthesia was achieved.
Jliiantitatiou o j antibodieJ
A modification of the enzyme-linked immunosorbent assay (ELISA) of ENCVALL & PERLMANN (1972) was applied for quantitation of chicken IgG and IgM antibodies against BSA and Brucella abortus organisms. Anti-heavy-chain antibodies against chicken IgG and IgM (anti-y and anti-p) were produced in sheep and purified as described earlier (VIL,JANEN et al. 197.5). The method for conjugation of these antibodies with alkaline phosphatase (Sigma Chemicals, St. Louis, Mo., USA) was essentially the same as described & PERLMANN (1972). by ENCVALL The antibody quantitations were carried out in disposable polystyrene cuvettes, which were combined to form 9-cuvette blocks to help handling and measurement (Finnpipette-Labsystems, Helsinki, Finland) (LEINIKKI & PASSILA1976). The antigens were diluted with 0.05 M phosphate buffered saline (PBS p H 7.5) for coupling into the cuvettes. Two hundred microliters of BSA (10 mg/ml) or formalin-killed Brucella abortus organisms (107/ml) were added into the cuvettes. The cuvette blocks were incubated a t 37°C for 3 h and washed three times with 0.9% NaCl with 0.05% Tween 20 (NaC1-Tw). In order to decrease t h e non-specific absorption of proteins onto the solid phase, it was saturated by incubating the cuvettes at 37°C for 16 h with 200 p I of I:< normal sheep
'Table 1 The experimental groups. Blood halothane concentration and rectal temperature of the chickens at the end of anaesthesia. Arithmetic meansf s.d.
Group Control 1 "A v/v halothane 2% v/v halothane 2% v/v halothane 2% v / v halothane
***
2 I1 1h 2 11 3h
345
Number of chickens
Blood halothane concentration (g/l)
Rectal temperature ('C)
8 8 8 7 7
-
0.133 fO.019 0.217 f0.017 0.224k 0.020 0.222 k 0.023
40.3 f0.8 40.2 f0.5 39.6 k 0.6 37.9f 1.4*** 35.7 k 2.5***
= P
